FMA:84050CytokineFMA:241981ChemokineFMA:86578InterleukinPR:000016456transmembrane protease serine 2PR:P07711cathepsin L1 (human)GO:0019015viral genomePR:000036197viral proteinPR:000003622angiotensin-converting enzyme 2GO:0031381viral RNA-directed RNA polymerase complexGO:0072516viral assembly compartmentGO:0019024ssRNA viral genomePR:000024938interferon alphaPR:000024939interferon betaGO:0019022RNA viral genomeGO:0002534cytokine production involved in inflammatory responseGO:0090195chemokine secretionGO:0006956complement activationGO:0061025membrane fusionGO:0075509endocytosis involved in viral entry into host cellGO:0046718viral entry into host cellGO:0005102receptor bindingD009026mortalityGO:0039694viral RNA genome replicationGO:0039690positive stranded viral RNA replicationGO:0019074viral RNA genome packagingGO:0009299mRNA transcriptionGO:0019081viral translationGO:0060337type I interferon signaling pathwayGO:0071360cellular response to exogenous dsRNA1increased3occurrence2decreasedSARS-CoV2020-03-01T10:42:462020-03-01T10:42:46HCoV-NL632021-02-07T07:01:232021-02-07T07:01:23Sars-CoV-2<p>Virus from the coronaviridae family related to SARS-CoV, 229E, NL63, OC43, HKU1 and MERS.</p>
<p>Transmitted by aerosols</p>
2021-02-23T04:50:402022-09-09T05:09:36Danger Associated Molecular Patters (DAMPs)2021-03-26T04:13:092021-03-26T04:13:09Pathogen Associated Molecular Patterns (PAMPs)2021-03-26T04:14:192021-03-26T04:14:19cell free mitochondrial DNA (mtDNA)2021-03-19T10:49:022021-03-29T07:07:24Stressor:624 SARS-CoV-22021-04-20T03:40:362021-04-20T03:40:3610090mouse10116ratsWCS_9606human9606Homo sapiens9974Manis javanica9615Canis familiaris9541Macaca fascicularis10036Mesocricetus auratus9669Mustela putorius furo9685Felis catus9666Mustela lutreolaWCS_452646Neovison vison9694Panthera tigrisWikiUser_22all speciesWCS_9606humans10095mice10090Mus musculus9666mink9685cat9544rhesus macaqueWCS_9615dogWikiUser_17mammalsIncreased, secretion of proinflammatory mediatorsIncreased proinflammatory mediatorsCellular<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Pro-inflammatory mediators are the chemical and biological molecules that initiate and regulate inflammatory reactions. Pro-inflammatory mediators are secreted following exposure to an inflammogen in a gender/sex or developmental stage independent manner. They are secreted during inflammation in all species. Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). Cell-derived pro-inflammatory mediators include cytokines, chemokines, and growth factors. Blood derived pro-inflammatory mediators include vasoactive amines, complement activation products and others. These modulators can be grouped based on the cell type that secrete them, their cellular localisation and also based on the type of immune response they trigger. For example, members of the interleukin (IL) family including <a href="https://bioregistry.io/genecards:IL2">IL-2</a>, <a href="https://bioregistry.io/genecards:IL4">IL-4</a>, <a href="https://bioregistry.io/genecards:IL7">IL-7</a>, <a href="https://bioregistry.io/genecards:IL9">IL-9</a>, <a href="https://bioregistry.io/genecards:IL15">IL-15</a>, <a href="https://bioregistry.io/genecards:IL21">IL-21</a>, <a href="https://bioregistry.io/genecards:IL3">IL-3</a>, <a href="https://bioregistry.io/genecards:IL5">IL-5</a> and Granulocyte-macrophage colony stimulating factor (<a href="https://bioregistry.io/genecards:CSF2">GM-CSF</a>) are involved in the adaptive immune responses. The pro-inflammatory cytokines include IL-1 family (<a href="https://bioregistry.io/genecards:IL1a">IL-1</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="color:blue"><u><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> </span></span></u></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL1b">IL-1</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL1ra">IL-1r</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL18">IL-18</a>, <a href="https://bioregistry.io/genecards:IL36a">IL-36</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL36b">IL-36</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL36g">IL-36</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">γ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL36Ra">IL-36R</a></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, <a href="https://bioregistry.io/genecards:IL37">IL-37</a>), <a href="https://bioregistry.io/genecards:IL6">IL-6 </a>family, Tumor necrosis factor (<a href="https://bioregistry.io/genecards:TNF">TNF</a>) family, <a href="https://bioregistry.io/genecards:IL17">IL-17</a>, and Interferon gamma (<a href="https://bioregistry.io/genecards:IFNg">IFN</a>-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">γ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">) (Turner et al., 2014). While <a href="https://bioregistry.io/genecards:IL4">IL-4</a> and <a href="https://bioregistry.io/genecards:IL5">IL-5</a> are considered T helper (Th) cell type 2 response, <a href="https://bioregistry.io/genecards:IFNg">IFN</a>-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">γ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"> is suggested to be Th1 type response.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Different types of pro-inflammatory mediators are secreted during innate or adaptive immune responses across various species (Mestas and Hughes, 2004). However, <a href="https://bioregistry.io/genecards:IL1">IL-1</a> family cytokines, <a href="https://bioregistry.io/genecards:IL4">IL-4</a>, <a href="https://bioregistry.io/genecards:IL5">IL-5</a>, <a href="https://bioregistry.io/genecards:IL6">IL-6</a>, <a href="https://bioregistry.io/genecards:TNFa">TNF</a>-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, IFN-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">γ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"> are the commonly measured mediators in experimental animals and in humans. Similar gene expression patterns involving inflammation and matrix remodelling are observed in human patients of pulmonary fibrosis and mouse lungs exposed to bleomycin (Kaminski, 2002). </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em><span style="color:red">Literature evidence for its perturbation:</span></em></strong></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">Several studies show increased proinflammatory mediators in rodent lungs and bronchoalveolar lavage fluid, and in cell culture supernatants following exposure to a variety of carbon nanotube (CNT) types and other materials</span>. Poland et al., 2008 showed that long and thin CNTs (>5 µm) can elicit asbestos-like pathogenicity through the continual release of pro-inflammatory cytokines and reactive oxygen species. Exposure to crystalline silica induces release of inflammatory cytokines (TNF-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">, IL-1, IL-6), transcription factors (Nuclear factor kappa B [NF-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">κ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">B], Activator protein-1 [AP-1]) and kinase signalling pathways in mice that contain NF-</span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">κ</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">B luciferase reporter (Hubbard et al., 2002). Boyles et al., 2015 found that lung responses to long multi-walled carbon nanotubes (MWCNTs) included high expression levels of pro-inflammatory mediators Monocyte Chemoattractant Protein 1 (MCP-1), Transforming growth factor beta 1 (TGF-β1), and TNF-α (Boyles et al., 2015). Bleomycin administration in rodents induces lung inflammation and increased expression of pro-inflammatory mediators (Park et al., 2019). Inflammation induced by bleomycin, paraquat and CNTs is characterised by the altered expression of pro-inflammatory mediators. A large number of nanomaterials induce expression of cytokines and chemokines in lungs of rodents exposed via inhalation (Halappanavar et al., 2011; Husain et al., 2015a). Similarities are observed in gene programs involving pro-inflammatory event is observed in both humans and experimental mice (Zuo et al., 2002).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">The selection of pro-inflammatory mediators for investigation varies based on the expertise of the lab, cell types studied and the availability of the specific antibodies.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><u>Real-time reverse transcription-polymerase chain reaction (qRT-PCR)</u> – will measure the abundance of cytokine mRNA in a given sample. The method involves three steps: conversion of RNA into cDNA by reverse transcription method, amplification of cDNA using the PCR, and the real-time detection and quantification of amplified products (amplicons) (Nolan T et al., 2006). Amplicons are detected using fluorescence, increase in which is directly proportional to the amplified PCR product. The number of cycles required per sample to reach a certain threshold of fluorescence (set by the user – usually set in the linear phase of the amplification, and the observed difference in samples to cross the set threshold reflects the initial amount available for amplification) is used to quantify the relative amount in the samples. The amplified products are detected by the DNA intercalating minor groove-binding fluorophore SYBR green, which produces a signal when incorporated into double-stranded amplicons. Since the cDNA is single stranded, the dye does not bind enhancing the specificity of the results. There are other methods such as nested fluorescent probes for detection, but SYBR green is widely used. RT-PCR primers specific to several pro-inflammatory mediators in several species including mouse, rat and humans, are readily available commercially.</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><u>Enzyme-linked immunosorbent assays (ELISA)</u> – permit quantitative measurement of antigens in biological samples. The method is the same as described for the MIE. Both ELISA and qRT-PCR assays are used in vivo and are readily applicable to in vitro cell culture models, where cell culture supernatants or whole cell homogenates are used for ELISA or mRNA assays. Both assays are straight forward, quantitative and require relatively a small amount of input sample. </span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Apart from assaying single protein or gene at a time, cytokine bead arrays or cytokine PCR arrays can also be used to detect a whole panel of inflammatory mediators in a multiplex method (Husain et al., 2015b). This method is quantitative and especially advantageous when the sample amount available for testing is scarce. Lastly, immunohistochemistry can also be used to detect specific immune cell types producing the pro-inflammatory mediators and its downstream effectors in any given tissue (Costa et al., 2017). Immunohistochemistry results can be used as weight of evidence; however, the technique is not quantitative and depending on the specific antibodies used, the assay sensitivity may also become an issue (Amsen and De Visser, 2009).</span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red"><u>Cell models</u> - of varying complexity have been used to assess the expression of pro-inflammatory mediators. Two dimensional submerged monocultures of the main fibrotic effector cells – lung epithelial cells, macrophages, and fibroblasts – have routinely been used <em>in vitro</em> due to the large literature base, and ease of use, but do not adequately mimic the <em>in vivo</em> condition (Sundarakrishnan <em>et al.,</em> 2018, Sharma <em>et al.,</em> 2016). Recently, the EpiAlveolar <em>in vitro</em> lung model (containing epithelial cells, endothelial cells, and fibroblasts) was used to predict the fibrotic potential of MWCNT, and researchers noted increases in the pro-inflammatory molecules TNF-</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">α</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">, IL-1</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">, and the pro-fibrotic TGF-</span></span></span><span style="font-size:11.0pt"><span style="font-family:"Arial",sans-serif">β</span></span><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red"> using ELISA (Barasova <em>et al.,</em> 2020). A similar, but less complicated co-culture model of immortalized human alveolar epithelial cells and idiopathic pulmonary fibrosis patient derived fibroblasts was used to assess pro-fibrotic signalling, and noted enhanced secretion of Platelet derived growth factor (PDGF) and Basic fibroblast growth factor (bFGF), as well as evidence for epithelial to mesenchymal transition of epithelial cells in this system (Prasad et al., 2014). Models such as these better capitulate the <em>in vivo</em> pulmonary alveolar capillary, but have lower reproducibility as compared to traditional submerged mono-culture experiments. </span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:red">Human, mouse, rat</span></span></span></p>
<p><span style="font-size:16px"><span style="font-family:Arial,Helvetica,sans-serif">Cytokines are the common pro-inflammatory mediators secreted following inflammogenic stimuli. Cytokines can be defined as diverse group of signaling protein molecules. They are secreted by different cell types in different tissues and in all mammalian species, irrespective of gender, age or sex. A lot of literature is available to support cross species, gender and developmental stage application for this KE. The challenge is the specificity; most cytokines exhibit redundant functions and many are pleotropic.</span></span></p>
CL:0000255eukaryotic cellHighMaleHighFemaleHighAdultsHighHighHigh<p><span style="color:#000000"><span style="font-family:Calibri">1. </span></span>Amsen D, de Visser KE, Town T. Approaches to determine expression of inflammatory cytokines. Methods Mol Biol. 2009;511:107-42. doi: 10.1007/978-1-59745-447-6_5. </p>
<p><span style="color:red"><span style="font-family:Calibri">2. </span></span>Barosova H, Maione AG, Septiadi D, Sharma M, Haeni L, Balog S, O'Connell O, Jackson GR, Brown D, Clippinger AJ, Hayden P, Petri-Fink A, Stone V, Rothen-Rutishauser B. Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS Nano. 2020 Apr 28;14(4):3941-3956. doi: 10.1021/acsnano.9b06860. </p>
<p><span style="color:#000000"><span style="font-family:Calibri">3. </span></span>Boyles MS, Young L, Brown DM, MacCalman L, Cowie H, Moisala A, Smail F, Smith PJ, Proudfoot L, Windle AH, Stone V. Multi-walled carbon nanotube induced frustrated phagocytosis, cytotoxicity and pro-inflammatory conditions in macrophages are length dependent and greater than that of asbestos. Toxicol In Vitro. 2015 Oct;29(7):1513-28. doi: 10.1016/j.tiv.2015.06.012. </p>
<p><span style="color:#000000"><span style="font-family:Calibri">4. </span></span>Costa PM, Gosens I, Williams A, Farcal L, Pantano D, Brown DM, Stone V, Cassee FR, Halappanavar S, Fadeel B. Transcriptional profiling reveals gene expression changes associated with inflammation and cell proliferation following short-term inhalation exposure to copper oxide nanoparticles. J Appl Toxicol. 2018 Mar;38(3):385-397. doi: 10.1002/jat.3548.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">5. </span></span>Halappanavar S, Jackson P, Williams A, Jensen KA, Hougaard KS, Vogel U, Yauk CL, Wallin H. Pulmonary response to surface-coated nanotitanium dioxide particles includes induction of acute phase response genes, inflammatory cascades, and changes in microRNAs: a toxicogenomic study. Environ Mol Mutagen. 2011 Jul;52(6):425-39. doi: 10.1002/em.20639. </p>
<p><span style="color:#000000"><span style="font-family:Calibri">6. </span></span>Hubbard AK, Timblin CR, Shukla A, Rincón M, Mossman BT. Activation of NF-kappaB-dependent gene expression by silica in lungs of luciferase reporter mice. Am J Physiol Lung Cell Mol Physiol. 2002 May;282(5):L968-75. doi: 10.1152/ajplung.00327.2001.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">7. </span></span>Husain M, Kyjovska ZO, Bourdon-Lacombe J, Saber AT, Jensen KA, Jacobsen NR, Williams A, Wallin H, Halappanavar S, Vogel U, Yauk CL. Carbon black nanoparticles induce biphasic gene expression changes associated with inflammatory responses in the lungs of C57BL/6 mice following a single intratracheal instillation. Toxicol Appl Pharmacol. 2015a Dec 15;289(3):573-88. doi: 10.1016/j.taap.2015.11.003.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">8. </span></span>Husain M, Wu D, Saber AT, Decan N, Jacobsen NR, Williams A, Yauk CL, Wallin H, Vogel U, Halappanavar S. Intratracheally instilled titanium dioxide nanoparticles translocate to heart and liver and activate complement cascade in the heart of C57BL/6 mice. Nanotoxicology. 2015b;9(8):1013-22. doi: 10.3109/17435390.2014.996192.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">9. </span></span>Kaminski N. Microarray analysis of idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol. 2003 Sep;29(3 Suppl):S32-6.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">10. </span></span>Mestas J, Hughes CC. Of mice and not men: differences between mouse and human immunology. J Immunol. 2004 Mar 1;172(5):2731-8. doi: 10.4049/jimmunol.172.5.2731.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">11. </span></span>Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR. Nat Protoc. 2006;1(3):1559-82. doi: 10.1038/nprot.2006.236.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">12. </span></span>Park SJ, Im DS. Deficiency of Sphingosine-1-Phosphate Receptor 2 (S1P<sub>2</sub>) Attenuates Bleomycin-Induced Pulmonary Fibrosis. Biomol Ther (Seoul). 2019 May 1;27(3):318-326. doi: 10.4062/biomolther.2018.131.</p>
<p><span style="color:#000000"><span style="font-family:Calibri">13. </span></span>Poland CA, Duffin R, Kinloch I, Maynard A, Wallace WA, Seaton A, Stone V, Brown S, Macnee W, Donaldson K. Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study. Nat Nanotechnol. 2008 Jul;3(7):423-8. doi: 10.1038/nnano.2008.111.</p>
<p><span style="color:red"><span style="font-family:Calibri">14. </span></span>Prasad S, Hogaboam CM, Jarai G. Deficient repair response of IPF fibroblasts in a co-culture model of epithelial injury and repair. Fibrogenesis Tissue Repair. 2014 Apr 29;7:7. doi: 10.1186/1755-1536-7-7. </p>
<p><span style="color:red"><span style="font-family:Calibri">15. </span></span>Sharma M, Nikota J, Halappanavar S, Castranova V, Rothen-Rutishauser B, Clippinger AJ. Predicting pulmonary fibrosis in humans after exposure to multi-walled carbon nanotubes (MWCNTs). Arch Toxicol. 2016 Jul;90(7):1605-22. doi: 10.1007/s00204-016-1742-7. </p>
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<p><span style="color:#000000"><span style="font-family:Calibri">17. </span></span>Turner MD, Nedjai B, Hurst T, Pennington DJ. Cytokines and chemokines: At the crossroads of cell signalling and inflammatory disease. Biochim Biophys Acta. 2014 Nov;1843(11):2563-2582. doi: 10.1016/j.bbamcr.2014.05.014. </p>
<p><span style="color:#000000"><span style="font-family:Calibri">18. </span></span>Zuo F, Kaminski N, Eugui E, Allard J, Yakhini Z, Ben-Dor A, Lollini L, Morris D, Kim Y, DeLustro B, Sheppard D, Pardo A, Selman M, Heller RA. Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6292-7. doi: 10.1073/pnas.092134099.</p>
2018-01-02T13:12:112023-04-21T09:51:32SARS-CoV-2 cell entry SARS-CoV-2 cell entry Cellular<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Coronavirus is recognized by the binding of S protein on the viral surface and angiotensin-converting enzyme 2 (ACE2) receptor on the cellular membrane, followed by viral entry via processing of S protein by transmembrane serine protease 2 (TMPRSS2) <span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Hoffmann et al., 2020b).</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif"> ACE2 is expressed on epithelial cells of the lung and intestine, and also can be found in the heart, kidney, adipose, and male and female reproductive tissues </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Lukassen et al., 2020, Lamers et al., 2020, Chen et al., 2020, Jing et al., 2020, Subramanian et al., 2020)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">. </span></span></span></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">SARS-CoV-2 is an enveloped virus characterized by displaying spike proteins at the viral surface (Juraszek et al., 2021). Spike is critical for viral entry (Hoffmann et al., 2020b) and is the primary target of vaccines and therapeutic strategies, as this protein is the immunodominant target for antibodies (Yuan et al., 2020, Ju et al., 2020, Robbiani et al., 2020, Premkumar et al., 2020, Liu et al., 2020). Spike is composed of S1 and S2 subdomains. S1 contains the N-terminal (NTD) and receptor-binding (RBD) domains, and the S2 contains the fusion peptide (FP), heptad repeat 1 (HR1) and HR2, the transmembrane (TM) and cytoplasmic domains (CD) (Lan et al., 2020). S1 leads to the recognition of the angiotensin-converting enzyme 2 (ACE2) receptor and S2 is involved in membrane fusion (Hoffmann et al., 2020b, Letko et al., 2020, Shang et al., 2020).</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">Upon binding to ACE2, the spike protein needs to be activated (or primed) through proteolytic cleavage (by a host protease) to allow membrane fusion. Fusion is a key step in viral entry as it is the way to release SARS-CoV-2 genetic material inside the cell. Cleavage happens between its spike’s S1 and S2 domains, liberating S2 that inserts its N-terminal domain into a host cell membrane and mediates membrane fusion </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Millet and Whittaker, 2018)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">.</span></span></span> Many proteases were identified to activate coronaviruses including furin, cathepsin L, trypsin-like serine proteases TMPRSS2, TMPRSS4, TMPRSS11, and human airway trypsin-like protease (HATs). These may operate at four different stages of the<a href="https://www.wikipathways.org/index.php/Pathway:WP4846"> virus infection cycle</a>: (a) pro-protein convertases (e.g., furin) during virus packaging in virus-producing cells, (b) extracellular proteases (e.g., elastase) after virus release into extracellular space, (c) cell surface proteases [e.g., type II transmembrane serine protease (TMPRSS2)] after virus attachment to virus-targeting cells, and (d ) lysosomal proteases (e.g., cathepsin L) after virus endocytosis in virus-targeting cells (Li, 2016).<span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif"> SARS-CoV-2 lipidic envelope may fuse with two distinct membrane types, depending on the host protease(s) responsible for cleaving the spike protein: (i) cell surface following activation by serine proteases such as TMPRSS2 and furin </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Hoffmann et al., 2020b)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">; or (ii) endocytic pathway within the endosomal–lysosomal compartments including processing by lysosomal cathepsin L </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Yang and Shen, 2020)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">. These flexibility for host cell factors mediating viral entry, highlights that the availability of factors existing in a cell type dictates the mechanism of viral entry </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Kawase et al., 2012)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">. When TMPRSS2 (or other serine proteases such as TMPRSS4 </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Zang et al., 2020)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif"> or human airway trypsin-like protease [HAT]</span></span></span> <span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Bestle et al., 2020a)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">) is expressed, fusion of the virus with the cell surface membrane is preferred </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Shirato et al., 2018)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">, while in their absence, the virus can penetrate the cell by endocytosis </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Kawase et al., 2012)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">. A third factor has also been shown to facilitate SARS-CoV-2 entry in cells that have ACE2 and even promote, although to very low levels, SARS-CoV-2 entry in cells that lack ACE2 and TMPRSS2 which is the neuropilin-1 (NRP-1) </span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">(Cantuti-Castelvetri et al., 2020)</span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-family:"Calibri",sans-serif">. This key event deals with SARS-CoV-2 entry in host cells and is divided in three categories: TMPRSS2, capthesin L and NRP-1.