PR:000003622angiotensin-converting enzyme 2PR:000016456transmembrane protease serine 2PR:P07711cathepsin L1 (human)GO:0019015viral genomePR:000036197viral proteinGO:0031381viral RNA-directed RNA polymerase complexGO:0072516viral assembly compartmentGO:0019024ssRNA viral genomeCHEBI:26523reactive oxygen speciesGO:0072559NLRP3 inflammasome complexGO:0005102receptor bindingGO:0061025membrane fusionGO:0075509endocytosis involved in viral entry into host cellGO:0046718viral entry into host cellGO:0039694viral RNA genome replicationGO:0039690positive stranded viral RNA replicationGO:0019074viral RNA genome packagingGO:0009299mRNA transcriptionGO:0019081viral translationGO:0034599cellular response to oxidative stressGO:0000302response to reactive oxygen speciesGO:0044546NLRP3 inflammasome complex assembly3occurrence1increasedSars-CoV-2<p>Virus from the coronaviridae family related to SARS-CoV, 229E, NL63, OC43, HKU1 and MERS.</p>
<p>Transmitted by aerosols</p>
2021-02-23T04:50:402022-09-09T05:09:36Stressor:624 SARS-CoV-22021-04-20T03:40:362021-04-20T03:40:369606Homo sapiens10090mouse9666Mustela lutreola9685Felis catus9694Panthera tigris9615Canis familiaris9974Manis javanica9541Macaca fascicularis10036Mesocricetus auratus9669Mustela putorius furoWCS_452646Neovison vison10090Mus musculusBinding 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>
<table cellspacing="0" class="MsoTableGrid" style="border-collapse:collapse; border:medium none; height:806px; width:1049px">
<tbody>
<tr>
<td style="background-color:#a6a6a6; border-bottom:1px solid black; border-left:1px solid black; border-right:1px solid black; border-top:1px solid black; vertical-align:top; width:146px">
<p style="text-align:center"> </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: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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
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<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>
2020-03-02T03:18:472023-04-03T04:03:07SARS-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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2020-03-01T10:29:312023-04-04T07:39:34Increased 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>
UBERON:0000062organCL:0000000cellHighUnspecificHighAll life stagesHighModerateModerate<p style="margin-left:48px"><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif"><span style="font-family:Arial,Helvetica,sans-serif">1. D. Kim<em> et al.</em>, The Architecture of SARS-CoV-2 Transcriptome. <em>Cell</em> <strong>181</strong>, 914-921 e910 (2020).</span></span></span></p>
<p style="margin-left:48px"><span style="font-family:Arial,Helvetica,sans-serif"><span style="font-size:11pt">2. E. J. Snijder<em> et al.</em>, Unique and Conserved Features of Genome and Proteome of SARS-coronavirus, an Early Split-off From the Coronavirus Group 2 Lineage. <em>Journal of Molecular Biology</em> <strong>331</strong>, 991-1004 (2003).</span></span></p>
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2021-03-25T19:55:122022-06-14T08:49:57Diminished protective oxidative stress responseDiminished Protective Response to ROSCellular<p>Oxidative stress is caused by an imbalance between the production of reactive oxygen and the detoxification of reactive intermediates. Reactive intermediates such as peroxides and free radicals can be very damaging to many parts of cells such as proteins, lipids, and DNA. Severe oxidative stress can trigger apoptosis and necrosis. (Ref. IPA, NRF2-mediated Oxidative Stress Response, version60467501, release date: 2020-11-19)</p>
