Increased SARS-CoV-2 production

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. 

Coronavirus is a class of viruses that have single-stranded positive-sense RNA genomes in their envelopes [D. Kim et al.]. The virus contains a 29.7 kB positive-sense RNA genome flanked by 5' and 3' untranslated regions of 265 and 342 nucleotides, respectively [E. J. Snijder et al.] that contain cis-acting secondary RNA structures essential for RNA synthesis [N. C. Huston et al.]. The genome just prior to the 5′ end contains the transcriptional regulatory sequence leader (TRS-L) [C.J. Budzilowicx et al.]. The SARS-CoV genome is polycistronic and contains 14 open reading frames (ORFs) that are expressed by poorly understood mechanisms [E. J. Snijder et al.]. Preceding each ORF there are other TRSs called the body TRS (TRS B). The 5′ two-thirds of the genome contains two large, overlapping, nonstructural ORFs and the 3′ third contains the remainder ORFs [H. Di et al.]. Genome expression starts with the translation of two large ORFs of the 5’ two-thirds: ORF1a of 4382 amino acids and ORF1ab of 7073 amino acid that occurs via a programmed (- 1) ribosomal frameshifting [E. J. Snider et al.], yielding pp1a and pp1ab. These two polyproteins are cleaved into 16 subunits by two viral proteinases encoded by ORF1a, nsp3, and nsp5 that contain a papain-like protease domain and a 3C-like protease domain [M. D. Sacco et al.]. The processing products are a group of replicative enzymes, named nsp1-nsp16, that assemble into a viral replication and transcription complex (RTC) associated with membranes of endoplasmic reticulum (ER) with the help of various membrane-associated viral proteins [S. Klein et al., E. J. Snijder et al., P. V'Kovski, et al.]. Besides replication, which yields the positive-sense gRNA, the replicase also 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. 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]. sgRNAs 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 [D. Kim et al.]. The gRNA is packaged by the structural proteins to assemble progeny virions.

Viral translation:

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 et al.]. 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 et al.]. Other elements of the genome include are shown below [V. B. O'Leary et al.]. The first event upon cell entry is the primary translation of the ORF1a and ORF1b gRNA to produce non-structural proteins (nsps).

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 et al.]. 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 et al.]. Two overlapping ORFs, ORF1a and ORF1b, generate continuous polypeptides, which are cleaved into a total of 16 so-called nsps [Y Finkel et al.]. 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 pp1a is approximately 1.4–2.2 times more expressed than pp1ab. After translation, the polyproteins are cleaved by viral proteases nsp3 and nsp5. Nsp5 protease can be referred to as 3C-like protease (3CL^pro) or as main protease (M^pro), as it cleaves the majority of the polyprotein cleavage sites. [H.A. Hussein et al.] Nsp1 cleavage is quick and nsp1 associates with host cell ribosomes and results in host cellular shutdown, suppressing host gene expression [M. Thoms et al.]. Fifteen proteins, nsp2–16 constitute the viral RTC. They are targeted to defined subcellular locations and establish a network with host cell factors. Nsp2–11 remodel host membrane architecture, mediate host immune evasion and provide cofactors for replication, whilst nsp12–16 contain the core enzymatic functions involved in RNA synthesis, modification and proofreading [P. V'Kovski et al.].  nsp-7 and nsp-8 form a complex priming the RNA-dependent RNA polymerase (RdRp or RTC) - nsp-12. nsp14 provides a 3′–5′ exonuclease activity providing RNA proofreading function. Nsp-10 composes the RNA capping machinery nsp-9. nsp13 provides the RNA 5′-triphosphatase activity. Nsp-14 is a N7-methyltransferase and nsp-16 the 2′-O-methyltransferase. 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 et al.]. Xia et al. [H. Xia et al.] 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.

Viral transcription and replication:

Viral transcription and replication occur at the viral replication organelle (RO) [E. J. Snijder et al.]. The RO is specifically formed during infection by reshaping ER and other membranes, giving rise to 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  [S. Klein et al., E. J. Snijder et al.]. There is some evidence that DMV accommodate viral replication which is based on radiolabelling viral RNA with nucleoside precursor ([5-³[H]uridine) and detection by EM autoradiography [E. J. Snijder et al.].