</span></span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>TMPRSS2 Spike cleavage:</strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TMPRSS2 (transmembrane serine protease 2, (<a href="https://www.ncbi.nlm.nih.gov/gene/7113" style="color:blue; text-decoration:underline">https://www.ncbi.nlm.nih.gov/gene/7113</a>) is a cell-surface protease (Hartenian et al., 2020) that facilitates entry of viruses into host cells by proteolytically cleaving and activating viral envelope glycoproteins. Viruses found to use this protein for cell entry include Influenza virus and the human coronaviruses HCoV-229E, MERS-CoV, SARS-CoV and SARS-CoV-2 (COVID-19 virus).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TMPRSS2 is a membrane bound serine protease also known as epitheliasin. TMPRSS2 belongs to the S1A class of serine proteases alongside proteins such as factor Xa and trypsin. Whilst there is evidence that TMPRSS2 autoclaves to generate a secreted protease, its physiological function has not been clearly identified. However, it is known to play a crucial role in facilitating entry of coronavirus particles into cells by cleaving the spike protein (Huggins, 2020).</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">After ACE2 receptor binding, SARS-CoV-2 S proteins can be subsequently cleaved and activated by host cell-surface protease at the S1/S2 and S2’ sites, generating the subunits S1 and S2 that remain non-covalently linked. Cleavage leads to activation of the S2 domain that drives fusion of the viral and host membranes (Hartenian et al., 2020, Walls et al., 2016). For other coronaviruses, processing of spike was proposed to be sequential with S1/S2 cleavage preceding that of S2. Cleavage at S1/S2 may be crucial for inducing conformational changes required for receptor binding or exposure of the S2 site to host proteases. </span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The S1/S2 site of SARS-CoV-2 S protein contains an insertion of four amino acids providing a minimal furin cleavage site (RRAR685↓) (that is absent in SARS-CoV). Interestingly, the furin cleavage site has been implicated in increased viral pathogensis (Bestle et al., 2020b, Huggins, 2020). <span style="color:black">Processing of the spike protein by furin at the S1/S2 cleavage site is thought to occur following viral replication in the endoplasmic reticulum Golgi intermediate compartment (ERGIC) </span><span style="color:black">(Hasan et al., 2020)</span><span style="color:black">. T</span>he spike S2’ cleavage site of SARS-CoV-2 possesses a paired dibasic motif with a single KR segment (KR815↓) (as SARS-CoV) that is recognized by trypsin-like serine proteases such as TMPRSS2. <strong><span style="color:black">The current data support a model for SARS-CoV-2 entry in which furin-mediated cleavage at the S1/S2 site pre-primes spike during biogenesis, facilitating the activation for membrane fusion by a second cleavage event at S2’ by TMPRSS2 following ACE2 binding</span></strong> <span style="color:black">(Bestle et al., 2020b, Johnson et al., 2020)</span><span style="color:black">.</span></span></span></p>
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<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Virus</span></span></p>
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<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">S1/S2 site</span></span></p>
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<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">S2’ site</span></span></p>
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<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">SARS-CoV-2</span></span></p>
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<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TNSP<strong>RRAR</strong>|SVA</span></span></p>
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<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">PSKPS<strong>KR</strong>|SFIEDL</span></span></p>
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<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">SARS-CoV </span></span></p>
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<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">S----LLR|STS</span></span></p>
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<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">PLKPT<strong>KR</strong>|SFIEDL</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Camostat mesylate, an inhibitor of TMPRSS2, blocks SARS-CoV-2 infection of lung cells like <span style="color:black">Calu-3 cells but not Huh7.5 and Vero E6 cells</span>. Cell entry was assessed using a viral isolate and viral pseudotypes (artificial viruses) expressing the COVID-19 spike (S) protein. The ability of the viral pseudotypes (expressing S protein from SARS-CoV and SARS-CoV-2) to enter human and animal cell lines was demonstrated, showing that SARS-CoV-2 can enter similar cell lines as SARS-CoV. Amino acid analysis and cell culture experiments showed that, like SARS-CoV, SARS-CoV-2 spike protein binds to human and bat angiotensin-converting enzyme 2 (ACE2) and uses a cellular protease TMPRSS2 for priming. Priming activates the spike protein to facilitate viral fusion and entry into cells. Cell culture experiments were performed using immortalized cell lines and primary human lung cells (Hoffmann et al., 2020b, Rahman et al., 2020).</span></span></p>
<p> </p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>Spike binding to neuropilin-1:</strong></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Neuropilin-1 (NRP1) is a transmembrane glycoprotein that serves as a cell surface receptor for semaphorins and various ligands involved in angiogenesis in vertebrates. NRP1 is expressed in neurons, blood vessels (endothelial cells), immune cells and many other cell types in the mammalian body (maternal fetal interface) and binds a range of structurally and functionally diverse extracellular ligands to modulate organ development and function (Raimondi et al., 2016). NRP1 is well described as a co-receptor for members of the class 3 semaphorins (SEMA3) or vascular endothelial growth factors (VEGFs) (Gelfand et al., 2014). Structurally, NRP1 comprises seven sub-domains, of which the first five are extracellular; two CUB domains (a1 and a2), two coagulation factor V/VIII domains (FV/VIII; b1 and b2) and a meprin, A5 μ-phosphatase domain (MAM; c). NRP1 contains only a short cytosolic tail with a PDZ-binding domain lacking internal signaling activity. The different ligand families bind to different sites of NRP1; SEMA3A binding requires the first three sub-domains of NRP1 (a1, a2, and b1), whereas binding of VEGF-A requires the b1 and b2 domains (Muhl et al., 2017). Additional studies conducted by means of in silico computational technology to identify and validate inhibitors of the interaction between NRP1 and SARS-CoV-2 Spike protein are reported in (Perez-Miller et al., 2020). Represents a schematic picture of VEGF-A triggered phosphorylation of VEGF-R2. Screening of NRP-1/VEGF-A165 inhibitors by in-cell Western (Perez-Miller et al., 2020).v NRP1 acts as a co-receptor for SARS-CoV-2. </span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">NRP1 is a receptor for <span style="color:black">furin-cleaved SARS-CoV-2 spike peptide </span><span style="color:black">(Cantuti-Castelvetri et al., 2020, Daly et al., 2020, Johnson et al., 2020)</span><span style="color:black">. Blockade of NRP1 reduces infectivity and entry, and alteration of the furin site leads to loss of NRP1 dependence, reduced replication in Calu3, augmented replication in Vero E6, and attenuated disease in a hamster pathogenesis disease model </span><span style="color:black">(Johnson et al., 2020)</span><span style="color:black">.</span> In fact, a small sequence of amino acids was found that appeared to mimic a protein sequence found in human proteins that interact with NRP1. The spike protein of SARS-CoV-2 binding with NRP1 aids viral infection of human cells. This was confirmed by applying a range of structural and biochemical approaches to establish that the spike protein of SARS-CoV-2 does indeed bind to NRP1. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic RRAR C-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors. It was reported that the S1 CendR motif directly bound NRP1 by X-ray crystallography and biochemical approaches. Blocking this interaction using RNAi or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture (Daly et al., 2020).</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">NRP1, known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, which was revealed by a monoclonal blocking antibody against NRP1. It was found that a SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells faced the nasal cavity (Cantuti-Castelvetri et al., 2020). Furthermore, it has been found that NRP1 is a new potential SARS<span style="font-family:"Cambria Math",serif">‑</span>CoV<span style="font-family:"Cambria Math",serif">‑</span>2 infection mediator implicated in the neurologic features and central nervous system involvement of COVID<span style="font-family:"Cambria Math",serif">‑</span>19. Preclinical studies have suggested that NRP1, a transmembrane receptor that lacks a cytosolic protein kinase domain and exhibits high expression in the respiratory and olfactory epithelium, may also be implicated in COVID<span style="font-family:"Cambria Math",serif">‑</span>19 by enhancing the entry of SARS<span style="font-family:"Cambria Math",serif">‑</span>CoV<span style="font-family:"Cambria Math",serif">‑</span>2 into the brain through the olfactory epithelium. NRP1 is also expressed in the CNS, including olfactory<span style="font-family:"Cambria Math",serif">‑</span>related regions such as the olfactory tubercles and paraolfactory gyri. Supporting the potential role of NRP1 as an additional SARS<span style="font-family:"Cambria Math",serif">‑</span>CoV<span style="font-family:"Cambria Math",serif">‑</span>2 infection mediator implicated in the neurologic manifestations of COVID<span style="font-family:"Cambria Math",serif">‑</span>19. Accordingly, the neurotropism of SARS<span style="font-family:"Cambria Math",serif">‑</span>CoV<span style="font-family:"Cambria Math",serif">‑</span>2 via NRP1<span style="font-family:"Cambria Math",serif">‑</span>expressing cells in the CNS merits further investigation (Davies et al., 2020).</span></span></p>
<p style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Up-regulation of NRP1 protein in diabetic kidney cells hints at its importance in a population at risk of severe COVID-19. Involvement of NRP-1 in immune function is compelling, given the role of an exaggerated immune response in disease severity and deaths due to COVID-19. NRP-1 has been suggested to be an immune checkpoint of T cell memory. It is unknown whether involvement and up-regulation of NRP-1 in COVID-19 may translate into disease outcome and long-term consequences, including possible immune dysfunction (Mayi et al., 2021).</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The main feature of NRP1 co-receptor is to form complexes with multiple other receptors. Hence, there is a competition between receptors to complex with NRP-1, which may determine their abilities both quantitatively and qualitatively to transduce signals. It is tempting to hypothesize that the occupancy of NRP-1 with one receptor may thus decrease its availability for virus entry. Recent proteomics work has shown that NRP-1 can form a complex with the α7 nicotinic receptor in mice. Both receptors are expressed in the human nasal and pulmonary epithelium (Mayi et al., 2021).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">NRP1, is highly expressed in the respiratory and olfactory epithelium; it is also expressed in the CNS, including olfactory related regions such as the olfactory tubercles and paraolfactory gyri (Davies et al., 2020).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">More information on tissue distribution and protein expression of NRP1 can be found in https://www.proteinatlas.org/ENSG000000992 50-NRP1</span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>Spike entry via <span style="color:black">lysosomal cathepsins and endocytosis</span>:</strong></span></span></p>
<p style="text-align:justify"><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Evidence shows the role of TMPRSS2 and other serine proteases in activating the coronavirus spike protein for plasma membrane fusion. However, studies using various cell culture systems showed that SARS-CoV2 could enter cells via an alternative endosomal–lysosomal pathway.</span> Evidence came from studies<span style="color:black"> demonstrating that lysosomotropic agents reduced SARS-CoV replication in cells lacking TMPRSS2 and other studies, using highly potent and specific small-molecule cathepsin inhibitors, to understand the role of cathepsins in processing and activating the spike for membrane fusion, mainly of cathepsin L (one of the 11 cathepsins) </span><span style="color:black">(Rossi et al., 2004, Simmons et al., 2005)</span><span style="color:black">. SARS-CoV-2 and other coronaviruses can establish infection through endosomal entry in commonly used in vitro cell culture systems. Of relevance, inhibitors of the endosomal pathway, as the cathepsin inhibitor Z-FA-FMK and PIKfyve inhibitor apilimod, blocked viral entry in Huh7.5 and Vero E6 cells but not Calu-3 cells.</span></span></span></p>
<p style="text-align:justify"><strong>Viral entry leads to delivery of virion proteins and translation of viral proteins immediately: </strong></p>
<p style="text-align:justify"><span style="font-size:14px">Coronavirus is a class of viruses that have single-stranded positive-sense RNA genomes in their envelopes [Kim D,<em> et al., 2020</em>]. The virus contains a <span style="color:#131413">29.7 kB positive-sense RNA genome flanked by 5' and 3' untranslated regions of 265 and 342 nucleotides, respectively</span><span style="color:black"> </span><span style="color:#131413">that contain cis-acting secondary RNA structures essential for RNA synthesis [</span>Huston N. C.<em> et al., 2021</em>]<span style="color:black">. T</span>he genome just prior to the 5′ end contains the transcriptional regulatory sequence leader (TRS-L) [Budzilowicx C.J., <em>et al., 1985</em>]. The SARS-CoV genome is polycistronic and contains 14 open reading frames (ORFs) that are expressed by poorly understood mechanisms [Snijder E. J., <em>et al.</em>, 2003]<span style="color:black">.</span> Preceding each ORF there are other TRSs called the body TRS (<span style="color:black">TRS B). </span>The <span style="color:black">5′ two-thirds of the </span>genome contains <span style="color:black">two large, overlapping, nonstructural ORFs and the 3′ third contains the remainder ORFs [Di H., <em>et al.</em>, 2018].</span> Genome expression starts with the translation of <span style="color:#131413">two large ORFs of the 5’ two-thirds: ORF1a of</span><span style="color:black"> 4382 amino acids and ORF1ab of 7073 amino acid that occurs via a</span><span style="color:#131413"> programmed (- 1) ribosomal frameshifting </span>[Snijder E. J., <em>et al.</em>, 2003]<span style="color:black">, yielding</span><span style="color:#131413"> pp1a and pp1ab</span><span style="color:black">. These two polyproteins are cleaved into 16 subunits by two viral proteinases encoded by ORF1a,</span> <span style="color:black">nsp3, and nsp5 that contain a papain-like protease domain and a 3C-like protease domain</span> [Sacco M. D. <em>et al., 2020</em>]<span style="color:#131413">. </span><span style="color:black">The processing products are a group of replicative enzymes, named nsp1-nsp16, that assemble into a viral replication a</span>nd transcription <span style="color:black">complex (RTC) associated with membranes of endoplasmic reticulum (ER) with the help of various membrane-associated viral proteins [</span>Klein<em> </em>S., <em>et al., 2021</em>, Snijder E. J.<em>, et al., 2020, </em>V'Kovski P. , <em>et al., 2021</em>]<span style="color:black">. This association leads to replication factories or organelles, that are originate new membranous structures that are observed by electron mciroscopy . They are a feature of all coronaviridae and the site of viral replication and transcription hidden from innate immune molecules.</span></span></p>
<p>SARS-CoV2 entry can be determined by many different ways:</p>
<p>1) quantitative RT-PCR specific to the subgenomic mRNA of the N transcript, in cells manipulated with host factors that express of not TMPRSS2, cathepsinL, neuropilin-1, hACE2 [Glowacka I, et al. (2011)], or exogenous addition of HAT or furin.</p>
<p>2) using spike-pseudotyped viral particles expressing GFP/luciferase/bgalactosidase and comparing with vesicular stomatitis virus G seudotyped particles expressing the same reporter analysed in manipulated cultured with cell lines, followed by determining fluorescence, biolumincescence, luciferase activity in cell lysates [Hoffmann M, et al. (2020)].</p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>TMPRSS2:</strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TMPRSS2 gene expression can be measured by RNAseq and microarray (Baughn et al., 2020).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Expression levels of TMPRSS2 can be measured by RNA in situ hybridization (RNA-ISH) (Qiao et al., 2020)</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>NRP-1:</strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Several methods have been identified in the literature for measuring and detecting NRP1 receptor binding. Briefly described:</span></span></p>
<ol>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">X-ray crystallography and biochemical approaches help to show that the S1 CendR motif directly bound NRP1 (1). Binding of the S1 fragment to NRP1 was assessed and ability of SARS-CoV-2 to use NRP1 to infect cells was measured in angiotensin-converting enzyme-2 (ACE-2)-expressing cell lines by knocking out NRP1 expression, blocking NRP1 with 3 different anti-NRP1 monoclonal antibodies, or using NRP1 small molecule antagonists </span><span style="color:black">(Centers for Disease Control and Prevention, 2020, Daly et al., 2020)</span><span style="color:black">.</span></span></span></li>
</ol>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Key findings (Centers for Disease Control and Prevention, 2020, Daly et al., 2020): </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">• The S1 fragment of the cleaved SARS-CoV-2 spike protein binds to the cell surface receptor neuropilin-1 (NRP1). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">• SARS-CoV-2 utilizes NRP1 for cell entry as evidenced by decreased infectivity of cells in the presence of: NRP1 deletion (p <0.01). Three different anti-NRP1 monoclonal antibodies (p <0.001). Selective NRP1 antagonist, EG00229 (p <0.01).</span></span></p>
<ol start="2">
<li><span style="font-size:11pt"><span style="color:black"><span style="font-family:"Calibri",sans-serif">Cell lines were modified to express ACE2 and TMPRSS2, the two known SARS-CoV-2 host factors, and NRP1 to assess the contribution of NRP1 to infection. Autopsy specimens from multiple airway sites were stained with antibodies against SARS-CoV-2 proteins, ACE2, and NRP1, to visualize co-localization of proteins (6, 15).</span></span></span></li>
</ol>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Key findings (Cantuti-Castelvetri et al., 2020, Centers for Disease Control and Prevention, 2020): </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">• Infectivity of cells expressing angiotensin converting enzyme-2 (ACE2, receptor for SARS-CoV-2), transmembrane protease serine-2 (TSS2, primes the Spike [S] protein), and neuropilin-1 (NRP1) with pseudovirus expressing the SARS-CoV-2 S1 protein was approximately 3-fold higher than in cells expressing either ACE2 or TSS2 alone (p<0.05).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">• Analysis of autopsy tissue from COVID-19 patients showed co-localization of the SARS-CoV-2 spike (S) protein and NRP1 in olfactory and respiratory epithelium.</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Virtual screen of nearly 0.5 million compounds against the NRP-1 CendR site, resulting in nearly 1,000 hits. A pharmacophore model was derived from the identified ligands, considering both steric and electronic requirements. Preparation of receptor protein and grid for virtual screening, docking of known NRP-1 targeting compounds, ELISA based NRP1-VEGF-A165 protein binding assay; more details on methodology in the referenced paper </span></span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">(Perez-Miller et al., 2020)</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TMPRSS2 vertebrates (Lam et al., 2020)</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">NRP1 in human & rodents (but also present in monkey and other vertebrates </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Lu and Meng, 2015)</span></span></p>
