The importance of Coronavirus RTC for antiviral signaling

Internalised viruses are recognised by pathogen recognition receptors (PRRs) which sense Pathogen-associated Molecular Patterns (PAMP). Generally, PRRs are located within the endosomes -as it the case for Toll-like receptor (TLR)-7 or MyD88 - or alternatively in the cytoplasm, as it the case for retinoic acid inducible gene I (RIG-I) like receptors, and melanoma differentiation associated gene 5 (MDA5). In the case of RNA viruses, viral RNA is recognised by proteins belonging to the cytoplasmic RNA-induced silencing complex (RISC), which consists of RIG-1, MDA-5, protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer. RIG-1, PACT, and MDA-5 recognize viral RNA and induce the expression of various cytokines, in particular Interferon(s) (IFN) and ultimately a broad array of IFN stimulated genes (ISG) as part of the antiviral response. In contrast, TRBP and Dicer process pre-microRNAs into mature microRNAs (miRNAs) by recruiting Argonaute 2 (Ago2) that target specific mRNA species -such as viral RNA- for regulation and targeting steroid-responsive promoters.

PACT, allowing the degradation of viral RNA as well as stimulating an immune response, can induce both TBRP and Dicer and thus targets viral microRNAs, viral small RNAs, and viral dsRNA.

ssRNA or dsRNA can induce an antiviral Interferon or NF-κB response via TLR or MAVS

In order to prevent host cell derived mRNAs from being recognised by cellular PRRs, cellular mRNA molecules not only possess a 5’ cap structure but are also methylated at their 5’ end; any RNA contained no 5’ cap, a non-methylated, or incompletely methylated 5’cap can therefore be recognised by cellular PAMP receptors and induce an antiviral response, in addition to viral dsRNA intermediates. In this system, RIG-1 preferentially recognizes short blunt end or 5’ triphosphate RNAs whereas MDA-5 recognizes dsRNA intermediates as well as non-methylated RNAs lacking 2’O methylated ribose. Antiviral signaling by these pathways can be inhibited by viruses using either the cellular machinery for capping viral RNAs, and thus prevent them from being recognised by the RISC, or alternatively encoding enzymes which cap the viral RNA in a manner similar to cellular enzymes.

In addition to stimulating the expression of antiviral genes, the recognition of single-stranded viral RNA by TLR-7 also induces the formation of autophagy vesicles via the interaction between the TLR adaptor MyD88 and the Beclin-1, the latter being required for the formation of the Phagosome/Autophagosomes.

Autophagy and the antiviral response

The formation of autophagy vesicles has been postulated to facilitate the presentation of viral antigens by the cellular MHC Class I (e.g. HSV-1 gB), MHC Class II complex (e.g. EBNA-1) and target viral RNA to endosomes (and thus being recognised by TLR-7) as well as facilitating the degradation of viral components. Indeed, infecting cells with a HSV-1 mutant deficient for the viral autophagy inhibitory ICP 34.5 protein not only fails to inhibit autophagy but also has decreased infectious titers, suggesting that autophagy degrades HSV-1 components in addition to facilitate the presentation of viral antigens.

Coronavirus RTC and  the antiviral response

In the case of Coronavirus infected cells, the major pathogen associated pattern recognised by the PARRs is the dsRNA intermediate and the viral ssRNA both which are located with the replication-transcription complexes (RTCs) derived from the ER and induced by the expression of the viral nsp-3,-, and -6 proteins as discussed earlier. In these RTCs, the viral RNAs are not only synthesized but also modified in order to prevent the induction of the antiviral response. In order to prevent the viral RNA from being recognised by RIG-1 or MDA-5, the Coronavirus RNA contain a 5’cap structure which is added to newly synthesized viral RNA within the RTC by the viral RNA-triphosphatase (nsp13), 2’O-methyltransferase (nsp16), as well as a N7-Methyltransferase (nsp14). Failure to methylate viral RNA in cells infected with a nsp16 deficient virus induces a IFN type 1, MDA-5 dependent, antiviral response concomitant with elevated levels of ISGs. In addition to bind MDA-5, uncapped viral RNA binds IFN induced protein with tetratricopeptide repeats (IFIT)-1, thus preventing the translation of viral RNA. So far the precise contribution of the viral N7-Methyltransferase remains unknown although the equivalent in West Nile Virus has been shown to be required for 2’O-methyltransferase mediated methylation of WNV viral RNA. The N-terminus of nsp14 encodes for the viral 3’ -to-5’ exoribo-nuclease (ExoN) which hydrolyses both ssRNA and dsRNA and excises nucleotide mismatches in dsRNA intermediates, thus providing proofreading of newly synthesized RNA. Accordingly, inhibiting or deleting ExoN increases the potency of antivirals such as 5-Fluorouracil.  Since both ExoN and 2’O-methyltransferase activity positively regulated by nsp10, inhibiting nsp10 might be an interesting target for antiviral therapy. In addition to ExoN, both the Arteriviridae and the Coronaviridae express another enzyme that can hydrolyze RNA, EndoU. In contrast to ExoN the function of EndoU (nsp15) is less well defined and it has been proposed that it might be involved in cleaving free -“mislocalised”-RNA in order to prevent the recognition by PRRs, although EndoU can be found both in RTCs as well as in the cytoplasm. One possibility, which the author of these lines suggests, is that EndoU might be associated with Endosomes that contain viral RNA (see below). These Endosomes might contain viral RNA as a result of the induction of the formation of autophagy vesicles by nsp-3,-4, and -6.