<p>The cellular defence/defense response to oxidative stress includes induction of detoxifying enzymes and antioxidant enzymes. Nuclear factor-erythroid 2-related factor 2 (Nrf2) binds to the antioxidant response elements (ARE) within the promoter of these enzymes and activates their transcription. Inactive Nrf2 is retained in the cytoplasm by association with an actin-binding protein Keap1. Upon exposure of cells to oxidative stress, Nrf2 is phosphorylated in response to the protein kinase C, phosphatidylinositol 3-kinase and MAP kinase pathways. After phosphorylation, Nrf2 translocates to the nucleus, binds AREs, and transactivates detoxifying enzymes and antioxidant enzymes, such as glutathione S-transferase, cytochrome P450, NAD(P)H quinone oxidoreductase, heme oxygenase, and superoxide dismutase. (Ref. IPA, NRF2-mediated Oxidative Stress Response, version60467501, release date: 2020-11-19)</p>
<p>Nrf2, a master regulator of oxidative stress through enhanced expression of anti-oxidant genes of glutathione and thioredoxin-antioxidant systems, has anti-inflammatory, anti-apoptotic, and antioxidant effects. Dimethyl fumarate (DMF), an activator of Nrf2, can decrease inflammation and reactive oxygen species (ROS) through the inhibition of NF-kappaB by inducing anti-oxidant enzymes (Jackson et al, 2014; Hassan et al, 2020; Timpani et al, 2021).</p>
<p>Inactivation of Nrf2 causes diminished protective responses to ROS.</p>
<p>Oxidative stress can be measured as follows:</p>
<p>1. Direct detection of reactive oxygen species (ROS)</p>
<p>ROS can be detected by intracellular ROS assay, in vitro ROS/RNS assay. Nitric oxide can be detected in intracellular nitric oxide assay (Ashoka et al, 2020).</p>
<p>Hydroxyl, peroxyl, or other ROS can be measured using a fluorescence probe, 2', 7'-Dichlorodihydrofluorescin diacetate (DCFH-DA), at fluorescence detection at 480 nm/530 nm.</p>
<p>Hydrogen peroxide (H2O2) can be detected with a colorimetric probe, which reacts with H2O2 in a 1:1 stoichiometry to produce a bright pink colored product, followed by the detection with a standard colorimetric microplate reader with a filter in the 540-570 nm range.</p>
<p>ROS can be detected by PEGylated bilirubin-coated iron oxide nanoparticles in whole blood (Lee et al, 2020).</p>
<p>2. Measurement of anti-oxidants</p>
<p>The level of catalase, glutathione, or superoxide dismutase can be measured as anti-oxidants. Catalase is an anti-oxidative enzyme that catalyses the resolution of hydrogen peroxide (H2O2) into H2O and O2. The chemiluminescence or fluorescence of HRP catalytic reaction can be detected with residual H2O2 and probes (DHBS+AAP, or ADHP (10-Acetyl-3, 7-dihydroxyphenoxazine)).</p>
<p>Anti-oxidant capacity is also one of the oxidative stress markers. Oxygen radical antioxidant capacity (ORAC), hydroxyl radical antioxidant capacity (HORAC), total antioxidant capacity (TAC), the cell-based exogenous antioxidant assay can be used for measuring the antioxidant capacity.</p>
<p>3. Detection of damages in protein, lipid, DNA or RNA</p>
<p>Oxidation of protein can be measured by the detection of protein carbonyl content (PCC), 3-nitrotyrosine, advanced oxidation protein products, or BPDE protein adduct. </p>
<p>DNA oxidation can be detected with 8-oxo-dG / 8-hydroxy-2'-deoxyguanosine (8-OHdG) by ELISA or HPLC (Chepelev et al, 2015; Valavanidis et al, 2009).</p>
<p>Lipid peroxides decompose to form malondialdehyde (MDA) and 4, hydroxynonenal (4-HNE), natural bi-products of lipid peroxidation. Lipid peroxidation can be monitored by thiobarbituric acid (TBA) reactive substances in biological samples. MDA and TBA form MDA-TBA adduct in a 1:2 stoichiometry and detected by colorimetric or fluorometric measurement.</p>
<p> </p>
<p>Response to ROS occurs in many cell types and tissues in all life stages and the broad range of mammals.</p>
UBERON:0000062organCL:0000000cellHighUnspecificHighAll life stagesHigh<p>Ashoka, A.H. et al. (2020), “Recent Advances in Fluorescent Probes for Detection of HOCl and HNO”, ACS omega, 5(4), 1730-1742. https://doi.org/10.1021/acsomega.9b03420.</p>
<p>Chepelev, N.L. et al. (2015), “HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, in Cultured Cells and Animal Tissues”, J Vis Exp, e52697-e52697, https://doi.org/10.3791/52697.</p>
<p>Hassan, S.M. et al. (2020), “The Nrf2 Activator (DMF) and Covid-19: Is there a Possible Role?”, Med Arch, 74(2), 134-138. https://doi.org/10.5455/medarh.2020.74.134-138.</p>