Viral replicative proteins and specific host factors are recruited to ROs [E. J. Snijder et al.]. RNA viral genome is transcribed into messenger RNA by the viral RTC [P. Ahlquist et al.]. 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 et al.]. Positive-sense (messenger-sense) RNA viruses replicate their genomes through negative-strand RNA intermediates [M. Schwartz et al.]. The intermediates comprise 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. The transcription of coronaviruses 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 Nidovirales 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 are unique features of the coronaviruses and arteriviruses [W. A. Miller and G. Koev.]. Of note, the produced genomic RNA represents a small fraction of the total vRNA [N. S. Ogando et al.].

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 [I. Sola, F. Almazan et al., I. Sola, J. L. Moreno, et al.]. 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 [H. Di et al.]. Therefore, the 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 [I. Sola, F. Almazan et al. I. Sola, J. L. Moreno, et al.]. 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 [X. Zhang et al.]. 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 [I. Sola et al. 2015, S. G. Sawicki et al., Y. Yang et al.]. 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 [Y. Yang]. The set of sg mRNAs is then translated to yield 29 identified different proteins [F. Wu et al.], although many papers have identified additional ORFs [D. Kim et al.. Y. Finkel et al., A. Vandelli et al.]. 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 et al.]. 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 5′ last third are found in higher amounts, and the nucleoprotein is the protein found in higher levels [Y. Finkel et al.].

Viral assembly:

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 [J. R. Cohen et al., A. Perrier et al.] and viral 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, forming viral ribonucleoprotein (vRNPs) complexes, before being selectively packaged into progeny virions [P. V'Kovski et al.], a step in which vRNPs bud into the lumen of the ER and the ER-Golgi intermediate compartment (ERGIC) [N. S. Ogando et al.]. 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.

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 et al., D. E. Gordon et al., 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 et al.], and this way organize viral transcription. Five studies have shown that N protein undergoes liquid-liquid phase separation (LLPS) in vitro [A. Savastano et al., H. Chen et al., C. Iserman et al., T. M. Perdikari et al., J. Cubuk et al.], dependent on its C-terminal domain (CTD) [H. Chen et al.]. 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 et al.], 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 et al., C. Iserman et al., C. R. Carlson et al.].

The ERGIC constitutes the main assembly site of coronaviruses [S. Klein et al., E. J. Snijder et al., L. Mendonca et al.] and budding events have been seen by EM studies. For SARS-CoV-2, virus-budding was mainly clustered in regions with a high vesicle density and close to ER- and Golgi-like membrane arrangements [S. Klein et al., E. J. Snijder et al., L. Mendonca et al.]. 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 [S. Klein et al.].

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 et al.]. This process results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation [S. Ghosh et al.].

Viral translation:

SARS-CoV-2 Nsp1 binds the ribosomal mRNA channel to inhibit translation [Schubert et al. 2020]

• Sucrose pelleting binding assay to verify Nsp1–40S complex formation
• In vivo translation assay
• Transient expression of FLAG-Nsp1 in HeLa cells and puromycin incorporation assay

SARS-CoV-2 disrupts splicing, translation, and protein trafficking [Banerjee et al. 2020]

• SARS-CoV-2 viral protein binding to RNA
• Interferon stimulation experiments
• Splicing assessment experiments
• IRF7-GFP splicing reporter, 5EU RNA labeling, capture of biotinylated 5EU labeled RNA

Membrane SUnSET assay for transport of plasma membrane proteins to the cell surface

Viral transcription:

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 et al.]. 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, et al.]. The viral mRNAs are reverse-transcribed with RT, followed by the amplification by PCR.

Viral replication:

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.

Viral production:

Plaque assays, infectivity assays, RT-qPCR to detect viral RNA in released virions, sequencing to detect mutations in the genome, electron microscopy.

Broad mammalian host range has been demonstrated based on spike protein tropism for and binding to ACE2 [Conceicao et al. 2020; Wu et al. 2020] and cross-species ACE2 structural analysis [Damas et al. 2020]. 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.

Very broad mammalian tropism: human, bat, cat, dog, civet, ferret, horse, pig, sheep, goat, water buffalo, cattle, rabbit, hamster, mouse

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