Although the Coronaviral nsps-3,-4, and -6 induce the formation of autophagy like (LC3-II negative) vesicles, so far the formation of autolysosomes and subsequent degradation of viral components in infected cells has not been demonstrated. Since the formation of LC3-II positive vesicles has been demonstrated in cells transfected with nsp6 derived from SARS-CoV, MHV, and avian IBV it might be possible that viral RNA derived from autophagy vesicles can be found in endosomes and induce TLR-7 mediated antiviral signaling and/or viral components transferred to multivesicular class II loading compartments. If this is the case, TLR-7/-8 mediated anitviral signaling might be inhibited by the orf4a protein (in the case of MERS-CoV and SARS-CoV) whereas the MHC- Class II mediated activation of cytotoxic T lymphocytes might not be inhibited and indeed contribute in particular to the disease outcome of SARS-CoV or MERS-CoV infected patients.  

Coronavirus nsp-3,-4, and -6 might contribute to the induction of autophagy vesicle induced antiviral response counteracted by orf4a

The infection of microglia cells with a neurotropic strain of MHV, MHV-JHM, indeed lead to a sustained up-regulation of both MHC Class I and Class II molecules not only during viral induced inflammation but also following viral clearance, similar to patients which have recovered from SARS. The formation of LC3-II positive vesicles therefore might induce an antiviral response that might be partially blocked by viral proteins. Some evidence suggests that the formation of LC3-II is more pronounced in cells transfected with SARS-CoV derived nsp6 compared to IBV derived nsp6 but if this has any implication for inducing an antiviral response has to be investigated.

Last but not least, a short note on STAT1 and 2 mediated signaling, STAT 1 and 2 signaling can be inhibited by SARS-CoV orf6 protein which in contrast to the above mentioned proteins is not residing within the RTC but at the ER and Golgi membrane where it sequesters STAT1 and 2 and thus prevents nuclear entry of these. In this case, orf6 therefore prevents the activation of IFN-β stimulated genes.

The emerging MERS-CoV has been shown to infect a wide variety of cells of the immune response, including dendritic cells, macrophages and T-Lymphocytes. It remains to be seen if the antiviral genes encoded by MERS-CoV are differ in their ability to block antiviral signaling compared to those found in SARS-CoV or HCoV-NL63. It also remains to be seen if the antiviral signaling in dromedary camels or in bats infected with MERS-CoV is less vulnerable to these proteins or not - not an easy task in dromedaries- and if the differences in the antiviral response account for the relatively benign outcome in these animals. In humans, it would be interesting to see if TLR-8 (restricted to myeloid dendrite cells, monocytes, and monocyte derived dendritic cells) is differently affected by MERS-CoV derived proteins than TLR-7 (plasmacytoid dendrite cells). One might argue that these are marginal questions, but I would argue that it might shed some light on the pathogenesis of MERS. 

The importance of antagonizing the antiviral response does not end at preventing the viral RNA from being recognised. A recent study indicated that the HCoV-OC43 Nucleocapsid protein binds microRNA9 and prevents a NF-κΒ dependent antiviral response and thus TLR-8 antiviral signaling; . In addition, the MERS-CoV derived orf4b and SARS-CoV N protein also block NF-κΒ. Moreover, the SARS-CoV nsp1 papain-like protease inhibits IRF3 induced expression of IFN-β. It remains of course to be seen if the mechanisms described for SARS-CoV or HCoV-OC43 also apply to MERS-CoV and more importantly if these mechanisms differ among various host species.