<p>Jackson, A.F. et al. (2014), “Case study on the utility of hepatic global gene expression profiling in the risk assessment of the carcinogen furan”, Toxicol Appl Pharmacol, 274, 63-77, https://doi.org/10.1016/j.taap.2013.10.019.</p>
<p>Lee, D.Y. et al. (2020), “PEGylated Bilirubin-coated Iron Oxide Nanoparticles as a Biosensor for Magnetic Relaxation Switching-based ROS Detection in Whole Blood”, Theranostics, 10(5), 1997-2007. https://doi.org/10.7150/thno.39662.</p>
<p>Timpani, C.A, E. Rybalka. (2021), “Calming the (Cytokine) Storm: Dimethyl Fumarate as a Therapeutic Candidate for COVID-19.”, Pharmaceuticals, 14(1), 15. https://doi.org/10.3390/ph14010015.</p>
<p>Valavanidis, A. et al. (2009), “8-hydroxy-2' -deoxyguanosine (8-OHdG): A critical biomarker of oxidative stress and carcinogenesis”, J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 27, 120-39. https://doi.org/10.1080/10590500902885684</p>
2021-04-20T03:34:232023-03-09T20:49:35NLRP3 inflammasome activity, increasedinflammasome activity, increasedMolecular<p><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">The NLRP3 inflammasome is a critical component of the innate immune system that mediates Caspase 1 (CASP1) activation. The NLRP3 inflammasome is a multimolecular complex composed of the sensor molecule NLRP3, the adaptor protein PYCARD (commonly called ASC), and pro-caspase 1 (Yang et al, 2019b). In activated inflammasome pro-caspase 1 is cleaved into active CASP1 which subsequently cleaves and thus activates highly pro-inflammatory cytokines interleukin-1B (IL1B) and IL18 leading to increased inflammation (Kelley et al, 2019). NLRP3 inflammasome activation can also induce pyroptosis, an inflammatory form of a cell death (Bergsbaken et al, 2009). Inflammasome activation is associated with COVID-19 disease severity and poor clinical outcome (Rodrigues et al, 2021). NLRP3 inflammasomes can assemble in many cell types, including macrophages, dendritic cells, neutrophils, B cells and T cells, epithelial cells, adipocytes, fibroblasts, astrocytes, cardiomyocytes, hepatocytes, etc (Enni et al, 2020; Ershaid et al, 2019; Wree et al, 2014; Wu et al, 2020; Yang et al, 2019a; Zheng et al, 2020).</span></span></p>
<p><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">In activated inflammasome pro-caspase 1 is cleaved into active CASP1 which then cleaves and thus activates IL1B and IL18. A common method of detection of activated inflammasome is the measurement of secreted IL1B and/or IL18 levels with the enzyme-linked immunosorbent assay (ELISA) using specific antibodies </span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">(Martinez<em> et al</em>, 2015; Piancone<em> et al</em>, 2018; Shi<em> et al</em>, 2018; Sun<em> et al</em>, 2017; Yaron<em> et al</em>, 2015)</span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">. </span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Formation of ASC oligomers reflects inflammasome activation thus ASC oligomers are often used to assess NLRP3 activation. Various methods of ASC oligomer or ASC specks detection is described in a thorough review from Zito and co-authors with references to the studies where the discussed methods are used </span></span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">(Zito<em> et al.</em>, 2020)</span></span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">. </span></span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">For measuring CASP1 activity as a result of inflammasome assembly and activation, Caspase 1 Fluorescein (FLICA) Assay can be used </span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">(Guo<em> et al</em>, 2018; Yaron<em> et al.