<p><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">The ability for SARS-CoV-2 to use multiple host pathways for viral entry, means that it is critical to map which viral entry pathway is prevalent in specific cell types. This is key for understanding coronavirus biology, but also use informed decisions to select cells for cell-based genetic and small-molecule screens and to interpret data. In fact, a combination of protease inhibitors that block both TRMPSS2 and cathepsin L is the most efficient combination to block coronavirus infection </span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Yamamoto et al., 2020, Shang et al., 2020, Shirato et al., 2018)</span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. In accordance, SARS-CoV-2 entry processes are highly dependent on endocytosis and endocytic maturation in cells that do not express TMPRSS2, such as VeroE6 or 293T cells </span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Murgolo et al., 2021, Kang et al., 2020, Mirabelli et al., 2020, Riva et al., 2020)</span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. However, even in these cells, heterologous expression of TMPRSS2 abrogates the pharmacological blockade of cathepsin inhibitors </span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Kawase et al., 2012, Hoffmann et al., 2020a)</span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. Treatment of SARS-CoV-2 with trypsin enables viral cell surface entry, even when TMPRSS2 is absent. Moreover, TMPRSS2 is more efficient to promote viral entry than cathepsins </span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Lamers et al., 2020)</span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">, as when both factors are present,d cathepsin inhibitors are less effective than TMPRSS2 inhibitors </span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">(Hoffmann et al., 2020b)</span></span></span></span><span style="font-family:"MinionPro-Regular",serif"><span style="color:black"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">. Therefore it is critical to map which cells contain the different types of proteases.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">In summary, TMPRSS2 appears to be expressed in a wide range of healthy adult organs, but in restricted cell types, including:</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">AT2 and clara cells of lungs</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">sinusoidal endothelium, and hepatocyte of the liver, </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">endocrine cells of the prostate, </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">goblet cells , and enterocytes of the small intestine, </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">intercalated cells, and the proximal tubular of the kidney.</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Ciliated, secretory and suprabasal of nasal</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">spermatogonial stem cells of testes</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">cyto tropoblast and peri vascular cells of placenta</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The nasal epithelium expresses various combinations of factors that, in principle, could facilitate SARS-CoV-2 infection, but it also expresses robust basal levels of RFs, which may act as a strong protective barrier in this tissue.</span></span></li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">There is a shift in TMPRSS2 regulation during nasal epithelium differentiation in young individuals that is not occurring in old individuals (Lin et al., 1999, Lucas et al., 2008, Singh et al., 2020). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Only a small minority of human respiratory and intestinal cells have genes that express both ACE2 and TMPRSS2. Amongst the ones that do, three main cell types were identified: A) lung cells called type II pneumocytes (which help maintain air sacs, known as alveoli); B) intestinal cells called enterocytes, which help the body absorb nutrients; and C) goblet cells in the nasal passage, which secrete mucus (Ziegler et al., 2020). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The clinical manifestations of COVID‐19 include not only complications from acute myocardial injury, elevated liver enzymes, and acute kidney injury in patients presenting to hospitals, but also gastrointestinal symptoms in community patients experiencing milder forms of the disease (Madjid et al., 2020, Pan et al., 2020). </span></span></p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>NRP-1:</strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">All life stages</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The expression of isoforms 1 (NRP1) and 2 (NRP2) does not seem to overlap. Isoform 1 is expressed by the blood vessels of different tissues. In the developing embryo it is found predominantly in the nervous system. In adult tissues, it is highly expressed in heart and placenta; moderately in lung, liver, skeletal muscle, kidney and pancreas; and low in adult brain. Isoform 2 is found in liver hepatocytes, kidney distal and proximal tubules. Expressed in colon and 234 other tissues with Low tissue specificity (UniProtKB). </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The expression of NRP1 protein in gastric cancer was not related to gender or age (Cao et al., 2020).</span></span></p>
<p> </p>
<p><strong><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Sex Applicability:</span></span></strong></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>TMPRSS2:</strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Androgen receptors (ARs) play a key role in the transcription of TMPRSS2 (Fig. 1). This may explain the predominance of males to COVID-19 infection, fatality, and severity because males tend to have a higher expression and activation of ARs than females, which is due to the presence of dihydrotestosterone (DHT).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Regulation of COVID-19 severity and fatality by sex hormones. Females have aromatase, the enzyme that converts androgen substrates into estrogen. On the other hand, males have steroid 5α reductase, the enzyme that is responsible for the conversion of testosterone into dihydrotestosterone (DHT). In case of males, DHT activates androgen receptor (AR) that binds to the androgen response element (ARE) present in the promoter of TMPRSS2 gene, leading to its transcription. This ultimately results into enhanced processing of viral spike protein for greater entry and spread of SARS-CoV-2 into host cells. On the other hand,in females, estrogen activates estrogen receptor (ER), which binds to the estrogen response element (ERE) present in the promoter of eNOS gene to drive its transcription and catalyze the formation of nitric oxide (NO) from L-arginine. This NO is involved in vasodilation as well as inhibition of viral replication. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>NRP-1:</strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">For more information difference of NRP1 expression between male and female see <a href="https://www.proteinatlas.org/ENSG00000099250-NRP1/tissue"><span style="color:blue">https://www.proteinatlas.org/ENSG00000099250-NRP1/tissue</span></a><span style="color:blue">.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The expression of NRP1 protein in gastric cancer was not related to gender, age. The expression of NRP1 protein in gastric cancer is closely correlated to clinical stage, tumor size, TNM stage, differentiation, and lymph node metastasis (Cao et al., 2020).</span></span></p>
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2020-03-01T10:29:312023-04-04T07:39:34Binding to ACE2Binding to ACE2Molecular<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">Angiotensin-converting enzyme 2 (<a href="https://www.genecards.org/cgi-bin/carddisp.pl?gene=ACE2">ACE2</a>) is an enzyme that can be found either attached to the membrane of the cells (mACE2) in many tissues and in a soluble form form (sACE2). </span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:12px">A table on ACE2 expression levels according to tissues <em>(Kim et al.)</em></span></span></p>
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<td style="background-color:#a6a6a6; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:9.0pt">Sample size</span></strong></span></span></p>
</td>
<td style="background-color:#a6a6a6; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:9.0pt">ACE2 mean expression</span></strong></span></span></p>
</td>
<td style="background-color:#a6a6a6; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:9.0pt">Standard deviation of expression</span></strong></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">Intestine</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">51</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">9.50</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">1.183</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">Kidney</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">129</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">9.20</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">2.410</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">Stomach</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">35</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">8.25</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">3.715</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">Bile duct</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">9</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">7.23</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">1.163</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">Liver</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">50</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">6.86</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">1.351</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">Oral cavity</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">32</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">6.23</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">1.271</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">Lung</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">110</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">5.83</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">0.710</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">Thyroid</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">59</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">5.65</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">0.646</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">Esophagus</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">11</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">5.31</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">1.552</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">Bladder</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">19</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">5.10</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">1.809</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">Breast</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">113</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">4.61</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">0.961</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">Uterus</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">25</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">4.37</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">1.125</span></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">Protaste</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:146px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">52</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">4.35</span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; vertical-align:top; width:147px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">1.905</span></span></span></p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><span style="color:#0070c0">ACE2 receptors in the brain (endothelial, neuronal and glial cells):</span></strong></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#0070c0">The highest ACE2 expression level in the brain was found in the pons and medulla oblongata in the human brainstem, containing the medullary respiratory centers (Lukiw et al., 2020). High ACE2 receptor expression was also found in the amygdala, cerebral cortex and in the regions involved in cardiovascular function and central regulation of blood pressure including the sub-fornical organ, nucleus of the tractus solitarius, paraventricular nucleus, and rostral ventrolateral medulla (Gowrisankar and Clark 2016; Xia and Lazartigues 2010). The neurons and glial cells, like astrocytes and microglia also express ACE-2. </span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#0070c0">In the brain, ACE2 is expressed in endothelium and vascular smooth muscle cells (Hamming et al., 2004), as well as in neurons and glia (Gallagher et al., 2006; Matsushita et al., 2010; Gowrisankar and Clark, 2016; Xu et al., 2017; de Morais et al., 2018) (from Murta et al., 2020). Astrocytes are the main source of angiotensinogen and express ATR1 and MasR; neurons express ATR1, ACE2, and MasR, and microglia respond to ATR1 activation (Shi et al., 2014; de Morais et al., 2018). </span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#1abc9c"><strong><em>ACE2 receptors in the intestines</em></strong></span></span></span></p>
<p dir="ltr" style="text-align:justify"><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#1abc9c"><span style="background-color:transparent">The highest levels of ACE2 are found at the luminal surface of the enterocytes, the differentiated epithelial cells in the small intestine, lower levels in the crypt cells and in the colon (Liang et al, 2020; Hashimoto et al., 2012, Fairweather et al. 2012; Kowalczuk et al. 2008). </span></span></span></span></p>
<p dir="ltr" style="text-align:justify"> </p>
<p dir="ltr" style="text-align:justify"> </p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Arial,sans-serif"><span style="color:black"><strong><span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif"><em>In vitro</em> methods supporting interaction between ACE2 and SARS-CoV-2 spike protein</span></span></strong></span></span></span></p>
<p style="text-align:justify"><span style="font-size:12pt"><span style="font-family:Arial,sans-serif"><span style="color:black"><span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif">Several reports using surface plasmon resonance (SPR) or biolayer interferometry binding (BLI) approaches. to study the interaction between recombinant ACE2 and S proteins have determined a dissociation constant (Kd) for SARS-CoV S and SARS-CoV-2 S as follow,</span></span></span></span></span></p>
<table cellspacing="0" class="Table" style="border-collapse:collapse; width:568px">
<tbody>
<tr>
<td style="background-color:#f7f7f7; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; height:28px; width:176px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:9.0pt">Reference</span></strong></span></span></p>
</td>
<td style="background-color:#f7f7f7; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:28px; width:102px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:9.0pt">ACE2 protein </span></strong></span></span></p>
</td>
<td style="background-color:#f7f7f7; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:28px; width:140px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:9.0pt">SARS-CoV S</span></strong></span></span></p>
</td>
<td style="background-color:#f7f7f7; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:28px; width:151px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:9.0pt">SARS-CoV2 S</span></strong></span></span></p>
</td>
<td style="background-color:#f7f7f7; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:28px; width:128px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:9.0pt">Method</span></strong></span></span></p>
</td>
<td style="background-color:#f7f7f7; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:1px solid black; height:28px; width:156px">
<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><strong><span style="font-size:9.0pt">Measured Kd</span></strong></span></span></p>
</td>
</tr>
<tr>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; width:176px">
<p><span style="font-size:11px"><span style="font-family:Arial,Helvetica,sans-serif">doi:<a class="id-link" href="https://doi.org/10.1126/science.abb2507" rel="noopener" target="_blank">10.1126/science.abb2507</a></span></span></p>
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:102px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">1–615 aa</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">306–577 aa</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"> </span></span></p>
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:128px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">SPR</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">325.8 nM</span></span></span></p>
</td>
</tr>
<tr>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"> </span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">1–1208 aa</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">14.7 nM</span></span></span></p>
</td>
</tr>
<tr>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; width:176px">
<p><span style="font-size:11px"><span style="font-family:Arial,Helvetica,sans-serif">doi:<a class="id-link" href="https://doi.org/10.1001/jama.2020.3786" rel="noopener" target="_blank">10.1001/jama.2020.3786</a></span></span></p>
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:102px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">19–615 aa</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">306–527 aa</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"> </span></span></p>
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:128px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">SPR</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">408.7 nM</span></span></span></p>
</td>
</tr>
<tr>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"> </span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">319–541 aa</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">133.3 nM</span></span></span></p>
</td>
</tr>
<tr>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; width:176px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><a href="https://elifesciences.org/articles/61390#bib67" style="color:blue; text-decoration:underline"><span style="font-size:9.0pt">Lan et al., 2020</span></a></span></span></p>
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:102px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">19–615 aa</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">306–527 aa</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"> </span></span></p>
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:128px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">SPR</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">31.6 nM</span></span></span></p>
</td>
</tr>
<tr>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"> </span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">319–541 aa</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">4.7 nM</span></span></span></p>
</td>
</tr>
<tr>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; width:176px">
<p><span style="font-size:11px"><span style="font-family:Arial,Helvetica,sans-serif">doi:<a class="id-link" href="https://doi.org/10.1016/j.cell.2020.02.058" rel="noopener" target="_blank">10.1016/j.cell.2020.02.058</a></span></span></p>
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:102px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">1–614 aa</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">306–575 aa</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"> </span></span></p>
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:128px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">BLI</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">1.2 nM</span></span></span></p>
</td>
</tr>
<tr>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"> </span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">328–533 aa</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">5 nM</span></span></span></p>
</td>
</tr>
<tr>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; width:176px">
<p><span style="font-size:11px"><span style="font-family:Arial,Helvetica,sans-serif">doi:<a class="id-link" href="https://doi.org/10.1126/science.abb2507" rel="noopener" target="_blank">10.1126/science.abb2507</a></span></span></p>
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:102px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">1–615 aa</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">306–577 aa</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"> </span></span></p>
</td>
<td rowspan="2" style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:128px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">BLI</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">13.7 nM</span></span></span></p>
</td>
</tr>
<tr>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:140px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"> </span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:151px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">319–591 aa</span></span></span></p>
</td>
<td style="background-color:white; border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; width:156px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt">34.6 nM</span></span></span></p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif">Pseudo typed vesicular stomatitis virus expressing SARS-CoV-2 S (VSV-SARS-S2) expression system can be used efficiently infects cell lines, with Calu-3 human lung adenocarcinoma epithelial cell line, CaCo-2 human colorectal adenocarcinoma colon epithelial cell line and Vero African grey monkey kidney epithelial cell line being the most permissive (Hoffmann et al., 2020; Ou et al., 2020). It can be measured using a wide variety of assays targeting different biological phases of infection and altered cell membrane permeability and cell organelle signaling pathway. Other assay measured alteration in the levels of permissive cell lines all express ACE2 or hACE2-expressing 293T cell (e.g. pNUO1-hACE2, pFUSE-hIgG1-Fc2), as previously demonstrated by indirect immunofluorescence (IF) or by immunoblotting are associated with ELISA(W Tai et al., nature 2020). To prioritize the identified potential KEs for selection and to select a KE to serve as a case study, further in-silico data that ACE2 binds to SARS-CoV-2 S is necessary for virus entry. The above analysis outlined can be used evidence-based assessment of molecular evidence as a MIE.</span></span></p>
<p> </p>
<p style="text-align:justify"> </p>
UBERON:0000062organCL:0000000cellHighMixedHighAdult, reproductively matureHighDuring development and at adulthoodHighHighHighModerateModerateLow<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">de Morais SDB, et al. Integrative Physiological Aspects of Brain RAS in Hypertension. Curr Hypertens Rep. 2018 Feb 26; 20(2):10.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Gallagher PE, et al. Distinct roles for ANG II and ANG-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes. Am J Physiol Cell Physiol. 2006 Feb; 290(2):C420-6.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Gowrisankar YV, Clark MA. Angiotensin II regulation of angiotensin-converting enzymes in spontaneously hypertensive rat primary astrocyte cultures. J Neurochem. 2016 Jul; 138(1):74-85.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Hamming I et al. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol. 2004 Jun;203(2):631-7.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Jakhmola S, et al. SARS-CoV-2, an Underestimated Pathogen of the Nervous System. SN Compr Clin Med. 2020.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Lukiw WJ et al. SARS-CoV-2 Infectivity and Neurological Targets in the Brain. Cell Mol Neurobiol. 2020 Aug 25;1-8.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Matsushita T, et al. CSF angiotensin II and angiotensin-converting enzyme levels in anti-aquaporin-4 autoimmunity. J Neurol Sci. 2010 Aug 15; 295(1-2):41-5.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Murta et al. Severe Acute Respiratory Syndrome Coronavirus 2 Impact on the Central Nervous System: Are Astrocytes and Microglia Main Players or Merely Bystanders? ASN Neuro. 2020. PMID: 32878468</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Shi A, et al. Isolation, purification and molecular mechanism of a peanut protein-derived ACE-inhibitory peptide. PLoS One. 2014; 9(10):e111188.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Xia, H. and Lazartigues, E. Angiotensin-Converting Enzyme 2: Central Regulator for Cardiovascular Function. Curr. Hypertens. 2010 Rep. 12 (3), 170– 175</span></span></span></p>
2020-03-02T03:18:472023-04-03T04:03:07Increase, the risk of acute respiratory failureIncrease, the risk of acute respiratory failureOrgan2020-03-10T02:05:092020-03-10T02:05:09Increased inflammatory immune responsesIncreased inflammatory immune responsesTissue2020-03-10T02:17:552021-01-21T03:35:32Increased MortalityIncreased MortalityPopulation<p><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="color:black">Increased mortality refers to an increase in the number of individuals dying in an experimental replicate group or in a population over a specific period of time.</span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="font-size:11pt"><span style="color:#212529"><span style="background-color:white">Mortality of animals is generally observed as cessation of the heart beat, breathing (gill or lung movement) and locomotory movements. Mortality is typically measured by observation. Depending on the size of the organism, instruments such as microscopes may be used. The reported metric is mostly the mortality rate: the number of deaths in a given area or period, or from a particular cause.</span></span></span></span></span></span></p>
<p style="text-align:start"><span style="font-size:medium"><span style="font-family:Calibri,sans-serif"><span style="color:#000000"><span style="font-size:11pt"><span style="color:#212529"><span style="background-color:white">Depending on the species and the study setup, mortality can be measured:</span></span></span></span></span></span></p>