Coronavirus derived proteins block the antiviral signaling at various stages

As the reader of these lines can see, there is much to be learned from a virus family which until 2003 has been considered to be only a marginal virus by many.  

Further reading

Kindler E, & Thiel V (2014). To sense or not to sense viral RNA-essentials of coronavirus innate immune evasion. Current opinion in microbiology, 20C, 69-75 PMID: 24908561 

Zinzula L, & Tramontano E (2013). Strategies of highly pathogenic RNA viruses to block dsRNA detection by RIG-I-like receptors: hide, mask, hit. Antiviral research, 100 (3), 615-35 PMID: 24129118 

Redfern AD, Colley SM, Beveridge DJ, Ikeda N, Epis MR, Li X, Foulds CE, Stuart LM, Barker A, Russell VJ, Ramsay K, Kobelke SJ, Li X, Hatchell EC, Payne C, Giles KM, Messineo A, Gatignol A, Lanz RB, O'Malley BW, & Leedman PJ (2013). RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators. Proceedings of the National Academy of Sciences of the United States of America, 110 (16), 6536-41 PMID: 23550157 

English, L., Chemali, M., Duron, J., Rondeau, C., Laplante, A., Gingras, D., Alexander, D., Leib, D., Norbury, C., Lippé, R., & Desjardins, M. (2009). Autophagy enhances the presentation of endogenous viral antigens on MHC class I molecules during HSV-1 infection Nature Immunology, 10 (5), 480-487 DOI: 10.1038/ni.1720 

English L, Chemali M, & Desjardins M (2009). Nuclear membrane-derived autophagy, a novel process that participates in the presentation of endogenous viral antigens during HSV-1 infection. Autophagy, 5 (7), 1026-9 PMID: 19556870 

Paludan C, Schmid D, Landthaler M, Vockerodt M, Kube D, Tuschl T, & Münz C (2005). Endogenous MHC class II processing of a viral nuclear antigen after autophagy. Science (New York, N.Y.), 307 (5709), 593-6 PMID: 15591165 

Cavignac Y, & Esclatine A (2010). Herpesviruses and autophagy: catch me if you can! Viruses, 2 (1), 314-33 PMID: 21994613

Taylor GS, & Rickinson AB (2007). Antigens and autophagy: the path less travelled? Autophagy, 3 (1), 60-2 PMID: 17102586 

Züst R, Cervantes-Barragan L, Habjan M, Maier R, Neuman BW, Ziebuhr J, Szretter KJ, Baker SC, Barchet W, Diamond MS, Siddell SG, Ludewig B, & Thiel V (2011). Ribose 2'-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5. Nature immunology, 12 (2), 137-43 PMID: 21217758

Chen Y, Su C, Ke M, Jin X, Xu L, Zhang Z, Wu A, Sun Y, Yang Z, Tien P, Ahola T, Liang Y, Liu X, & Guo D (2011). Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex. PLoS pathogens, 7 (10) PMID: 22022266 

Cao J, & Zhang X (2012). Comparative in vivo analysis of the nsp15 endoribonuclease of murine, porcine and severe acute respiratory syndrome coronaviruses. Virus research, 167 (2), 247-58 PMID: 22617024 

Li T, Xie J, He Y, Fan H, Baril L, Qiu Z, Han Y, Xu W, Zhang W, You H, Zuo Y, Fang Q, Yu J, Chen Z, & Zhang L (2006). Long-term persistence of robust antibody and cytotoxic T cell responses in recovered patients infected with SARS coronavirus. PloS one, 1 PMID: 17183651

Hamo L, Stohlman SA, Otto-Duessel M, & Bergmann CC (2007). Distinct regulation of MHC molecule expression on astrocytes and microglia during viral encephalomyelitis. Glia, 55 (11), 1169-77 PMID: 17600339 

Frieman M, Yount B, Heise M, Kopecky-Bromberg SA, Palese P, & Baric RS (2007). Severe acute respiratory syndrome coronavirus ORF6 antagonizes STAT1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/Golgi membrane. Journal of virology, 81 (18), 9812-24 PMID: 17596301 

Lai FW, Stephenson KB, Mahony J, & Lichty BD (2014). Human coronavirus OC43 nucleocapsid protein binds microRNA 9 and potentiates NF-κB activation. Journal of virology, 88 (1), 54-65 PMID: 24109243