</em>, 2015)</span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">. FLICA allows active CASP1 enzyme fluorescent labelling that can be analyzed using fluorescence microscopy, </span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">fluorescence plate reader or</span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif"> flow cytometry. </span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif"><span style="color:black">Furthermore, </span></span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">co-immunoprecipitation assays can be used for specific protein interaction detection (e.g. NLRP3 and ASC/PYCARD) as a result of NLRP3 inflammasome assembly </span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">(Zito<em> et al.</em>, 2020)</span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<p><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">Most of the studies on NLRP3 inflammasome activation are derived from mouse and human tissue experiments. The NLRP3 inflammasome activation and downstream inflammatory response is comprehensively documented in and thus applicable for Homo sapiens </span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">(Zito<em> et al</em>, 2020)</span></span><span style="font-size:12.0pt"><span style="font-family:"Calibri",sans-serif">.</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Farag NS, Breitinger U, Breitinger HG, El Azizi MA (2020) Viroporins and inflammasomes: A key to understand virus-induced inflammation. <em>Int J Biochem Cell Biol</em> 122: 105738</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Guo C, Fu R, Wang S, Huang Y, Li X, Zhou M, Zhao J, Yang N (2018) NLRP3 inflammasome activation contributes to the pathogenesis of rheumatoid arthritis. <em>Clin Exp Immunol</em> 194: 231-243</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Martinez GJ, Robertson S, Barraclough J, Xia Q, Mallat Z, Bursill C, Celermajer DS, Patel S (2015) Colchicine Acutely Suppresses Local Cardiac Production of Inflammatory Cytokines in Patients With an Acute Coronary Syndrome. <em>J Am Heart Assoc</em> 4: e002128</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Piancone F, Saresella M, Marventano I, La Rosa F, Santangelo MA, Caputo D, Mendozzi L, Rovaris M, Clerici M (2018) Monosodium Urate Crystals Activate the Inflammasome in Primary Progressive Multiple Sclerosis. <em>Front Immunol</em> 9: 983</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Shah A (2020) Novel Coronavirus-Induced NLRP3 Inflammasome Activation: A Potential Drug Target in the Treatment of COVID-19. <em>Front Immunol</em> 11: 1021</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Shi J, Zhao W, Ying H, Zhang Y, Du J, Chen S, Li J, Shen B (2018) Estradiol inhibits NLRP3 inflammasome in fibroblast-like synoviocytes activated by lipopolysaccharide and adenosine triphosphate. <em>Int J Rheum Dis</em> 21: 2002-2010</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Sun X, Hao H, Han Q, Song X, Liu J, Dong L, Han W, Mu Y (2017) Human umbilical cord-derived mesenchymal stem cells ameliorate insulin resistance by suppressing NLRP3 inflammasome-mediated inflammation in type 2 diabetes rats. <em>Stem Cell Res Ther</em> 8: 241</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Xu H, Chitre SA, Akinyemi IA, Loeb JC, Lednicky JA, McIntosh MT, Bhaduri-McIntosh S (2020) SARS-CoV-2 viroporin triggers the NLRP3 inflammatory pathway. <em>bioRxiv</em>: 2020.2010.2027.357731</span></span></p>
<p><span style="font-size:11pt"><span style="font-family:Calibri,sans-serif">Yaron JR, Gangaraju S, Rao MY, Kong X, Zhang L, Su F, Tian Y, Glenn HL, Meldrum DR (2015) K(+) regulates Ca(2+) to drive inflammasome signaling: dynamic visualization of ion flux in live cells. <em>Cell Death Dis</em> 6: e1954</span></span></p>
<p><span style="font-size:11.0pt"><span style="font-family:"Calibri",sans-serif">Zito G, Buscetta M, Cimino M, Dino P, Bucchieri F, Cipollina C (2020) Cellular Models and Assays to Study NLRP3 Inflammasome Biology. <em>Int J Mol Sci</em> 21</span></span></p>