<ul>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:11pt"><span style="color:#212529"><span style="background-color:white">in the lab by recording mortality during exposure experiments</span></span></span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:11pt"><span style="color:#212529"><span style="background-color:white">in dedicated setups simulating a realistic situation such as mesocosms or drainable ponds for aquatic species</span></span></span></span></span></li>
<li><span style="font-size:12pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:11pt"><span style="color:#212529"><span style="background-color:white">in the field, for example by determining age structure after one capture, or by capture-mark-recapture efforts. The latter is a method commonly used in ecology to estimate an animal population's size where it is impractical to count every individual.</span></span></span></span></span></li>
</ul>
<p>All living things are susceptible to mortality.</p>
ModerateUnspecificHighAll life stagesHigh2016-11-29T18:41:242022-07-08T07:32:26Toll Like Receptor (TLR) DysregulationTLR Activation/DysregulationMolecular<p><strong>Background</strong></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Toll-like receptors (TLRs) are a family of 13 conserved transmembrane receptors that are at the forefront of directing innate and adaptive immune responses against invading bacteria, fungi, viruses and parasites (Akira 2003, Takeda, Akira 2004, Pasare, Medzhitov 2005, Tal, Adini et al. 2020, van der Made, Simons et al. 2020). Upon activation TLRs initiate overlapping and distinct signaling pathways in various cell types such as macrophages (MP), conventinal DC (cDC), plasmacytoid DC (pDC), lamina propria DC (LPDC), and inflammatory monocytes (iMO). Engagement of TLR with specific stressors (e.g. PAMPs and DAMPs) induces conformational changes of TLRs that allow homo- or heterophilic interactions of TLRs and recruitment of adaptor proteins such as MyD88, TIRAP, TRIF, and TRAM to control intracellular signalling pathways leading to the synthesis and secretion of appropriate cytokines and chemokines by cells of the immune system. TLRs have various biological roles both in pathogen combat and tissue homeostasis. </span></span></p>
<p><strong><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">This KE is first developed in context of COVID-19 CIAO project. </span></span></strong></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">The key gatekeepers in detecting and combating viral infections are TLR3, TLR7, TLR8 and TLR9 and these are predominantly localized in intracellular compartments. In the setting of COVID-19, multiple TLRs are likely relevant in viral combat. Literature covering TLR triggering via </span></span><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">SARS-CoV-2 derived PAMPS (Pathogen Associated Molecular Patterns) include:</span></span></p>
<ul style="margin-left:40px">
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR7 and TLR8 (+TLR3, TLR4, TLR6) (Khanmohammadi and Rezaei, 2021)</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR1, TLR4 and TLR6 activated by SARS-CoV-2 spike proteins (Choudhury A <em>et al</em>, 2020)</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR9: Less CpG suppression in coronavirus compared to other viruses, for SARS-CoV-2 in the Envelope (E) open reading frame (E-ORF) and ORF10 (Ng et al., 2004; Digard et al. 2020) and multidisciplinary links described in Bezemer and Garssen, 2021</span></span></li>
</ul>
<p><strong><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR dysregulation can be multi-fold: </span></span></strong></p>
<ol>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Underperformance of TLR function leading to poor pathogen combat. This is covered in AOP 378</span></span></li>
</ol>
<ul style="margin-left:40px">
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">COVID-19 patients having poor TLR function (due to polymorphisms) could potentially have less viral clearance capability and more adverse events leading to more severe disease and mortality. This has been shown for TLR7 loss of function polymorphisms (van der Made et al 2020). </span></span><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Knowledge Gap: it is not known if loss of function of other TLRs has a worse outcome in COVID-19 patients.</span></span></li>
</ul>
<ol start="2">
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Overperformance of TLR function contributing to exaggerated immune response/cytokine storm/thrombosis/progression into ARDS and MOD. This is covered in AOP377</span></span></li>
</ol>
<ul style="margin-left:40px">
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR7 and TLR9 expression, measured by RNAseq gene analysis, is more elevated in black Americans than white Americans, which is proposed to explain in part the racial disparity in Covid-19 mortality rates via TLR mediated DC activation (Tal et al. 2020)</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">genetic mutations leading to TLR9 gain of function in human is associated with immune-mediated disease and with a higher incidence of ICU acquired infection (Chatzietal.,2018;Ng et al.,2010).</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Higher presence of host derived TLR stressors in vulnerable patients can contribute to TLR overstimulation/dysregulation. (Bezemer and Garssen, 2021)</span></span></li>
</ul>
<p><strong>Different classes of "stressors" act on TLR activation/dysregulation</strong></p>
<ul style="margin-left:40px">
</ul>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">1. Pathogen associated molecular patterns (PAMPs). TLRs can sense PAMPS during infection or upon exposure to stressors containing micro-organisms or fragments thereof (e.g. cigarette smoke, bioaerosols, house dust mite)</span></span></p>
<ul style="margin-left:40px">
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR1 is activated by bacterial Lipopeptides</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR2 is activated by bacterial lipoproteins and glycolipids</span></span>, TLR2 can form conformations with TLR1 and TLR6 to distinguish between diacyl and triacyl lipopeptides.</li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR3 is activated by viral double stranded RNA(dsRNA)</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR4 is activated by Bacterial LPS</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR5 is activated by Bacterial f</span></span><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">lagellig</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR6 is activated by Bacterial lipopeptides and Fungal zymosan</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR7 and 8 recognize viral single stranded RNA(ssRNA) and bacterial RNA. </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR9 recognizes RNA and DNAmotifs that are rich in unmethylated Cytosine-phosphate-Guanine (CpG) sequences. CpG-motifs are higher expressed in the bacterial and viral genome compared to the vertebrate genome (Hemmi et al., 2000). </span></span></li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">2. host derived Damage-Associated Molecular Patterns (DAMPS). Note that in the context and nomenclature of AOP these DAMPS cannot be labeld as "stressors" since they are derived from inside and not from outside, however these "pseudostressors" do act on the TLR receptors in similar way as PAMPs </span></span></p>
<ul style="margin-left:40px">
<li>
<p>TLR2 and TLR4 are activated by heat shock proteins 60 and 70 (HSP60 and HSP70); extracellular matrix components (ECM); oligosaccharides of hyaluronic acid (HA) and heparan sulfate (HS) (Piccinini AM and Midwood KS, 2010).</p>
</li>
<li>
<p>high-mobility group protein B1 (HMGB1) triggers TLR2, TLR4 and TLR9</p>
</li>
<li><span style="font-size:11pt">Oxidative injury/Oxidized phospholipids triggers TLR4 mediated NET formation</span></li>
<li><span style="font-size:11pt">Human mitochondrial DNA (mtDNA), evolutionary derived from endosymbiont bacteria, contains unmethylated CpG-motifs and triggers inflammatory responses directly via TLR9 during injury and/or infection (Zhang et al., 2010).</span></li>
<li><span style="font-size:11pt">Altered self-ligands, called carboxy-alkyl-pyrroleprotein adducts (CAPs), that are generated during oxidative stress, are known to aggravate TLR9/MyD88 pathway activation (Zhanget al., 2010;Panigrahi et al., 2013). CAPs have been shown to promote platelet activation, granule secretion, and aggregation in vitro and thrombosis in vivo (Panigrahi et al., 2013). </span></li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">3. synthetic TLR triggers/blockers (agonists/antagonists) for therapeutic purposes. Examples include CpG-ODNs triggering TLR9 for vaccin adjuvants/cancer treatment/immuno-modulation</span></span></p>
<p> </p>
<p><strong><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Several Modulating factors can contribute to TLR activation/dysregulation</span></span></strong></p>
<ul style="margin-left:40px">
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Co-infection and Trauma (for instance ventilator induced damage) can induce increased levels of TLR9 stressor, mtDNA, which is known to drive worse outcome at ICU in setting of other disorders. </span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">High levels of Visceral Fat, can increase TLR9 expression levels ánd circulating mtDNA</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Aging triggers both </span></span>immunosenescence<span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"> and inflammaging in part via impaired TLR function versus inappropriate triggering via increases of circulating DAMPS (Shaw et al 2011)</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Genetic polymorphisms can lead to TLR dysregulation (TLR9 gain of function and TLR7 loss of function with worse outcome at ICU Chatzi et al 2018, van der Made et al 2020, Chen et al 2011, )</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Circulating DAMPS such as mtDNA levels increase with age which is a familiar trait contributing to chronic inflammation, so called“inflamm-aging”in elderly people (Pinti et al., 2014).</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Vitamin D inhibits expression levels of TLR9</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Men, higher testosterone, higher TLR4</span></span></li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>Patient specific Ex vivo analysis </strong></span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Levels of TLR specific stressors (for instance for TLR9, cell free DNA/RNA, mtDNA) are measurable in biological samples (serum, plasma)</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR gain of function and loss of function polymorphisms are measurable</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR expression levels on different cell types and different tissues are measurable by mRNA analysis and by protein analysis</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR function in response to stressors is measurable by analysing components of downstream cascades and read outs of inflammatory mediators (IL6, IL8, IL10, Il17, INF, TNFalpha, etc). This can be done by ex vivo stimulations of cells isolated from patients for instance PBMCs. </span></span></li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong>In vitro/ in vivo models</strong></span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR Reporter assays</span></span></li>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">TLR knock-out mice</span></span></li>
</ul>
<p><strong>Cell applicability</strong><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">: TLRs are broadly expressed on various cell types. Examples include: epithelial cells, macrophages, neutrophils, platelets, dendritic cells, NK cells, Tcells, Bcells, neurons, Adipocytes. </span></span></p>
<p><strong><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Tissue/organ level </span></span></strong><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">: TLRs are broadly expressed in all vital tissues/organs: lung, heart, liver, spleen, kidney, brain, muscle, gut, skin</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:12.0pt">Taxonomic Applicability</span></strong>: TLRs are well conserved across species but between species variations are reported in terms of sensitivity towards stressors. For instance certain CpG-ODNs have a stronger TLR9 activating potential in mice than in human and vice versa. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Life Stages</span></span></strong>: TLRs are expressed in all life stages but age variation of level of TLR activation/dysregulation are reported. In elderly immunoscenescence and inflammation are both linked to TLR dysregulation</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif">Sex Applicability</span></span></strong>: Male and female subjects both express functionally active TLRs but sex differences have been reported. For instance certain TLR gain and/or loss of function polymorphisms have higher prevalence in men. Example of TLR7 loss of function (van der Made et al 2020) and TLR9 gain of function (Gao et al 2018, Traub et al 2012, Elsherif et al 2019). Higher testosterone in men has also been linked to higher TLR4 expression.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">TLR7 is located in a region on the X-chromosome which have a high chance of escaping inactivation leading to higher expression levels in women. Estrogens trigger TLR7, which is higher in women. Exposure of Peripheral blood mononuclear cells (PBMC) to TLR7 ligands will cause a higher production of type I IFN (IFN-a) in female cells than male cells. (Kovats, 2015; Takahashi and Iwasaki, 2021; Libert et al., 2010; Scully et al., 2020) </span></span></p>
ModerateMixedHighMaleHighBirth to < 1 monthHighOld AgeHighAll life stagesHighHighHigh<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">AKIRA, S., 2003. Toll-like receptor signaling. <em>Journal of Biological Chemistry, </em><strong>278</strong>(40), pp. 38105-38108.</span></span></span></span></p>
<div>
<div>Gillina F. G. Bezemer, Seil Sagar, Jeroen van Bergenhenegouwen, Niki A. Georgiou, Johan Garssen, Aletta D. Kraneveld and Gert Folkerts</div>
</div>
<div>Dual role of TLRs in asthma and COPD. <em>Pharmacological Reviews</em> April 1, 2012, 64 (2) 337-358; DOI: https://doi.org/10.1124/pr.111.004622</div>
<div> </div>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">BEZEMER, G.F.G. and GARSSEN, J., 2021. TLR9 and COVID-19: A Multidisciplinary Theory of a Multifaceted Therapeutic Target. <em>Frontiers in pharmacology, </em><strong>11</strong>, pp. 601685.</span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">KAWAI, T. and AKIRA, S., 2011. Toll-like Receptors and Their Crosstalk with Other Innate Receptors in Infection and Immunity. <em>Immunity, </em><strong>34</strong>(5), pp. 637-650.</span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">PASARE, C. and MEDZHITOV, R., 2005. Toll-like receptors: Linking innate and adaptive immunity. <em>Mechanisms of Lymphocyte Activation and Immune Regulation X: Innate Immunity, </em><strong>560</strong>, pp. 11-18.</span></span></span></span></p>
<p>Piccinini AM, Midwood KS. DAMPening inflammation by modulating TLR signalling. <em>Mediators Inflamm</em>. 2010;2010:672395. doi:10.1155/2010/672395</p>
<p>Shaw AC, Panda A, Joshi SR, Qian F, Allore HG, Montgomery RR. Dysregulation of human Toll-like receptor function in aging. <em>Ageing Res Rev</em>. 2011;10(3):346-353. doi:10.1016/j.arr.2010.10.007</p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">TAKEDA, K. and AKIRA, S., 2004. TLR signaling pathways. <em>Seminars in immunology, </em><strong>16</strong>(1), pp. 3-9.</span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">TAL, Y., ADINI, A., ERAN, A. and ADINI, I., 2020. Racial disparity in Covid-19 mortality rates - A plausible explanation. </span></span><em><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Clinical immunology (Orlando, Fla.), </span></span></em><strong><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">217</span></span></strong><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">, pp. 108481.</span></span></span></span></p>
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">VAN DER MADE, C.I., SIMONS, A., SCHUURS-HOEIJMAKERS, J., VAN DEN HEUVEL, G., MANTERE, T., KERSTEN, S., VAN DEUREN, R.C., STEEHOUWER, M., VAN REIJMERSDAL, S.V., JAEGER, M., HOFSTE, T., ASTUTI, G., COROMINAS GALBANY, J., VAN DER SCHOOT, V., VAN DER HOEVEN, H., HAGMOLEN OF TEN HAVE, W., KLIJN, E., VAN DEN MEER, C., FIDDELAERS, J., DE MAST, Q., BLEEKER-ROVERS, C.P., JOOSTEN, L.A.B., YNTEMA, H.G., GILISSEN, C., NELEN, M., VAN DER MEER, J.W.M., BRUNNER, H.G., NETEA, M.G., VAN DE VEERDONK, F.L. and HOISCHEN, A., 2020. </span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Presence of Genetic Variants Among Young Men With Severe COVID-19. </span></span><em><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Jama, </span></span></em><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif">Kovats, Cell Immunol. 2015 April; 294(2): 63–69; </span></span></p>
<p>Takahashi and Iwasaki, Science. 2021 Jan 22;371(6527):347-348</p>
<div>Libert et al., Nat Rev Immunol. 2010 Aug;10(8):594-604</div>
<p>Scully EP, et al. Nat Rev Immunol. 2020. PMID: 32528136</p>
<p> </p>
2021-03-26T03:48:512021-11-23T16:54:16Increased SARS-CoV-2 productionSARS-CoV-2 productionCellular<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">This KE1847 "Increase coronavirus production" deals with how the genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is translated, replicated, and transcribed in detail, and how the genomic RNA (gRNA) is packaged, and the virions are assembled and released from the cell. </span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Coronavirus is a class of viruses that have single-stranded positive-sense RNA genomes in their envelopes [D. Kim<em> et al.</em>]. The virus contains a <span style="color:#131413">29.7 kB positive-sense RNA genome flanked by 5' and 3' untranslated regions of 265 and 342 nucleotides, respectively</span><span style="color:black"> </span><span style="color:#131413">[</span>E. J. Snijder<em> et al.</em><span style="color:#131413">] that contain cis-acting secondary RNA structures essential for RNA synthesis [</span>N. C. Huston<em> et al.</em>]<span style="color:black">. T</span>he genome just prior to the 5′ end contains the transcriptional regulatory sequence leader (TRS-L) [C.J. Budzilowicx <em>et al.</em>]. The SARS-CoV genome is polycistronic and contains 14 open reading frames (ORFs) that are expressed by poorly understood mechanisms [E. J. Snijder <em>et al.</em>]<span style="color:black">.</span> Preceding each ORF there are other TRSs called the body TRS (<span style="color:black">TRS B). </span>The <span style="color:black">5′ two-thirds of the </span>genome contains <span style="color:black">two large, overlapping, nonstructural ORFs and the 3′ third contains the remainder ORFs [H. Di <em>et al.</em>].</span> Genome expression starts with the translation of <span style="color:#131413">two large ORFs of the 5’ two-thirds: ORF1a of</span><span style="color:black"> 4382 amino acids and ORF1ab of 7073 amino acid that occurs via a</span><span style="color:#131413"> programmed (- 1) ribosomal frameshifting [E. J. Snider <em>et al.</em>]</span><span style="color:black">, yielding</span><span style="color:#131413"> pp1a and pp1ab</span><span style="color:black">. These two polyproteins are cleaved into 16 subunits by two viral proteinases encoded by ORF1a,</span> <span style="color:black">nsp3, and nsp5 that contain a papain-like protease domain and a 3C-like protease domain</span> [M. D. Sacco <em>et al.</em>]<span style="color:#131413">. </span><span style="color:black">The processing products are a group of replicative enzymes, named nsp1-nsp16, that assemble into a viral replication a</span>nd transcription <span style="color:black">complex (RTC) associated with membranes of endoplasmic reticulum (ER) with the help of various membrane-associated viral proteins [</span>S. Klein<em> et al.</em>, E. J. Snijder<em> et al., </em>P. V'Kovski, <em>et al.</em>]<span style="color:black">. Besides replication, which yields the positive-sense gRNA, the replicase also</span> <span style="color:black">mediates transcription leading to the synthesis of a nested set of subgenomic (sg) mRNAs to express all ORFs downstream of ORF1b that encode structural and accessory viral proteins. </span>These localize to the 3′ one-third of the genome, as stated above, and result in a 3′ coterminal nested set of 7–9 mRNAs that share ~70–90 nucleotide (nt) in the 5′ leader and that is identical to the 5′ end of the genome [P. Liu, and J. Leibowitz]. s<span style="color:black">gRNAs encode conserved structural proteins (spike protein [S], envelope protein [E], membrane protein [M], and nucleocapsid protein [N]), and several accessory proteins. SARS-CoV-2 is known to have at least six accessory proteins (3a, 6, 7a, 7b, 8, and 10). Overall the virus is predicted to express 29 proteins [</span>D. Kim<em> et al.</em>]<span style="color:black">. The gRNA is packaged by the structural proteins to assemble progeny virions.</span></span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Viral translation:</strong></span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">SARS-CoV-2 is an enveloped virus with a single-stranded RNA genome of ~30 kb, sequence orientation in a 5’ to 3’ direction typical of positive sense and reflective of the resulting mRNA [D. Kim<em> et al.</em>]. The SARS-CoV-2 genome contains a 5’-untranslated region (UTR; 265 bp), ORF1ab (21,289 bp) holding two overlapping open reading frames (13,217 bp and 21,289 bp, respectively) that encode two polyproteins [D. Kim<em> et al.</em>]. Other elements of the genome include are shown below [V. B. O'Leary <em>et al.</em>]. <strong>The first event upon cell entry is the primary translation of the ORF1a and ORF1b gRNA to produce non-structural proteins (nsps).</strong></span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">This is completely dependent on the translation machinery of the host cell. Due to fewer rare “slow-codons”, SARS-CoV-2 may have a higher protein translational rate, and therefore higher infectivity compared to other coronavirus groups [V. B. O'Leary <em>et al.</em>]. The ORF1a produces polypeptide 1a (pp1a, 440–500 kDa) that is cleaved into nsp-1 through nsp-11. A -1 ribosome frameshift occurs immediately upstream of the ORF1a stop codon, to allow translation through ORF1b, yielding 740–810 kDa polypeptide pp1ab, which is cleaved into 15 nsps [D. Kim<em> et al.</em>]. Two overlapping ORFs, ORF1a and ORF1b, generate continuous polypeptides, which are cleaved into a total of 16 so-called nsps [Y Finkel <em>et al.</em>]. Functionally, there are five proteins from pp1ab (nsp-12 through nsp-16) as nsp-1-11 are duplications of the proteins in pp1a due to the ORF overlap. The <span style="color:black">pp1a is approximately 1.4–2.2 times more expressed than pp1ab. </span>After translation, the polyproteins are cleaved by viral proteases nsp3 and nsp5. Nsp5 <span style="color:black">protease can be referred to as 3C-like protease (3CL<sup>pro</sup>) or as main protease (M<sup>pro</sup>), as it cleaves the majority of the polyprotein cleavage sites. [H.A. Hussein </span><em>et al.</em><span style="color:black">] Nsp1 cleavage is quick and nsp1 associates with host cell ribosomes and results in host cellular shutdown, </span><span style="color:#231f20">suppressing host gene expression </span><span style="color:#000000">[</span>M. Thoms<em> et al.]</em><span style="color:black">. Fifteen proteins, nsp2–16 constitute the viral RTC. They are targeted to defined subcellular locations and establish a network with host cell factors.</span> N<span style="color:black">sp2–11 remodel host membrane architecture, mediate host immune evasion and provide cofactors for replication, w</span>hilst <span style="color:black">nsp12–16 contain the core enzymatic functions involved in RNA synthesis, modification and proofreading [</span>P. V'Kovski <em>et al.</em>]<span style="color:black">. </span>nsp-7 and nsp-8 form a complex priming the RNA-dependent RNA polymerase (RdRp or RTC) - nsp-12. <span style="color:black">nsp14 provides a 3′–5′ exonuclease activity providing RNA proofreading function.</span> Nsp-10 composes the RNA <span style="color:black">capping machinery</span> nsp-9. <span style="color:black">nsp13 provides the RNA 5′-triphosphatase activity</span>. Nsp-14 is a <em><span style="color:black">N</span></em><span style="color:black">7-methyltransferase and nsp-16 the 2′-<em>O</em>-methyltransferase. </span>Many of the nsps have multiple functions and many viral proteins are involved in innate immunity inhibition. Nsp-3 is involved in vesicle formation along with nsp-4 and nsp-6 where viral replication occurs. Interactions between SARS-CoV-2 proteins and human RNAs thwart the IFN response upon infection: nsp-16 binds to U1 and U2 splicing RNAs to suppress global mRNA splicing; nsp-1 binds to 40S ribosomal RNA in the mRNA entry channel of the ribosome to inhibit host mRNA translation; nsp-8 and nsp-9 bind to the 7SL RNA to block protein trafficking to the cell membrane [A. K. Banerjee<em> et al.</em>]. Xia et al. [H. Xia<em> et al.</em>] found that nsp-6 and nsp-13 antagonize IFN-I production via distinct mechanisms: nsp-6 binds TANK binding kinase 1 (TBK1) to suppress interferon regulatory factor 3 (IRF3) phosphorylation, and nsp-13 binds and blocks TBK1 phosphorylation.</span></span></p>
<p> </p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Viral transcription and replication:</strong></span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Viral transcription and replication occur at the viral replication organelle (RO) [E. J. Snijder<em> et al.</em>]. The RO is specifically formed during infection by reshaping ER and other membranes, giving rise to <span style="color:black">small spherular invaginations, and large vesiculotubular clusters, consisting of single- and/or double-membrane vesicles (DMV), convoluted membranes (CM) and double-membrane spherules invaginating from the ER [</span>S. Klein<em> et al., </em>E. J. Snijder<em> et al.</em>]<span style="color:black">. There is some evidence that DMV accommodate viral replication which is based on radiolabelling viral RNA with nucleoside precursor ([5-<sup>3</sup>[H]uridine) and detection by EM autoradiography</span> <span style="color:#000000">[</span>E. J. Snijder<em> et al.</em>]<span style="color:black">.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">Viral replicative proteins and specific host factors are recruited</span> to ROs [E. J. Snijder<em> et al.</em>]. RNA viral genome is transcribed into messenger RNA by the viral RTC [P. Ahlquist <em>et al.</em>]. Viral RTC act in combination with other viral and host factors involved in selecting template RNAs, elongating RNA synthesis, differentiating genomic RNA replication from mRNA transcription, modifying product RNAs with 5’ caps or 3’ polyadenylate [P. Ahlquist <em>et al.</em>]. Positive-sense (messenger-sense) RNA viruses replicate their genomes through negative-strand RNA intermediates [M. Schwartz<em> et al.</em>]. The intermediates comprise <span style="color:black">full-length negative-sense complementary copies of the genome, which functions as templates for the generation of new positive-sense gRNA, and a nested set of sg mRNAs that lead to the expression of proteins encoded in all ORFs downstream of ORF1b. </span>The transcription of coronaviruses <span style="color:black">is a discontinuous process that produces nested 3′ and 5′ co-terminal sgRNAs. Of note, the synthesis of sg mRNAs is not exclusive to the order <em>Nidovirales</em> but a discontinuous minus-strand synthesis strategy to produce a nested set of 3′ co-terminal sg mRNAs with a common 5′ leader in infected cells</span> <span style="color:black">are unique features of the <em>coronaviruses</em> and <em>arteriviruses</em> [</span>W. A. Miller and G. Koev.]<span style="color:black">. Of note, the produced genomic RNA represents a small fraction of the total vRNA [</span>N. S. Ogando<em> et al.</em>]<span style="color:black">.</span></span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">The discontinuous minus-strand synthesis of a set of nested sg mRNAs happens during the synthesis of the negative-strand RNA, by an interruption mechanism of the RTC as it reads the TRS-B preceding each gene in the 3′ one-third of the viral genome [</span>I. Sola, F. Almazan <em>et al., </em>I. Sola, J. L. Moreno, <em>et al.</em>]<span style="color:black">. The synthesis of the negative-strand RNA stops and is re-initiated at the TRS-L of the genome sequence close from the 5′ end of the genome [</span>H. Di <em>et al.</em>]<span style="color:black">. Therefore, t</span><span style="color:black">he mechanism by which the leader sequence is added to the 5' end requires that the RTC switches template by a jumping mechanism. This interruption process involves the interaction between complementary TRSs of the nascent negative-strand RNA TRS-B and the positive-strand gRNA at the positive-sense TRS-L. The TRS-B site has a 7-8 bp conserved core sequence (CS) that facilitates RTC template switching as it hybridizes with a near complementary CS in the TRS-L [</span>I. Sola, F. Almazan <em>et al. </em>I. Sola, J. L. Moreno, <em>et al.</em>]<span style="color:black">.</span> <span style="color:black">Upon re-initiation of RNA synthesis at the TRS-L region, a negative-strand copy of the leader sequence is added to the nascent RNA to complete the synthesis of negative-strand sgRNAs. This means that all sg mRNAs as well as the genomic RNA share a common 5' sequence, named leader sequence [</span>X. Zhang et al.]<span style="color:black">. This programmed template switching leads to the generation of sg mRNAs with identical 5' and 3' sequences, but alternative central regions corresponding to the beginning of each structural ORF [</span>I. Sola <em>et al.</em> 2015, S. G. Sawicki <em>et al.</em>, Y. Yang <em>et al.</em>]<span style="color:black">. Of note, the existence of TRSs also raises the possibility that these sites are at the highest risk of recombining through TRS-B mediated template switching [</span>Y. Yang]<span style="color:black">.</span> <span style="color:black">The set of sg mRNAs is then translated to yield </span>29 identified different proteins [F. Wu<em> et al.</em>], although many papers have identified additional ORFs [D. Kim<em> et al.. </em>Y. Finkel<em> et al., </em>A. Vandelli<em> et al.</em>]. The translation of the linear single-stranded RNA conducts to the generation of the following proteome: 4 are structural proteins, S, N, M, and E; 16 proteins nsp: the first 11 are encoded in ORF1a whereas the last 5 are encoded in ORF1ab. In addition, 9 accessory proteins named ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c, and ORF10 have been identified [F. Wu<em> et al.</em>]. At the beginning of infection, there is the predominant expression of the nsp that result from ORF1a and ORF1ab, however, at 5 hpi, the proteins encoded by the <span style="color:black">5′ last third are found in higher amounts, and the nucleoprotein is the protein found in higher levels [</span>Y. Finkel<em> et al.</em>]<span style="color:black">.</span></span></span></p>
<p style="text-align:justify"> </p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><strong>Viral assembly:</strong></span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">The final step of viral production requires virion assembly and this process is not well explored for SARS-CoV-2. For example, the role of the structural proteins of SARS-CoV-2 in virus assembly and budding in not known. In general, all beta-coronavirus structural proteins assemble at the endoplasmic reticulum (ER)-to-Golgi compartment [</span>J. R. Cohen <em>et al.</em><em>, </em>A. Perrier<em> et al.</em>]<span style="color:black"> and v</span>iral assembly requires two steps: Genome packaging which is a process in which the SARS-CoV-2 gRNA must be coated by the viral protein nucleoprotein (N) protein, <span style="color:black">forming viral ribonucleoprotein (vRNPs) complexes, </span>before being selectively packaged into progeny virions [P. V'Kovski <em>et al.</em>], a step in which vRNPs<span style="color:black"> bud into the lumen of the ER and the ER-Golgi intermediate compartment (ERGIC) [</span>N. S. Ogando<em> et al.</em>]<span style="color:black">. This results in viral particles enveloped with host membranes containing viral M, E, and S transmembrane structural proteins that need to be released from the cell.</span> </span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">SARS-CoV-2 gRNA packaging involves the N protein. The N protein of human coronaviruses is highly expressed in infected cells. It is considered a multifunctional protein, promoting efficient sub-genomic viral RNA transcription, viral replication, virion assembly, and interacting with multiple host proteins [P. V'Kovski <em>et al.</em>, D. E. Gordon<em> et al.</em>, R. McBride, and M. van Zyl, B. C.]. In relation to transcription and replication, the N protein could provide a cooperative mechanism to increase protein and RNA concentrations at specific localizations S. Alberti, and S. Carra, S. F. Banani <em>et al.</em>], and this way organize viral transcription. Five studies have shown that N protein undergoes liquid-liquid phase separation (LLPS) <em>in vitro</em> [A. Savastano <em>et al.</em>, H. Chen<em> et al.</em>, C. Iserman<em> et al.</em>, T. M. Perdikari<em> et al.</em>, J. Cubuk<em> et al.</em>], dependent on its C-terminal domain (CTD) [H. Chen<em> et al.]</em>. It has been hypothesised that N could be involved in replication close to the ER and in packaging of gRNA into vRNPs near the ERGIC where genome assembly is thought to take place [A. Savastano<em> et al.</em>], but so far this is still speculative. Phosphorylation of N could adjust the physical properties of condensates differentially in ways that could accommodate the two different functions of N: transcription and progeny genome assembly [A. Savastano <em>et al.</em>, C. Iserman<em> et al., </em>C. R. Carlson<em> et al.</em>]. </span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black">The ERGIC constitutes the main assembly site of coronaviruses [</span>S. Klein<em> et al.</em><span style="color:black"><em>, </em></span>E. J. Snijder<em> et al.</em>, L. Mendonca<em> et al.</em>]<span style="color:black"> and budding events have been seen by EM studies.</span> For SARS-CoV-2, v<span style="color:black">irus-budding was mainly clustered in regions with a high vesicle density and close to ER- and Golgi-like membrane arrangements [</span>S. Klein<em> et al.</em><span style="color:black"><em>, </em></span>E. J. Snijder<em> et al.</em>, L. Mendonca<em> et al.</em>]<span style="color:black">. The ectodomain of S trimers were found facing the ERGIC lumen and not induce membrane curvature on its own, therefore proposing that vRNPs and spike trimers</span> <span style="color:black">[</span>S. Klein<em> et al.</em>]<span style="color:black">.</span> </span></span></p>
<p style="text-align:justify"><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Finally, it has been shown that SARS-CoV-2 virions de novo formed traffic to lysosomes for unconventional egress by Arl8b-dependent lysosomal exocytosis [S. Ghosh<em> et al.</em>]. This process results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation [S. Ghosh<em> et al.</em>].</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"><strong>Viral translation:</strong></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation [Schubert </span></span><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black"><em>et al.</em></span></span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"> 2020]</span></span></p>
<ul>
<li><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">Sucrose pelleting binding assay to verify Nsp1–40S complex formation</span></span></li>
<li><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">In vivo translation assay</span></span></li>
<li><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">Transient expression of FLAG-Nsp1 in HeLa cells and puromycin incorporation assay</span></span></li>
</ul>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">SARS-CoV-2 disrupts splicing, translation, and protein trafficking [Banerjee </span></span><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black"><em>et al.</em></span></span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"> 2020]</span></span></p>
<ul>
<li><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">SARS-CoV-2 viral protein binding to RNA</span></span></li>
<li><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">Interferon stimulation experiments</span></span></li>
<li><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">Splicing assessment experiments</span></span></li>
<li><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">IRF7-GFP splicing reporter, 5EU RNA labeling, capture of biotinylated 5EU labeled RNA</span></span></li>
</ul>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">Membrane SUnSET assay for transport of plasma membrane proteins to the cell surface</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"><strong>Viral transcription:</strong></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11.0pt">The mRNA transcripts are detected by the real-time reverse transcription-PCR (RT-PCR) assay. Several methods targeting the mRNA transcripts have been developed, which includes the RT-PCR assays targeting RdRp/helicase (Hel), spike (S), and nucleocapsid (N) genes of SARS-CoV-2 [Chan <em>et al.</em>]. RT-PCR assays detecting SARS-CoV-2 RNA in saliva include quantitative RT-PCR (RT-qPCR), direct RT-qPCR, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) [Nagura-Ikeda M, <em>et al.</em>]. The viral mRNAs are reverse-transcribed with RT, followed by the amplification by PCR.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"><strong>Viral replication:</strong></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">viral replication is measured by RT-qPCR in infected cells, formation of liquid organelles is assessed in vitro reconstitution systems and in infected cells. Labelling by radioactive nucleosides.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"><strong>Viral production:</strong></span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11.0pt">Plaque assays, infectivity assays, RT-qPCR to detect viral RNA in released virions, sequencing to detect mutations in the genome, electron microscopy.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">Broad mammalian host range has been demonstrated based on spike protein tropism for and binding to ACE2 [Conceicao </span></span><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black"><em>et al.</em></span></span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"> 2020; Wu </span></span><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black"><em>et al.</em></span></span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"> 2020] and cross-species ACE2 structural analysis [Damas </span></span><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:black"><em>et al.</em></span></span></span><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt"> 2020]. No literature has been found on primary translation and molecular interactions of nsps in non-human host cells, but it is assumed to occur if the virus replicates in other species.</span></span></p>
<p><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11.0pt">Very broad mammalian tropism: human, bat, cat, dog, civet, ferret, horse, pig, sheep, goat, water buffalo, cattle, rabbit, hamster, mouse</span></span></p>
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<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">30. D. E. Gordon<em> et al.</em>, A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. <em>Nature</em> <strong>583</strong>, 459-468 (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">31. R. McBride, M. van Zyl, B. C. Fielding, The coronavirus nucleocapsid is a multifunctional protein. <em>Viruses</em> <strong>6</strong>, 2991-3018 (2014).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">32. S. Alberti, S. Carra, Quality Control of Membraneless Organelles. <em>Journal of Molecular Biology</em> <strong>430</strong>, 4711-4729 (2018).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">33. S. F. Banani, H. O. Lee, A. A. Hyman, M. K. Rosen, Biomolecular condensates: organizers of cellular biochemistry. <em>Nature Reviews Molecular Cell Biology</em> <strong>18</strong>, 285-298 (2017).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">34. A. Savastano, A. I. de Opakua, M. Rankovic, M. Zweckstetter, Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates. (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">35. H. Chen<em> et al.</em>, Liquid-liquid phase separation by SARS-CoV-2 nucleocapsid protein and RNA. <em>Cell Res</em>, (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">36. C. Iserman<em> et al.</em>, Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate. <em>bioRxiv</em>, (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">37. T. M. Perdikari<em> et al.</em>, SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins. <em>bioRxiv</em>, (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">38. J. Cubuk<em> et al.</em>, The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA. <em>bioRxiv</em>, (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">39. C. Iserman<em> et al.</em> (Cold Spring Harbor Laboratory, 2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">40. C. R. Carlson<em> et al.</em>, Phosphoregulation of phase separation by the SARS-CoV-2 N protein suggests abiophysical basis for its dual functions. <em>Molecular Cell</em>, (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">41. L. Mendonca<em> et al.</em>, SARS-CoV-2 Assembly and Egress Pathway Revealed by Correlative Multi-modal Multi-scale Cryo-imaging. <em>bioRxiv</em>, (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">42. S. Ghosh<em> et al.</em>, beta-Coronaviruses Use Lysosomes for Egress Instead of the Biosynthetic Secretory Pathway. <em>Cell</em> <strong>183</strong>, 1520-1535 e1514 (2020).</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">43. Schubert, K., Karousis, E.D., Jomaa, A. <em>et al.</em> SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation. <em>Nat Struct Mol Biol</em> <strong>27, </strong>959–966 (2020). </span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">44. Chan, Jasper Fuk-Woo et al. Improved Molecular Diagnosis of COVID-19 by the Novel, Highly Sensitive and Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-PCR Assay Validated <em>In Vitro</em> and with Clinical Specimens. J Clin Microbiol. 2020:58(5)e00310-20. doi:10.1128/JCM.00310-20</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">45. Nagura-Ikeda M, Imai K, Tabata S, et al. Clinical Evaluation of Self-Collected Saliva by Quantitative Reverse Transcription-PCR (RT-qPCR), Direct RT-qPCR, Reverse Transcription-Loop-Mediated Isothermal Amplification, and a Rapid Antigen Test To Diagnose COVID-19. J Clin Microbiol. 2020;58(9):e01438-20. doi:10.1128/JCM.01438-20</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">46. Conceicao C, Thakur N, Human S, Kelly JT, Logan L, Bialy D, et al. (2020) The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins. PLoS Biol 18(12): e3001016. https://doi.org/10.1371/journal.pbio.3001016</span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">47. Damas J, Hughes GM, Keough KC, Painter CA, Persky NS, Corbo M, Hiller M, Koepfli KP, Pfenning AR, Zhao H, Genereux DP, Swofford R, Pollard KS, Ryder OA, Nweeia MT, Lindblad-Toh K, Teeling EC, Karlsson EK, Lewin HA. Broad host range of SARS-CoV-2 predicted by comparative and structural analysis of ACE2 in vertebrates. Proc Natl Acad Sci U S A. 2020 Sep 8;117(36):22311-22322. doi: 10.1073/pnas.2010146117. Epub 2020 Aug 21. PMID: 32826334; PMCID: PMC7486773.</span></span></p>
2021-03-25T19:55:122022-06-14T08:49:57Interferon-I antiviral response, antagonized by SARS-CoV-2IFN-I response, antagonizedCellular<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">SARS-CoV-2 is an enveloped virus with a single-stranded RNA genome of ~30 kb, sequence orientation in a 5’ to 3’ direction typical of positive sense and reflective of the resulting mRNA <span style="font-size:14px">(</span></span></span><span style="font-size:14px">doi:<a class="article-header__doi__value" href="https://doi.org/10.1016/j.cell.2020.04.011">https://doi.org/10.1016/j.cell.2020.04.01</a></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:14px">). The SAR</span>S-CoV-2 genome contains a 5’-untranslated region (UTR; 265 bp), <a href="https://www.ncbi.nlm.nih.gov/gene/?term=ORF1a+SARS-CoV-2">ORF1ab</a> (21,289 bp) holding two overlapping open reading frames (13,217 bp and 21,289 bp, respectively) that encode two polyproteins (Kim et al. 2020; O’Leary et al. 2020). Viral transcription and replication is explained in depth in <a href="https://aopwiki.org/events/1847" style="color:blue; text-decoration:underline">KE1847</a>. Briefly, the first event upon cell entry is the primary translation of the ORF1a and ORF1b genomic RNA to produce non-structural proteins (NSPs). The ORF1a produces polypeptide 1a (pp1a, 440–500 kDa) that is cleaved into NSP-1 through NSP-11. A -1-ribosome frameshift occurs immediately upstream of the ORF1a stop codon, to allow translation through ORF1b, yielding 740–810 kDa polypeptide pp1ab, which is cleaved into 15 NSPs (duplications of NSP1-11 and five additional proteins, NSP12-16). Viral proteases NSP3 and NSP5 cleave the polypeptides through domains functioning as a papain-like protease and a 3C-like protease, respectively <span style="font-size:14px">(</span></span></span><span style="font-size:14px">doi:<a class="article-header__doi__value" href="https://doi.org/10.1016/j.cell.2020.04.011">https://doi.org/10.1016/j.cell.2020.04.01</a></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:14px">). </span> The NSPs, structural proteins, and accessory proteins are encoded by 10 ORFs in the SARS-CoV-2 RNA genome. They may have multiple functions during viral replication as well as in evasion of the host innate immune response, thus augmenting viral replication and spread<strong> (Amor et al. 2020). </strong>Extensive protein-protein interaction <strong>(Gordon et al. 2020) </strong>and viral protein-host RNA interaction networks have been demonstrated between the viral NSPs and accessory proteins and host molecules. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">This key event is focused on the specific viral:host protein interactions within the infected cell that are involved in the <a href="https://www.wikipathways.org/index.php/Pathway:WP4868">IFN-I antiviral response pathways</a>. IFN-I is the main component of the innate immune system that is suppressed by the SARS-CoV-2 coronavirus early in infection. </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The primary form of host intracellular virus surveillance detects viral components to induce an immediate systemic type I interferon (IFN) response. Cellular RNA sensors called pattern recognition receptors (PRRs) such as RIG-I, MDA5 and LGP2 detect the presence of viral RNAs and promote nuclear translocation of the transcription factor IRF3, leading to transcription, translation, and secretion of IFN-α and IFN-β. This in turn leads to interaction with the IFN receptor (IFNAR), phosphorylation of STAT1 and 2, and transcription and translation of hundreds of antiviral genes <strong>(Quarleri and Delpino, 2021).</strong></span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Interactions between SARS-CoV-2 proteins and human RNAs thwart the IFN response upon infection: NSP1 binds to 40S ribosomal RNA in the mRNA entry channel of the ribosome to inhibit host mRNA translation; NSP8 and NSP9 displace signal recognition particle proteins (SRP54, 27 and 19) to bind to the 7SL RNA and block protein trafficking to the cell membrane (Banerjee et al. 2020; Gordon et al. 2020). Xia et al. (2020) found that NSP6 and NSP13 antagonize IFN-I production via distinct mechanisms: NSP6 binds TANK binding kinase 1 (TBK1) to suppress interferon regulatory factor 3 (IRF3) phosphorylation, and NSP13 binds and blocks TBK1 phosphorylation. NSP14 induces lysosomal degradation of type 1 IFNAR to prevent STAT activation (Hayn et al. 2021). ORF6 hijacks KPNA2 to block IRF3, and Nup98/RAE1 to block STAT nuclear import, to silence IFN-I gene expression (Xia and Shi, 2020). ORF7a suppresses STAT2 phosphorylation and ORF7b suppresses STAT1 and STAT2 phosphorylation to block ISGF3 complex formation with IRF9 (Xia and Shi, 2020). ORF8 interacts and downregulates MHC-I (Zhang et al 2020), and has been reported to block INF</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">β</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> expression, but the mechanism has not been identified (Rashid et al. 2021; Li et al. 2020). ORF9b antagonizes Type I Interferons by targeting multiple components of RIG-I/MDA-5-MAVS, TOMM70, NEMO and cGAS-STING signalling (Han et al. 2020; Jiang et al. 2020; Wu et al. 2021; Gordon et al 2020).</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Following is a table of the current state of knowledge of SARS-CoV-2 protein putative functions in relation to IFN-I antiviral response antagonism.</span></span></p>