2021-06-25T05:21:292021-06-25T08:29:40PyroptosisPyroptosisCellular<p>Pyroptosis is an inflammatory form of programmed cell death. Pore-forming protein gasdermins (GSDM) are crucial factors for pyroptosis execution whereby GSDMD and GSDME are most deeply studied [1]. Pyroptosis is initiated through inflammasome activation resulting to activation of caspase-1 (CASP1). Active CASP1 cleaves GSDMD, and also cleaves and thus activates pro-inflammatory cytokines interleukin-1B (IL1B) and IL18 [2]. N-terminal cleaved domain of GSDMD forms pores in the plasma membrane leading to cell swelling and pyroptotic cell death [3, 4]. IL1B and IL18 are released through the pores contributing to inflammatory and pyroptotic processes.</p>
<p>A common way to measure pyroptosis is through enzymatic assays for detection of lactate hydrogenase (LDH). LDH is released when plasma membrane is damaged and is a common measure of cytotoxicity. LDH release assays using commercially available kit and a quick, cost-effective method adapted from Decker et al [8] are described by Rayamajhi and colleagues [9]. Den Hartigh and Fink also describe LDH release assay as well as fluorescent microscopy method for visualization of loss of membrane integrity during pyroptosis [10]. Pyroptosis initiation can be inferred from CASP1 activation hence the CASP1 Fluorescein Assay (FLICA) is also used by researchers as detection method [11, 12]. Furthermore, Hoechst 33342 (chromatin condensation detection) and propidium iodide (probe for membrane damage) double staining can be used for pyroptotic cell detection although this method also detects other cell death types (e.g. apoptotis) [11, 13].</p>
<p>1. Yu, P., et al., Pyroptosis: mechanisms and diseases. Signal Transduct Target Ther, 2021. 6(1): p. 128. </p>
<p>2. Kelley, N., et al., The NLRP3 Inflammasome: An Overview of Mechanisms of Activation and Regulation. Int J Mol Sci, 2019. 20(13). </p>
<p>3. He, W.T., et al., Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion. Cell Res, 2015. 25(12): p. 1285-98. </p>
<p>4. Sborgi, L., et al., GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death. Embo j, 2016. 35(16): p. 1766-78. </p>
<p>5. Farag, N.S., et al., Viroporins and inflammasomes: A key to understand virus-induced inflammation. Int J Biochem Cell Biol, 2020. 122: p. 105738. </p>
<p>6. Shah, A., Novel Coronavirus-Induced NLRP3 Inflammasome Activation: A Potential Drug Target in the Treatment of COVID-19. Front Immunol, 2020. 11: p. 1021. </p>
<p>7. Xu, H., et al., SARS-CoV-2 viroporin triggers the NLRP3 inflammatory pathway. bioRxiv, 2020: p. 2020.10.27.357731. </p>
<p>8. Decker, T. and M.L. Lohmann-Matthes, A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J Immunol Methods, 1988. 115(1): p. 61-9. </p>
<p>9. Rayamajhi, M., Y. Zhang, and E.A. Miao, Detection of pyroptosis by measuring released lactate dehydrogenase activity. Methods Mol Biol, 2013. 1040: p. 85-90. </p>
<p>10. den Hartigh, A.B. and S.L. Fink, Pyroptosis Induction and Detection. Curr Protoc Immunol, 2018. 122(1): p. e52. </p>
<p>11. Chen, A., et al., Rosuvastatin protects against coronary microembolization-induced cardiac injury via inhibiting NLRP3 inflammasome activation. Cell Death Dis, 2021. 12(1): p. 78. </p>
<p>12. Wang, Y., et al., Caspase-1-Dependent Pyroptosis of Peripheral Blood Mononuclear Cells Is Associated with the Severity and Mortality of Septic Patients. Biomed Res Int, 2020. 2020: p. 9152140. </p>
<p>13. Chen, A., et al., Liraglutide attenuates NLRP3 inflammasome-dependent pyroptosis via regulating SIRT1/NOX4/ROS pathway in H9c2 cells. Biochem Biophys Res Commun, 2018. 499(2): p. 267-272. </p>
2022-01-18T03:05:052022-01-19T05:03:39SARS-CoV-2 infection leading to pyroptosisSARS-CoV2 to pyroptosisUnder development: Not open for comment. Do not cite<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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<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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<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-2</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">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>
2021-06-25T05:14:422023-04-29T13:02:19