<p> </p>
<table cellspacing="0" class="Table" style="border-collapse:collapse; width:594px">
<tbody>
<tr>
<td style="border-bottom:3px double black; border-left:none; border-right:none; border-top:1px solid black; height:21px; vertical-align:top; width:58px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Gene</span></span></span></span></p>
</td>
<td style="border-bottom:3px double black; border-left:none; border-right:none; border-top:1px solid black; height:21px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Protein</span></span></span></span></p>
</td>
<td style="border-bottom:3px double black; border-left:none; border-right:none; border-top:1px solid black; height:21px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Function</span></span></span></span></p>
</td>
<td style="border-bottom:3px double black; border-left:none; border-right:none; border-top:1px solid black; height:21px; vertical-align:top; width:266px">
<p style="text-align:center"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Role in early innate immune evasion</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:21px; vertical-align:top; width:58px">
<p> </p>
</td>
<td style="height:21px; vertical-align:top; width:108px">
<p> </p>
</td>
<td style="height:21px; vertical-align:top; width:162px">
<p> </p>
</td>
<td style="height:21px; vertical-align:top; width:266px">
<p> </p>
</td>
</tr>
<tr>
<td style="height:21px; vertical-align:top; width:58px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF1a</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">NSP1</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">NSP1 antagonizes interferon<br />
induction to suppress host antiviral<br />
response. </span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">DNA Polymerase Alpha Complex: Regulates the activation of IFN-I through cytosolic<br />
RNA-DNA synthesis (POLA1/2-PRIM1/2) and primes DNA replication in the nucleus (Gordon et al. 2020; Chaudhuri et al. 2020). Can also inhibit host gene expression by binding to ribosomes and modifying host mRNAs (Shi et al. 2020; Schubert et al. 2020; Thoms et al. 2020). </span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:21px; vertical-align:top; width:58px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">NSP2</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">While not essential for viral replication, deletion of NSP2 diminishes viral growth and RNA synthesis</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Translation repression through binding GIGYF2and EIF4E2 (4EHP) (Gupta et al. 2021)</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:21px; vertical-align:top; width:58px">
<p> </p>
</td>
<td style="height:21px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">NSP3</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Papain-like protease (Plpro); Cleaves the ORF1a and 1ab polypeptides</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Suppresses IFN-I: Cleaves IRF3 (Moustaqil et al. 2021); binds/cleaves ISG15 (Rui et al. 2021; Shin et al. 2020; Liu et al. 2021; Klemm et al. 2020)</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:21px; vertical-align:top; width:58px">
<p> </p>
</td>
<td style="height:21px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">NSP5</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">3C-like protease (3CLpro);</span></span> <span style="font-size:10.0pt"><span style="color:black">Cleaves the ORF1a and 1ab polypeptides </span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Binds STING (Rui et al. 2021)</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:21px; vertical-align:top; width:58px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"> </span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">NSP6</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Limits autophagosome expansion</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Suppresses IFN-I expression: Binds TBK-1 to supress IRF3 phosphorylation (Xia et al. 2020; Quarleri and Delpino, 2021) </span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:21px; vertical-align:top; width:58px">
<p> </p>
</td>
<td style="height:21px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">NSP7</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">In complex with NSP8 forms primase as part of multimeric RNA-dependent RNA replicase (RdRp)</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:266px">
<p> </p>
</td>
</tr>
<tr>
<td style="height:21px; vertical-align:top; width:58px">
<p> </p>
</td>
<td style="height:21px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">NSP8</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Replication complex with NSP7, NSP9 and NSP12</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Binds SRP72/54/19 (Gordon et al. 2020) and 7SL RNA to block IFN membrane transport (Banerjee et al. 2020)</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:21px; vertical-align:top; width:58px">
<p> </p>
</td>
<td style="height:21px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">NSP9</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Replication complex with NSP7, NSP8 and NSP12</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Binds SRP and 7SL RNA with NSP8 to block IFN membrane transport (Banerjee et al. 2020)</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:21px; vertical-align:top; width:58px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF1b</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">NSP13</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Helicase and triphosphatase that initiates the first step in viral mRNA capping.</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Binds TBK1 (Xia et al. 2020)</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:21px; vertical-align:top; width:58px">
<p> </p>
</td>
<td style="height:21px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">NSP14</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:162px">
<p> </p>
</td>
<td style="height:21px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Induces lysosomal degradation of IFNAR1 (Hayn et al. 2021)</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:21px; vertical-align:top; width:58px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF2</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Spike (S)</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ACE2 interaction, cell entry</span></span></span></span></p>
</td>
<td style="height:21px; vertical-align:top; width:266px"> </td>
</tr>
<tr>
<td style="height:35px; vertical-align:top; width:58px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF3a</span></span></span></span></p>
</td>
<td style="height:35px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF3a</span></span></span></span></p>
</td>
<td style="height:35px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Interacts with M, S, E and 7a; form viroporins; immune evasion</span></span></span></span></p>
</td>
<td style="height:35px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Binds STING (Rui et al 2021)</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:20px; vertical-align:top; width:58px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF4</span></span></span></span></p>
</td>
<td style="height:20px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Envelope (E)</span></span></span></span></p>
</td>
<td style="height:20px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Viral assembly and budding</span></span></span></span></p>
</td>
<td style="height:20px; vertical-align:top; width:266px"> </td>
</tr>
<tr>
<td style="height:20px; vertical-align:top; width:58px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF5</span></span></span></span></p>
</td>
<td style="height:20px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Membrane (M)</span></span></span></span></p>
</td>
<td style="height:20px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Viral assembly</span></span></span></span></p>
</td>
<td style="height:20px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Interacts with RIG-I and MAVS sensors of viral RNA (Fu et al 2020)</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:70px; vertical-align:top; width:58px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF6</span></span></span></span></p>
</td>
<td style="height:70px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF6</span></span></span></span></p>
</td>
<td style="height:70px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Viral pathogenesis and virulence; interacts with ORF8; promotes RNA polymerase activity</span></span></span></span></p>
</td>
<td style="height:70px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Hijacks the nuclear importin Karyopherin a 2 (KPNA2) to block IRF3 (Xia and Shi, 2020) and Nup98/RAE1 to block STAT nuclear import (Miorin et al. 2020; Kato et al. 2020), leading to the silence of downstream ISGs</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:43px; vertical-align:top; width:58px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF7a</span></span></span></span></p>
</td>
<td style="height:43px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF7a</span></span></span></span></p>
</td>
<td style="height:43px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Interacts with S, ORF3a; immune evasion</span></span></span></span></p>
</td>
<td style="height:43px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Suppresses STAT2 phosphorylation to block IFN-I response (Xia and Shi, 2020).</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:35px; vertical-align:top; width:58px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF7b</span></span></span></span></p>
</td>
<td style="height:35px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF7b</span></span></span></span></p>
</td>
<td style="height:35px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Structural component of virion</span></span></span></span></p>
</td>
<td style="height:35px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Suppresses STAT1 and STAT2 phosphorylation to block IFN-I response (Xia and Shi, 2020)</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:70px; vertical-align:top; width:58px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF8</span></span></span></span></p>
</td>
<td style="height:70px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF8</span></span></span></span></p>
</td>
<td style="height:70px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Immune evasion</span></span></span></span></p>
</td>
<td style="height:70px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Interacts and downregulates MHC-I (Zhang et al. 2020). May inhibit type I interferon (IFN-β) and interferon-stimulated response element (ISRE) (Rashid et al. 2020; Li et al. 2020)</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:20px; vertical-align:top; width:58px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF9</span></span></span></span></p>
</td>
<td style="height:20px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Nucleocapsid (N)</span></span></span></span></p>
</td>
<td style="height:20px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Stabilizes viral RNA</span></span></span></span></p>
</td>
<td style="height:20px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Attenuates stress granule formation: G3BP1/2 (Chen et al. 2020; Cascarina et al. 2020); G3BP1 also interacts with RIG-I (Kim et al. 2019) and STAT1/2 (Mu et al. 2020)</span></span></span></span></p>
</td>
</tr>
<tr>
<td style="height:70px; vertical-align:top; width:58px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF9b</span></span></span></span></p>
</td>
<td style="height:70px; vertical-align:top; width:108px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">ORF9b</span></span></span></span></p>
</td>
<td style="height:70px; vertical-align:top; width:162px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Immune evasion</span></span></span></span></p>
</td>
<td style="height:70px; vertical-align:top; width:266px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-size:10.0pt"><span style="color:black">Membrane protein antagonizes Type I Interferons by targeting multiple components of RIG-I/MDA-5-MAVS, TOMM70, NEMO, and cGAS-STING signaling pathways (Fu et al. 2020; Chen et al. 2020; Han et al. 2020; Jiang et al. 2020; Wu et al. 2021; Gordon et al 2020)</span></span></span></span></p>
</td>
</tr>
</tbody>
</table>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Detection of IFN-I suppression involves measuring gene promoter/transcription activation (luciferase assays), gene up/down regulation (quantitative PCR), protein-protein interaction (immunoprecipitation, immunoblotting) or in-situ co-location of viral and host proteins (immunofluorescent or confocal microscopy) in cell culture. Examples of methods used include the following:</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Interferon I decrease (Xia et al. 2020):</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">IFN-I production and signaling luciferase reporter assays</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Co-immunoprecipitation and western blot</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Indirect immunofluorescence assays</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">DNA assembly and RNA transcription of a luciferase replicon for SARS-CoV-2</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Replicon RNA electroporation and luciferase reporter assay</span></span></li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">SARS-CoV-2 ORF9b inhibits RIG-I-MAVS antiviral signaling (Wu et al. 2021)</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Viral- and host-protein-specific antibodies</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Immunoprecipitation</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Immunofluorescent microscopy</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Dual-luciferase reporter assays</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Fluorescence quantification immunoblotting</span></span></li>
</ul>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">SARS-CoV-2-Human Protein-Protein Interaction Map (Gordon et al. 2020)</span></span></p>
<ul>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cloning and expression of viral proteins via plasmid transfection into HEK293T cell line</span></span></li>
<li><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Protein affinity purification using MagStrep beads with detection by anti-strep western blot of cell lysate</span></span></li>
<li><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Global analysis of SARS-CoV-2 host interacting proteins using affinity purification-mass spectrometry</span></span></li>
</ul>
<p> </p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Broad mammalian host range based on spike protein tropism for and binding to ACE2 (Conceicao et al. 2020; Wu et al. 2020) and cross-species ACE2 structural analysis (Damas et al. 2020). Some literature found on non-human hosts indicates that NSPs and accessory proteins can interact in a similar manner with bird (chicken) and other mammal proteins in the IFN-I pathway (Moustaqil et al. 2021; Rui et al. 2021).</span></span></p>
UBERON:0000062organCL:0000066epithelial cellHighUnspecificHighAll life stagesHighHighHighHighModerateHigh<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Amor et al. 2020. Innate immunity during SARS-CoV-2: evasion strategies and activation trigger hypoxia and vascular damage. Clinical and Experimental Immunology, 202: 193–209. doi: 10.1111/cei.13523 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Andres et al. 2020. SARS-CoV-2 ORF9c Is a Membrane-Associated Protein that Suppresses Antiviral Responses in Cells. bioRxiv preprint doi: <a href="https://doi.org/10.1101/2020.08.18.256776" style="color:blue; text-decoration:underline">https://doi.org/10.1101/2020.08.18.256776</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Banerjee et al. 2020. SARS-CoV-2 disrupts splicing, translation, and protein trafficking to supress host defenses. Cell 183, 1325–1339. <a href="https://doi.org/10.1016/j.cell.2020.10.004" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.cell.2020.10.004</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Cascarina and Ross, 2020. A proposed role for the SARS-CoV-2 nucleocapsid protein in the </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">formation and regulation of biomolecular condensates. The FASEB Journal, 34:9832–9842. DOI: 10.1096/fj.202001351 </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Chaudhuri, A. 2021. Comparative analysis of non-structural protein 1 of SARS-CoV2 with SARS-CoV1 and MERS-CoV: An in-silico study. Journal of Molecular Structure, Volume 1243, 130854, https://doi.org/10.1016/j.molstruc.2021.130854.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Chen et al. 2021. SARS-CoV-2 Nucleocapsid Protein Interacts with RIG-I and Represses RIG-Mediated IFN-β Production. Viruses. 13(1):47. <a href="https://doi.org/10.3390/v13010047" style="color:blue; text-decoration:underline">https://doi.org/10.3390/v13010047</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Conceicao et al. 2020. The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins. PLoS Biol 18(12): e3001016. <a href="https://doi.org/10.1371/journal.pbio.3001016" style="color:blue; text-decoration:underline">https://doi.org/10.1371/journal.pbio.3001016</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Damas et al. 2020. Broad host range of SARS-CoV-2 predicted by comparative and structural analysis of ACE2 in vertebrates. PNAS vol. 117 no. 36:22311–22322 <a href="http://www.pnas.org/cgi/doi/10.1073/pnas.2010146117" style="color:blue; text-decoration:underline">www.pnas.org/cgi/doi/10.1073/pnas.2010146117</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Fu et al. 2021. SARS-CoV-2 membrane glycoprotein M antagonizes the MAVS-mediated innate antiviral response. Cell Mol Immunol 18: 613–620. https://doi.org/10.1038/s41423-020-00571-x</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gordon et al. 2020. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature 483:459-473. <a href="https://doi.org/10.1038/s41586-020-2286-9" style="color:blue; text-decoration:underline">https://doi.org/10.1038/s41586-020-2286-9</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gupta et al. 2021. CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes. bioRxiv 2021.05.10.443524; doi: https://doi.org/10.1101/2021.05.10.443524</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Han et al. 2020. SARS-CoV-2 ORF9b Antagonizes Type I and III Interferons by Targeting Multiple Components of RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING Signaling Pathways. bioRX <a href="https://doi.org/10.1101/2020.08.16.252973" style="color:blue; text-decoration:underline">https://doi.org/10.1101/2020.08.16.252973</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hayn et al. 2021. Systematic functional analysis of SARS-CoV-2 proteins uncovers viral innate immune antagonists and remaining vulnerabilities. Cell Reports 35, 109126. <a href="https://doi.org/10.1016/j.celrep.2021.109126" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.celrep.2021.109126</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Jiang et al. 2020. SARS-CoV-2 Orf9b suppresses type I interferon responses by targeting TOM70. Cellular & Molecular Immunology 17:998–1000; https://doi.org/10.1038/s41423-020-0514-8</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Kato et al. 2021. Overexpression of SARS-CoV-2 protein ORF6 dislocates RAE1 and NUP98 from the nuclear pore complex. Biochemical and Biophysical Research Communications 536:59-66 <a href="https://doi.org/10.1016/j.bbrc.2020.11.115" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.bbrc.2020.11.115</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Kim et al. 2019. The stress granule protein G3BP1 binds viral dsRNA and RIG-I to enhance interferon-β response. J. Biol. Chem. 294(16): 6430–6438. DOI 10.1074/jbc.RA118.005868</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Kim et al. 2020. The Architecture of SARS-CoV-2 Transcriptome. Cell 181, 914–921. <a href="https://doi.org/10.1016/j.cell.2020.04.011" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.cell.2020.04.011</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Li et al. 2020. The ORF6, ORF8 and nucleocapsid proteins of SARS-CoV-2 inhibit type I interferon signaling pathway. Virus Research vol. 286. <a href="https://doi.org/10.1016/j.virusres.2020.198074" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.virusres.2020.198074</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Liu et al. 2021. ISG15-dependent activation of the sensor MDA5 is antagonized by the SARS-CoV-2 papain-like protease to evade host innate immunity. Nature Microbiol 6: 467–478. <a href="https://doi.org/10.1038/s41564-021-00884-1" style="color:blue; text-decoration:underline">https://doi.org/10.1038/s41564-021-00884-1</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Moustaqil et al. 2021. SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species, Emerging Microbes & Infections, 10:1, 178-195. <a href="https://doi.org/10.1080/22221751.2020.1870414" style="color:blue; text-decoration:underline">https://doi.org/10.1080/22221751.2020.1870414</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mu et al. 2020. SARS-CoV-2 N protein antagonizes type I interferon signaling by suppressing phosphorylation and nuclear translocation of STAT1 and STAT2. Cell Discov 6, 65. <a href="https://doi.org/10.1038/s41421-020-00208-3" style="color:blue; text-decoration:underline">https://doi.org/10.1038/s41421-020-00208-3</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">O’Leary et al. 2020 Unpacking Pandora from Its Box: Deciphering the Molecular Basis of the SARS-CoV-2 Coronavirus. Int. J. Mol. Sci. 2021, 22, 386. <a href="https://doi.org/10.3390/ijms22010386" style="color:blue; text-decoration:underline">https://doi.org/10.3390/ijms22010386</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Quarleri and Delpino, 2020. Type I and III IFN-mediated antiviral actions counteracted by SARS-CoV-2 proteins and host inherited factors. Cytokine & Growth Factor Reviews, 58: 55-65. <a href="https://doi.org/10.1016/j.cytogfr.2021.01.003" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.cytogfr.2021.01.003</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Rashid et al. The ORF8 protein of SARS-CoV-2 induced endoplasmic reticulum stress and mediated immune evasion by antagonizing production of interferon beta. Virus Research 296, 198350. <a href="https://doi.org/10.1016/j.virusres.2021.198350" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.virusres.2021.198350</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Ren et al. 2020. The ORF3a protein of SARS-CoV-2 induces apoptosis in cells. Cellular & Molecular Immunology 17:881–883; <a href="https://doi.org/10.1038/s41423-020-0485-9" style="color:blue; text-decoration:underline">https://doi.org/10.1038/s41423-020-0485-9</a></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Rui et al. 2021. Unique and complementary suppression of cGAS-STING and RNA sensing-triggered innate immune responses by SARS-CoV-2 proteins. Sig Transduct Target Ther 6, 123. <a href="https://doi.org/10.1038/s41392-021-00515-5" style="color:blue; text-decoration:underline">https://doi.org/10.1038/s41392-021-00515-5</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Schubert et al. 2020. SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation. Nature Structural & Molecular Bio. 27:959-966. <a href="https://doi.org/10.1038/s41594-020-0511-8" style="color:blue; text-decoration:underline">https://doi.org/10.1038/s41594-020-0511-8</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Shin et al. 2020. Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity. Nature 587: 657–662. <a href="https://doi.org/10.1038/s41586-020-2601-5" style="color:blue; text-decoration:underline">https://doi.org/10.1038/s41586-020-2601-5</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Thoms et al. 2020. Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2. Science 369(6508): 1249-1255. DOI: 10.1126/science.abc8665</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Wu et al. 2021. SARS-CoV-2 ORF9b inhibits RIG-I-MAVS antiviral signaling by interrupting K63-linked ubiquitination of NEMO. Cell Reports 34, 108761. <a href="https://doi.org/10.1016/j.celrep.2021.108761" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.celrep.2021.108761</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Wu et al. 2020. Broad host range of SARS-CoV-2 and the molecular basis for SARS-CoV-2 binding to cat ACE2. Cell Discovery 6:68. <a href="https://doi.org/10.1038/s41421-020-00210-9" style="color:blue; text-decoration:underline">https://doi.org/10.1038/s41421-020-00210-9</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Xia et al. 2020. Evasion of Type I Interferon by SARS-CoV-2. Cell Reports 33, 108234. <a href="https://doi.org/10.1016/j.celrep.2020.108234" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.celrep.2020.108234</a> </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Xia and Shi, 2020. Antagonism of Type I Interferon by Severe Acute Respiratory Syndrome Coronavirus 2. Journal of Interferon & Cytokine Research v.40, no. 12 DOI:10.1089/jir.2020.0214 </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Zhang et al. 2020. The ORF8 Protein of SARS-CoV-2 Mediates Immune Evasion through Potently Downregulating MHC-I. bioRxiv preprint doi: <a href="https://doi.org/10.1101/2020.05.24.111823" style="color:blue; text-decoration:underline">https://doi.org/10.1101/2020.05.24.111823</a></span></span></p>
2021-07-09T14:22:552023-04-03T15:20:422c7949b2-a7f6-4c1e-8025-dfe3bd05273c3ec60b38-ac3c-4d18-b776-b17a08ec1e3a<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif">This KER deals with the evidence supporting the individual weight that the surface protein of SARS-CoV-2 spike needs to bind:ACE2, and of being cleaved in two different sites, for viral entry to occur. Viral entry is essential for initiating a cascade of events leading to COVID19.</span></span></p>
<p> </p>
<p> </p>
<p> </p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><strong><em><span style="color:#0070c0">Binding of SARS-CoV-2 S protein to ACE2 receptors present in the brain (endothelial, neuronal and glial cells) :</span></em></strong></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#0070c0">The highest ACE2 expression level in the brain was found in the pons and medulla oblongata in the human brainstem, containing the medullary respiratory centers, and this may in part explain the susceptibility of many COVID-19 patients to severe respiratory distress (Lukiw et al., 2020). High ACE2 receptor expression was also found in the amygdala, cerebral cortex and in the regions involved in cardiovascular function and central regulation of blood pressure including the sub-fornical organ, nucleus of the tractus solitarius, paraventricular nucleus, and rostral ventrolateral medulla (Gowrisankar and Clark 2016; Xia and Lazartigues 2010). The neurons and glial cells, like astrocytes and microglia also express ACE-2, thus highlighting the vulnerability of the nervous system to SARS-CoV-2 infection. Additionally, they also express transmembrane serine protease 2 (TMPRSS2) and furin, which facilitate virus entry into the host (Jakhmola et al. 2020).</span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#0070c0">Once inside the brain, the virus can infect the neural cells, astrocytes, and microglia. These cells express ACE-2, thus initiating the viral budding cycle followed by neuronal damage and inflammation (Jakhmola et al. 2020). Specifically in the brain, ACE2 is expressed in endothelium and vascular smooth muscle cells (Hamming et al., 2004), as well as in neurons and glia (Gallagher et al., 2006; Matsushita et al., 2010; Gowrisankar and Clark, 2016; Xu et al., 2017; de Morais et al., 2018) (from Murta et al., 2020).</span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#0070c0">Astrocytes are the main source of angiotensinogen and express ATR1 and MasR; neurons express ATR1, ACE2, and MasR, and microglia respond to ATR1 activation (Shi et al., 2014; de Morais et al., 2018). </span></span></span></p>
<p><span style="font-size:12px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="color:#1abc9c"><strong><em>Binding of S protein to ACE2 receptors present in the intestines</em></strong></span></span></span></p>
HighUnspecificHighAll life stagesHigh<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><strong><span style="color:#0070c0">COVID19 References related to CNS:</span></strong></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">de Morais SDB, et al. Integrative Physiological Aspects of Brain RAS in Hypertension. Curr Hypertens Rep. 2018 Feb 26; 20(2):10.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Gallagher PE, et al. Distinct roles for ANG II and ANG-(1-7) in the regulation of angiotensin-converting enzyme 2 in rat astrocytes. Am J Physiol Cell Physiol. 2006 Feb; 290(2):C420-6.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Gowrisankar YV, Clark MA. Angiotensin II regulation of angiotensin-converting enzymes in spontaneously hypertensive rat primary astrocyte cultures. J Neurochem. 2016 Jul; 138(1):74-85.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Hamming I et al. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol. 2004 Jun;203(2):631-7.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Jakhmola S, et al. SARS-CoV-2, an Underestimated Pathogen of the Nervous System. SN Compr Clin Med. 2020.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Lukiw WJ et al. SARS-CoV-2 Infectivity and Neurological Targets in the Brain. Cell Mol Neurobiol. 2020 Aug 25;1-8.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Matsushita T, et al. CSF angiotensin II and angiotensin-converting enzyme levels in anti-aquaporin-4 autoimmunity. J Neurol Sci. 2010 Aug 15; 295(1-2):41-5.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Murta et al. Severe Acute Respiratory Syndrome Coronavirus 2 Impact on the Central Nervous System: Are Astrocytes and Microglia Main Players or Merely Bystanders? ASN Neuro. 2020. PMID: 32878468</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Shi A, et al. Isolation, purification and molecular mechanism of a peanut protein-derived ACE-inhibitory peptide. PLoS One. 2014; 9(10):e111188.</span></span></span></p>
<p><span style="font-size:11pt"><span style="font-family:"Calibri",sans-serif"><span style="color:#0070c0">Xia, H. and Lazartigues, E. Angiotensin-Converting Enzyme 2: Central Regulator for Cardiovascular Function. Curr. Hypertens. 2010 Rep. 12 (3), 170– 175</span></span></span></p>
2020-03-02T03:19:152023-02-07T23:24:14f3ec58f7-6c74-4e3f-a750-042df045078f0712f722-87c5-464f-b17b-cd88e327c2ec2021-04-16T04:29:332021-04-16T04:29:330712f722-87c5-464f-b17b-cd88e327c2ec88f0f792-3e32-49d5-972f-c9a3751ff411<p>The engagement of TLR with Pathogen-Associated Molecular Patterns (PAMPs) and host derived<br />
damage-associated molecular patterns (DAMPs) induces conformational changes<br />
of TLRs that allow recruitment of adaptor proteins such as MyD88, TIRAP, TRIF, and TRAM<br />
to control intracellular signaling pathways, including ERK, p38 and NF-�B, driving the<br />
synthesis and secretion of cytokines and chemokines [Kawai, T.; Akira, S. Signaling to NF-kappaB by Toll-like receptors. Trends Mol. Med. 2007, 13, 460–469] [DOI:<a class="article-header__doi__value" href="https://doi.org/10.1016/j.molmed.2007.09.002">https://doi.org/10.1016/j.molmed.2007.09.002</a>]. In a healthy state the amount and type of proinflammatory mediators are appropriate to required circumstances (e.g. defence against invading pathogens) and resolution of inflammation is promoted to reestablish homeostasis. Dysregulated TLR Activation can result from e.g. overabundance of PAMPS/DAMPS or over-, or under-expression of TLR protein intra- and/or extracellularly or over- or under-expression of downstream proteins. These circumstances can be modulated by a number of factors including biological/intrinsic factors (e.g. age, sex, genetic factors), pre-existing co-morbidities, lifestyle factors, environmental factors and therapeutic interventions. In context of an adverse outcome the resulting dysregulated over- or under-activation of TLRs contributes to dysproportional amounts of proinflammatory mediators (overproduction or underproduction) halting back defence and homeostasis.</p>
<p>Evidence in support of this KER in context of COVID-19:</p>
<p> </p>
2021-03-29T06:48:592023-02-07T23:52:0388f0f792-3e32-49d5-972f-c9a3751ff411e5f32b44-33d7-4ff7-838f-c191ba6e11b92020-03-10T02:18:402020-03-10T02:18:40e5f32b44-33d7-4ff7-838f-c191ba6e11b9fe4f8cae-2dbf-41fa-ba95-0950c1b3b9642020-03-10T02:19:232020-03-10T02:19:23fe4f8cae-2dbf-41fa-ba95-0950c1b3b96412fcdfa1-2629-4485-9175-96da14c16ceb2020-05-13T09:39:052020-05-13T09:39:053ec60b38-ac3c-4d18-b776-b17a08ec1e3a8fc4d1cc-b330-4628-8435-fc48240252c1<p><span style="font-size:16px">Upon entry of a virus into the host cell (KE1738), the virus is unpackaged from the structural nucleocapsid (N), envelope (E), and membrane (M) proteins. <span style="font-family:Calibri"><span style="color:black">The viral RNA is detected by Pattern Recognition Receptor (PRR) proteins including RIG-I and MDA5</span></span> but the M proteins can interact with these PRRs directly, and block this initial host reaction (Fu et al., 2021). The viral genomic RNA can then be translated directly at the host ribosomes. The viral proteins are processed through cleavage by viral protease enzymes. This releases a repertoire of non-structural proteins (NSPs) and accessory open reading frame (ORF) proteins that has evolved, for example in the SARS-CoV-2 virus, to bind and block the proteins in the interferon I (IFN-I) antiviral cascade (KE1901). <span style="font-family:"Calibri",sans-serif">The normal function of the host’s IFN-I response to other viruses is the expression of IFN-I which in turn stimulates the expression of many interferon-stimulated gene (ISG) proteins with antiviral functions. The SARS-CoV-2 antagonism of the IFN-I pathway delays or curtails the expression of IFN-I and ISG proteins. </span></span></p>
<p>Empirical evidence supporting this relationship is described below.</p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">This relationship is concerned with how entry of the virus into the host cell and subsequent release and transcription of viral proteins affects the downstream innate immune response. In particular, literature suggests the main pathway antagonized is the expression of type I interferons (IFN-I), consisting primarily of IFNα and IFNβ, and IFN-I stimulated genes (ISGs) (Banerjee et al., 2020; Blanco-Melo et al., 2020; Cheemarla et al., 2021; Xia et al., 2020; Sharif-Askari et al., 2022). Although there are few studies with evidence for cell entry leading directly to reduced IFN expression (Xia et al., 2020; Hatton et al. 2021), several studies demonstrate individual viral protein interactions with and blocking of host proteins in the IFN-I pathway or ISG proteins (Schubert et al. 2020; Thoms et al. 2020; Rui et al. 2021; Shin et al. 2020; Liu et al. 2021; Mostaqil et al., 2021; Xia et al. 2020; Quarleri and Delpino, 2021; Xia and Shi, 2020; Miorin et al. 2020; Kato et al. 2020; Fu et al. 2020; Chen et al. 2020; Han et al. 2020; Jiang et al. 2020; Wu et al. 2021; Gordon et al 2020; see below and also key event 1901). These studies provide the biological rationale that SARS-CoV-2 entry into the host cell causes interactions between viral proteins and known protein components of the host IFN-I antiviral response, resulting in inhibition of IFN-I and ISG expression.</span></span></p>
<p> </p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Empirical evidence in support of temporal concordance comes from patient reports, showing that interferon expression is delayed by SARS-CoV-2 compared to other viruses like influenza, which is also described as an untuned or imbalanced response between interferons being initially low in moderate to severe cases (Banco-Melo et al. 2020; Galani et al., 2021; Hadjadj et al., 2020; Hatton et al., 2021; Rouchka et al., 2021). This indicates that SARS-CoV-2 stressors are suppressing the interferon response and highlights an important point regarding the difference between SARS-CoV-2 and other viruses in the stressors produced upon viral entry. Other viruses, as well as non-viral compounds used in research (e.g., polyinosinic:polycytidylic acid or poly[I:C]) enter the cell and stimulate the normal functional operation of the immune response, while SARS-CoV-2 blocks the response at multiple points, acting as a true prototypical stressor. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hatton et al. (2021) used human nasal epithelium differentiated at the air-liquid interface (ALI) cultures (organoids) with several cell types. Secretory cells were the cell type with the highest expression of viral transcripts, with ciliated and deuterosomal cells also showing expression. The SARS-CoV-2-infected secretory and ciliated cells also had many downregulated ISGs. Compared to SARS-CoV-2, influenza A virus induced significantly higher levels of IFN-I (IFNβ) and IFN-III (IFNλ1) at 6 and 24 hours post infection, as well as ISGs Ubiquitin specific peptidase 18 (USP18), radical s-adenosyl methionine domain containing 2 (RSAD2), and ubiquitin-like protein ISG15 at 24 hours post infection (Hatton et al., 2021). </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Individual stressors from the virus were investigated by Xia et al. (2020) using an IFN-</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">β</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"> promoter luciferase assay. HEK293T cells were co-transfected with luciferase reporter plasmids, the specific viral protein expressing plasmid, and stimulator plasmid RIG-I (2CARD). Of the viral proteins tested (NSPs 1, 2, 4-16, S, N, E, M, and ORFs 3a, 3b, 6, 7a, 7b, 8, and 10), four proteins (NSPs 1, 6, and 13 and ORF6) significantly reduced INF-</span></span><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">β induction compared to the control (empty vector). A similarly conducted ISRE-promoter luciferase assay showed significant inhibition of the IFN-I signaling pathway (normally resulting in induction of ISGs) by NSPs 1, 6, 7, 13 and 14, ORFs 3a, 6, </span></span><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">7a and 7b, and M protein (Xia et al., 2020). See Xia et al. (2020) and Xia and Shi (2020) for schematics depicting the actions of the SARS-CoV-2 proteins on the protein components of the IFN-I antiviral response pathway.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">SARS-CoV-2 stressor proteins and the IFN-I pathway responses were investigated individually in the following studies:</span></span></p>
<table border="1" class="Table" style="border:solid windowtext 1px">
<tbody>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:103px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Viral protein stressor</strong></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:94px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Host protein</strong></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:60px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Crystal Structure PDB</strong></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:318px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>KER findings: Binding, Stressor/IFN-I or ISG expression</strong></span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:103px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">N (nucleocapsid)</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:94px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>RIG-I</strong>: Retinoic acid-inducible gene I</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:60px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Not available (NA)</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:318px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Significant reductions in IFNβ mRNA induction were seen when SARS-CoV-2 N protein was co-transfected into A549 cells with RIG-I, MAVS, or TBK1, and similar transfections resulted in IFNβ promoter activity reduction in poly(I:C)-stimulated HEK293T cells (Chen et al., 2020). </span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:103px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NSP3 Papain-like protease (Plpro)</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:94px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>MDA5</strong>: Melanoma differentiation-associated gene 5</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:60px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NA</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:318px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sun et al. (2022) determined that SARS-CoV-2 and avian coronavirus infectious bronchitis virus (IBV) NSP3 PLpro N-terminal domain directly interacts with MDA5 to inhibit IFNβ expression when co-transfected in HEK293T cells.</span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:103px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">M (membrane)</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:94px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>MAVS</strong>: Mitochondiral antiviral signaling protein</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:60px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NA</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:318px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Fu et al. (2020) found M interaction with MAVS (as determined by coimmunoprecipitation and in vitro pull-down assay) interferes with recruitment of downstream pathway proteins TRAF, TBK1, and IRF3, inhibiting IFNβ1 promoter, IFN-stimulated response element (ISRE), and NFκB promoter activity in a dose-dependent manner. The M protein inhibited the transcription of ISGs (ISG56, CXCL10, and TNF) based on mRNA levels, and inhibited IFNβ and TNFα secretion based on measures of these proteins in HEK293 cell culture.</span></span></p>
</td>
</tr>
<tr>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:103px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NSP3 Papain-like protease (Plpro)</span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; width:94px">
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>ISG15</strong>: Ubiquitin-like interferon stimulated gene 15</span></span></p>
</td>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><a href="https://www.rcsb.org/structure/6YVA" style="color:blue; text-decoration:underline">6YVA</a> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Shin et al. (2020) generated a crystal structure and found that SARS-CoV-2 Plpro preferentially cleaves ISG15. ISG15 functions in antiviral immunity to directly inhibit viral replication (Perng and Lenschow, 2018).</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">ORF9b</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>TOMM70</strong>: Translocase of outer mitochondrial membrane</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><a href="https://www.rcsb.org/structure/7KDT" style="color:blue; text-decoration:underline">7KDT</a> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gordon et al. (2020) showed interaction between TOMM70 and ORF9b via affinity purification-mass spectrometry (AP-MS). TOMM70-ORF9b interaction is supported by several studies (Gao et al., 2021; Brandherm et al., 2021; Ayinde et al., 2022). Jiang et al. (2020) used a dual luciferase reporter assay to show human IFN-β promoter activity was significantly reduced in the presence SARS-CoV-2 Orf9b compared to controls.</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">ORF6</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>Nup98-RAE1</strong>: Nuclear pore complex 98-ribonucleic acid export 1</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><a href="https://www.rcsb.org/structure/7VPG" style="color:blue; text-decoration:underline">7VPG</a>, <a href="https://www.rcsb.org/structure/7VPH" style="color:blue; text-decoration:underline">7VPH</a> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Gordon et al. (2020) showed interaction between ORF6 and the host Nup98-RAE1 protein pair via AP-MS. The interaction was confirmed by Miorin et al., 2020 and Li et al., 2021 (see crystal structures). Miorin et al. (2020) also demonstrate that upon treatment with recombinant IFN-I in HEK293T cells, Nup98 binding to SARS-CoV-2 Orf6 blocks translocation of STAT1 into the nucleus, resulting in suppression of ISRE-dependent gene expression.</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">ORF6</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>KPNA2</strong>: Karyopherin subunit alpha 1</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NA</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Using co-immunoprecipitation, Xia et al. (2020) showed that ORF6 selectively bound with KPNA2. Expression of ORF6 blocked nuclear translocation of IRF3, suggesting that ORF6 inhibited IFN-β production by binding to KPNA2 to block IRF3 nuclear translocation.</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">N (nucleocapsid)</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>G3BP1/2</strong>: GTPase-activating protein SH3 domain–binding protein</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><a href="https://www.rcsb.org/structure/7SUO" style="color:blue; text-decoration:underline">7SUO</a> </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Biswal et al. (2022) solved the X-ray crystal structure of the G3BP1 N-terminal nuclear transport factor 2-like domain bound to the first intrinsically disordered region of SARS-CoV-2 N protein.</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">ORF9b</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>NEMO</strong>: Nuclear factor kappa-B (NF-κB) essential modulator</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NA</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">The interaction of the N-terminus of ORF9b with NEMO upon viral infection interrupts its K63-linked polyubiquitination, thereby inhibiting viral-RNA-induced IFNβ1 activation in HEK293T cells in an ORF9b-dose-dependent manner (Wu et al., 2021)</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NSP5 (3CLpro)</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>NEMO</strong></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><a href="https://www.rcsb.org/structure/7T2U" style="color:blue; text-decoration:underline">7T2U</a></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Hameedi et al. (2022) solved the X-ray crystal structure of 3CLpro bound to NEMO and characterized 3CLpro cleavage of NEMO. </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NSP1</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>POLA</strong>1: DNA polymerase alpha 1, catalytic subunit</span></span></p>
<p><strong><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">40S ribosomal subunit</span></span></strong></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><a href="https://www.rcsb.org/structure/7OPL" style="color:blue; text-decoration:underline">7OPL</a> </span></span></p>
<p> </p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"><a href="https://www.rcsb.org/structure/6ZOJ" style="color:blue; text-decoration:underline">6ZOJ</a></span></span><u><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:blue">, </span></span></span></u><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"><a href="https://www.rcsb.org/structure/6zok" style="color:blue; text-decoration:underline">6ZOK</a></span></span><u><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:blue">, </span></span></span></u><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif"><a href="https://www.rcsb.org/structure/6zol" style="color:blue; text-decoration:underline">6ZOL</a></span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Kilkenny et al., 2021 demonstrate that components of the host DNA polymerase α (Pol α)–primase complex or primosome directly bind with SARS-CoV-2 NSP1. They also provide a cryo-electron microscopy structure of NSP1 bound to the primosome. </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Schubert et al. (2020) provide cryo-EM structures of NSP1 bound to the 40S ribosome subunit, inhibiting translation of host proteins.</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NSP6, </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NSP13</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>TBK1</strong>: TANK-binding kinase 1</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NA</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sui et al. (2022) show that NSP13 recruits TBK1 to an aggregation of ubiquitinated proteins (p62) for autophagic degradation, resulting in inhibition of IFNβ production, and that NSP13 impaired IRF3 luciferase reporter activity induced by TBK1 in a dose-dependent manner. </span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Xia et al. (2020) co-transfected HEK293T cells with plasmids containing TBK1 and either nsp6 or nsp13. Only NSP13 inhibited TBK1 phosphorylation, and did so in a dose-dependent manner, but both NSP6 and NSP13 suppressed IRF3 phosphorylation. Both NSP6 and NSP13 bind TBK1, as shown by co-immunoprecipitation. NSP6 binds to TBK1 without affecting TBK1 phosphorylation but this decreases IRF3 phosphorylation, while NSP13/TBK1 binding inhibits TBK1 phosphorylation. In both cases, IFN-β production is reduced (Xia et al., 2020).</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NSP5 (3CLpro), ORF3a</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>STING</strong>: Stimulator of interferon genes</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NA</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Rui et al. (2021) SARS-CoV-2 ORF3a and 3CLpro inhibited IFNβ promoter activity through cyclic GMP-AMP synthase (cGAS)-STING pathways, specifically through interaction with STING, as indicated by co-immunoprecipitation. 3CLpro also bound to STING and specifically inhibited K63-ubiquitin-mediated modification of STING, which is required for signaling and downstream expression of IFN-I. </span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NSP3 Papain-like protease (Plpro)</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>IRF3</strong>: Interferon regulatory factor 3</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NA</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mostaquil et al. (2020) showed with a fluorescent-based cleavage assay that NSP3 (Plpro) cleaves IRF-3, and thereby reduces IRF-3 available for induction of IFN-I expression.</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">N (nucleocapsid)</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><strong>STAT1/STAT2</strong>: Signal transducer and activator of transcription</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">NA</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Mu et al. (2020) used Sendai virus (SeV)-induced ISRE-promoter activation via the luciferase reporter assay to determine that SARS-CoV-2 N protein can inhibit the phosphorylation of STAT1 and STAT2 resulting in decrease in ISG production. They also showed through co-immunoprecipitation that N interacts with both STAT1 and STAT2, and that N inhibits STAT1/2 phosphorylation by blocking interactions with kinases including JAK1.</span></span></p>
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<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">There are uncertainties based on differing disease outcomes, especially associated with timing of IFN increase or suppression under different cell culture circumstances and in different people infected with SARS-CoV-2. Effectiveness of IFN treatment is still uncertain due to some studies evaluating IFN along with other drugs (Sodeifian et al., 2021).</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) are ISGs that have been implicated in SARS-CoV-2 entry as well as antiviral activity (Prelli Bozzo et al., 2021), in addition to the fact that the SARS-CoV-2 entry receptor ACE2 is an IFN-I stimulated gene (Ziegler et al., 2020). These are some of the paradoxes that confound transcriptomic studies that determine up- or downregulation of IFNs and ISGs in response to infection, and responses are highly dependent on the time points sampled. Efforts to address uncertainties around when and under what circumstances IFNs and ISGs either promote or supress the virus are ongoing. </span></span></p>
<p>The current quantitative understanding of this relationship is described below.</p>
HighUnspecificHighAll life stagesHigh<p><u>Sex and age applicability</u></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">It has been shown that in human populations males are more likely to suffer severe infections and deaths due to COVID-19 than females. However, in the viral entry and infection phase, one study found that women of working age had higher infection rates than men, but the suggested cause was higher contact rates among women (Doerre and Doblhammer, 2022). Contact rate increase is an important transmission factor but would not constitute a gender-based biological difference in viral entry or IFN-I pathway antagonism. A biological basis for females having higher levels of Type I IFN has been proposed concerning Toll-like receptor (TLR) 7. TLR7 is expressed in plasmacytoid dendritic cells (pDCs), an immune cell type that on infection with SARS-CoV-2 migrates from peripheral blood into the respiratory tract epithelium. TLR7 stimulates higher IFN-I production in pDCs in women than in men (Van der Sluis et al. 2022). It is proposed that this is due to the TLR7 gene being on the X chromosome, and that X inactivation in males is incomplete regarding the TLR7 gene, creating a double gene-dose effect in females (Spiering and de Vries, 2021). In a mouse SARS-CoV model, XY males had more adverse outcomes than XX females and XXY males (Gadi et al. 2020). Additionally, loss-of-function TLR7 mutations have been identified that are associated with increased COVID-19 severity (Szeto et al. 2021). However, these results focus on disease outcome as the endpoint, where factors beyond the initial antiviral response could be involved. Also note that the nasal and upper respiratory tract (URT) epithelial cells express ACE2 receptors for SARS-CoV-2 entry while the pDCs do not, relying on viral endocytosis (Van der Sluis et al. 2022). There is not a clear picture in the literature of the timing of pDC arrival in the epithelium after exposure, and the role of TLR7 in sex differences is currently hypothetical (Spiering and de Vries, 2021). </span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">In a large study modelling URT viral load dynamics drawn from measurements in 605 human subjects, variations over 5 orders of magnitude in URT viral load from the time of symptom onset was not explained by age, sex, or severity of illness. Additionally, these variables did not explain modelling results concerning control of viral load by immune responses in the early (innate) or late (adaptive) phases (Challenger et al. 2022). Other sources also support that rate of infection and measured viral load does not differ by gender (e.g., Arnold et al. 2022; Qi et al. 2021; Cheemarla et al. 2021). Therefore, evidence exists that the components of cell entry and the early antiviral response are not influenced by gender specific differences such as sex hormone levels or sex chromosomes to the extent of affecting viral load. Elderly people are more susceptible to severe disease than children and young adults, but Challenger et al. (2022) found no evidence indicating that life stage increases infectability or early viral load generation. However, Sharif-Askari et al. (2022) reported that children had higher expression of IFN-I and associated ISGs than adults.</span></span></p>
<p><span style="font-size:16px"><u><span style="font-family:"Calibri",sans-serif">Taxonomic applicability</span></u></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Generally, most mammals are likely susceptible to the SARS-CoV-2 virus based on reports of naturally and experimentally infected animals (See AO 1939). No infections have been reported in other classes of vertebrates. Other than bioinformatic studies on the ACE2 sequence across vertebrates however, there have been few studies on the mechanisms of susceptibility to infection of non-human hosts. Three studies were found on protein targets in the IFN-I innate immune response pathway that included other vertebrates. Rui et al. (2021) showed that SARS-CoV-2 3CLpro and ORF3a inhibit vertebrate (human, mouse, and chicken) STING ability to induce IFNβ promoter activity in a dose-dependent manner in HEK293T cells transfected with IFNβ-luciferase reporter plasmid vectors, together with tagged STING and cGAS vectors and increasing amounts of the SARS-CoV-2 3CLpro or ORF3a expression vectors. This study shows that the vulnerability of the host IFN-I pathway protein components to inhibition by SARS-CoV-2 protein stressors is not limited to humans, however Rui et al. (2021) did not determine the specific amino acids involved in the STING-ORF3a or STING-3CLpro interactions. Mostaquil et al. (2020) studied the cleavage site of IRF3 by PLpro (SARS-CoV-2 NSP3) and compared sequences across mammals. They determined that the IRF3 cleavage site in mammalian species in the taxonomic orders of primates, carnivora, artiodactyla, chiroptera (bats) and a few other mammals was conserved and would generally be susceptible to cleavage, and therefore IFN-I antagonism, but rodentia IRF3 would likely not be susceptible. Hameedi et al. (2022) compared molecular dynamic simulations of 3CLpro cleavage of NEMO in humans and mice showing a decrease in the average number of contacts between mNEMO and 3CLpro compared to hNEMO. Also, hNEMO may be more strongly bound to the catalytic site, and the mNEMO/3CLpro interaction appears more prone to destabilization (Hameedi et al., 2022).</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Arnold C.G., Libby, A., Vest, A. et al. 2022. Immune mechanisms associated with sex-based differences in severe COVID-19 clinical outcomes. <em>Biol Sex Differ</em> <strong>13</strong>, 7. <a href="https://doi.org/10.1186/s13293-022-00417-3" style="color:blue; text-decoration:underline">https://doi.org/10.1186/s13293-022-00417-3</a> </span></span></p>
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<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Ziegler CGK, Allon SJ, Nyquist SK, Mbano IM, … HCA Lung Biological Network. 2020. SARS-CoV-2 Receptor ACE2 Is an Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in Specific Cell Subsets across Tissues. Cell. 28;181(5):1016-1035.e19. <a href="https://doi.org/10.1016/j.cell.2020.04.035" style="color:blue; text-decoration:underline">https://doi.org/10.1016/j.cell.2020.04.035</a> </span></span></p>
2021-10-24T17:10:382023-04-03T15:49:338fc4d1cc-b330-4628-8435-fc48240252c1f3ec58f7-6c74-4e3f-a750-042df045078f2021-10-24T17:11:372021-10-24T17:11:37Binding of SARS-CoV-2 to ACE2 receptor leading to acute respiratory distress associated mortalitySARS-CoV-2 leads to acute respiratory distress<p> </p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif">Young Jun Kim<sup>1</sup>, Yongoh Lee<sup>1</sup>, Jihun Yang<sup>2</sup>, Chung Seok<sup>2,3</sup> Brigitte Landesmann<sup>4</sup>, Laure-Alix Clerbaux<sup>4</sup> , Penny Nymark<sup>5</sup>, Jukka Sund<sup>6</sup>, Filipovska, Julija<sup>7,</sup> Shihori Tanabe<sup>8</sup>, Gillian Bezemer<sup>9</sup>, Maria Joao Amorim<sup>10 </sup>and HyunJoon Kong<sup>11</sup> </span></span></p>
<p><span style="font-size:14px"><span style="font-family:Arial,Helvetica,sans-serif"><sup>1</sup>Korea Institute of Science and Technology (KIST) Europe, Saarbrücken 66123, Germany, <sup>2</sup> NEXT&BIO, South Korea, <sup> 3</sup>KU-KIST Graduate School of Converging Science and Technology, South Korea <sup>4</sup> F3 Chemical Safety and Alternative Methods Unit incorporating EURL ECVAM, Directorate F – Health, Consumers and Reference Materials Joint Research Centre, European Commission <sup>5</sup> Institute of Environmental Medicine, Karolinska Institutet, Solna, Sweden <sup>6</sup> F.3 unit, EURL-ECVAM Joint Research Centre, European Commission <sup>7 </sup>Independent, North Macedonia <sup>8</sup> Division of Risk Assessment, Center for Biological Safety and Research National Institute of Health Sciences, Japan <sup>9</sup> Impact Station, Universiteit Utrecht, Nederland <sup>10 </sup>Instituto Gulbenkian de Ciência, Portugal <sup>11 </sup>University of Illinois at Urbana-Champaign, USA</span></span></p>
Open for comment. Do not citeUnder DevelopmentIncluded in OECD Work Plan1.96<p>Inhalation of substances, including viral particles, the RNA virus capsid (S) glycoprotein binds the cellular receptor angiotensin-converting enzyme 2 (ACE2) and mediates fusion of the viral and cellular membranes through a pre- to postfusion conformation transition. The S protein is cleaved into S1 and S2 units by a human cell-derived protease (proteolytic enzyme) that is assumed to be Furin.S1 units then bind to its receptor, ACE2. The other fragment, S2, is cleaved by TMPRSS2, a human cell surface serine protease, resulting in cell membrane fusion. The S protein binds the catalytic domain of ACE2 with high affinities likewise, COVID-19 shares 79.6% homology of SARS-CoV and 96% identical at the whole-genome level to a bat coronavirus. The binding of the coronavirus S protein to ACE2 triggers a conformational change in the S protein of the coronavirus, allowing for proteolytic digestion by host cell proteases called TMPRSS2. The AOP reports the S glycoprotein of viral capsid in complex with its host cell receptor ACE2 resulted in acute respiratory distress associated with mortality by cytokine storms and enhanced inflammation in pulmonary tissue. S-glycoprotein of the virus uses ACE2 to get into cells that are found on the surface of epithelial cells in Kidney, Heart, Liver and Lung. However, there is an unexplored relationship for ACE2 levels between fibrotic hypersensitivity and Renin-Angiotensin Pathway which caused acute respiratory distress associated with mortality.</p>
<p>The <em>ACE2</em> gene encodes the angiotensin-converting enzyme-2, which has been proved to be the receptor for both the SARS-coronavirus (SARS-CoV) and the human respiratory coronavirus. ACE2 is a key component of blood pressure regulation in the renin-angiotensin system. Angiotensin (Ang) converting enzyme 2 (ACE2) is a homolog of ACE.<u><a href="https://pubmed.ncbi.nlm.nih.gov/15109615-ace2-a-new-regulator-of-the-renin-angiotensin-system/"> </a> </u><a href="https://pubmed.ncbi.nlm.nih.gov/15109615-ace2-a-new-regulator-of-the-renin-angiotensin-system/"><span style="color:#000000">ACE2 negatively regulates</span></a><u> </u>the renin-angiotensin system (RAS) by converting Ang II to Ang-(1-7) and AngI to Ang(1-9). The higher levels of receptor expression achieved by the expression of recombinant ACE2 could be relevant for cell-cell fusion. The underlying mechanisms remain to be elucidated and could play a role in the entry of the cell-free virus into cells and finally increase the acute respiratory distress associated with mortality.</p>
<p><span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif">Receptor recognition is an essential determinant of molecular level in this AOP. ACE2 was reported as an entry receptor for SARS-CoV-2. The viral entry process is mediated by the envelope-embedded surface-located spike (S) glycoprotein. Jun Lan and Walls, A.C et al (Nature 581, 215–220; Cell 180, 281–292) demonstrated a critical initial step of infection at the molecular level from the interaction of ACE2 and S protein. ACE2 has shown that receptor binding affinity to S protein is nM range. To elucidate the interaction between the SARS-CoV-2 RBD and ACE2 at a higher resolution, they also determined the structure of the SARS-CoV-2 RBD–ACE2 complex using X-ray crystallography.</span></span> <span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif">The expression and distribution of the ACE2 in human body may indicate the potential infection of SARS-CoV-2. Through the developed single-cell RNA sequencing (scRNA-Seq) technique and single-cell transcriptomes based on the public database, researchers analyzed the ACE2 RNA expression profile at single-cell resolution. High ACE2 expression was identified in type II alveolar cells (Zou, X. et al.</span></span> <span style="font-size:9.0pt"><span style="font-family:"Times New Roman",serif">Front. Med.2020)</span></span></p>
<p><span style="font-size:12px"><span style="font-family:"Times New Roman",serif">SARS-CoV-2 belongs to the Coronaviridae family, which includes evolutionary related enveloped (+) strand RNA viruses of vertebrates, such as seasonal common coronaviruses, SARS-CoV and CoV-NL63, SARS-CoV (Kim Young Jun et al)</span></span></p>
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<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:white">Human viruses strains</span></span></span></span></p>
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<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:white">Genus</span></span></span></span></p>
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<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:white">Major cell receptor</span></span></span></span></p>
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<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:white">First report</span></span></span></span></p>
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<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:white">Animal reservoir</span></span></span></span></p>
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<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:white">Intermediate host</span></span></span></span></p>
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<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:white">Pathology</span></span></span></span></p>
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<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:white">Diagnostic test</span></span></span></span></p>
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<p style="text-align:center"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:white">Evidence</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">HCoV-NL63</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">Alphacoronavirus</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">ACE2</span></span></span></span></p>
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<p style="text-align:right"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">2004</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">Bat</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">Unknown</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">Mild respiratory tract illness</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">RT-PCR, IF, ELISA, WB</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">Strong</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">SARS-CoV</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">Betacoronavirus</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">ACE2</span></span></span></span></p>
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<p style="text-align:right"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">2003</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">Bat</span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom; width:204px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">Pangolin</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">Severe acute respiratory syndrome</span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom; width:252px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">RT-PCR, IF, ELISA, WB</span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom; width:182px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">Strong</span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom; width:206px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">SARS-CoV-2</span></span></span></span></p>
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<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom; width:172px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">Betacoronavirus</span></span></span></span></p>
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<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">ACE2</span></span></span></span></p>
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<p style="text-align:right"><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">2020</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom; width:172px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">Bat</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom; width:204px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">Pangolin</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom; width:340px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">Severe acute respiratory syndrome</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom; width:252px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">RT-PCR, IF, ELISA, WB</span></span></span></span></p>
</td>
<td style="border-bottom:1px solid black; border-left:none; border-right:1px solid black; border-top:none; height:19px; vertical-align:bottom; width:182px">
<p><span style="font-size:12pt"><span style="font-family:"Times New Roman",serif"><span style="font-size:9.0pt"><span style="color:black">Strong</span></span></span></span></p>
</td>
</tr>
</tbody>
</table>
<p>Increased mortality is one of the most common regulatory assessment endpoints, along with reduced growth and reduced reproduction.</p>
adjacentHighHighadjacentNot SpecifiedModerateadjacentNot SpecifiedHighadjacentLowHighadjacentLowModerateadjacentNot SpecifiedModerateadjacentNot SpecifiedHighadjacentNot SpecifiedHighHighMixedHighConception to < FetalModerate<p>This AOP not only contributes new tools to study entry of the viral particles or Inhalation of stressors into cells and localize its receptor-binding domain of ACE2 but also could serve in the development of novel vaccine immunogens against TMPRSS2 proteases which may inhibit cell entry of COVID-19.</p>
HighModerateHigh<ol>
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</ol>
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