COVID-19 and antiviral singling: role of orf4b and M protein

Coronaviruses (CoV) are positive sense RNA viruses with a genome size of 29-32 kb with four genera (Alpha-, Beta-, Gamma- and Delta CoV) belonging to the family of the Coronaviridae within the order of Nidovirales, with the Betacoronaviruses further divided into four lineages (A-D).

Table: Classification of known CoV

Most CoV identified until now are causing severe disease only in animals including agricultural important animals such as chicken, cattle, and pigs. To date only six human CoV (HCoV) have been identified, namely HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Severe Respiratory Syndrome (SARS)-CoV and most recently Middle Eastern Respiratory Syndrome (MERS)-CoV, although a SARS-like CoV, WIV1-CoV, replicates in primary human epithelial cells at low levels as well as in mice expressing the human SARS-CoV receptor ACE2 albeit at lower levels compared to SARS-CoV.  

In contrast to SARS-CoV and COVID-19, the infection with HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1only causes mild upper respiratory tract infections, whereas the infection with COVID-19 causes severe lower respiratory infection and renal failure which may lead to the death of infected individuals.

Although the natural host for COVID-19 has not been conclusively identified, bats might act as a natural reservoir of COVID-19 (similar to Hendra and Nipah Virus) as evidenced by studies indicating the presence of MERS-like CoV in bats from Saudi Arabia, Europe, Africa and Asia. Furthermore, COVID-19 can enter and replicate both in cell lines derived from bats and humans and the closely related Bat CoV-HKU4 can enter cells by binding to DPP4, the receptor for COVID-19. In Jamaican fruit bats (Artibeus jamaicensis) experimentally infected with COVID-19, clinical signs are absent despite viral replication and shedding in the respiratory and intestinal tract for up to 9 days p.i. as well as the induction of an antiviral response (see below for discussion).

Despite the ability of MERS and MERS-like CoV to infect both human and bat cell lines, bats are unlikely to be the source for the current outbreak of MERS in the Arabian Peninsula based on findings that livestock –in particular dromedary camels- have a high seroprevalence for COVID-19. In addition, recent studies showed that DPP4 is expressed at high levels in the upper respiratory tract of dromedary camels (but not humans) and that sequences of COVID-19 isolated from dromedary camels are highly similar to sequence from human derived viral isolates. Finally, camel derived COVID-19 can efficiently replicate in human cells. 

                    COVID-19 and the Interferon response

The polycistronic single stranded positive sense viral RNA of COVID-19 encodes for two polyproteins, orf1a and orf1b, within the 5’ end which are cleaved into a total of 16 non-structural proteins (nsp) including the viral RNA dependent RNA polymerase whereas the 3’ end of the viral RNA encodes for the structural proteins, namely spike (S), membrane (M), envelope (E) protein as well as lineage specific proteins.

Figure: Processing of the orf1ab into several nsp

Following viral entry and release of the viral RNA, COVID-19 replicates in the cytoplasm of cell, although the viral N protein is imported into the nucleus and localizes to the nucleolus. During viral replication, replication centers containing viral proteins and dsRNA are formed in the ER-Golgi intermediate compartment (ERGIC) in a process that might involve the induction of autophagy and/or viral induced rearrangements of the ER membrane that promote the formation of vesicular structures.

Figure: Outline of CoV replication cycle

The infection of a variety of cell lines with SARS-CoV and COVID-19 has been reported to induce the expression of type I interferons (IFN) including IFN-α and IFN-β, suggesting that both SARS-CoV and COVID-19 induce antiviral signaling via the recognition of viral RNA by PRR and PAMPS. Furthermore, both SARS-CoV and COVID-19 express proteins that counteract the IFN pathway and thus contribute to the severity of infection. In general, COVID-19 RNA is recognized by viral sensors of the retinoic acid-inducible gene-I (RIG-1)-like receptor (RLR) family melanoma differentiation gene 5 (MDA5) that upon activation by viral dsRNA intermediates, induce the nuclear translocation of both IFN regulatory factor (IRF)-3 and nuclear factor κB (NF-κB), both of which induce the expression of IFN-β.  RIG-1 signaling induced by the viral RNA as well as by Poly (I:C) however can be inhibited by several viral proteins, including ORF4a, ORF4b, ORF5 and M. In the case of ORF4a, the viral protein binds dsRNA and thus inhibits the activation of RIG-1 and MDA-5, whereas the nuclear localization of ORF4b inhibits the induction of IFN-β as well as the expression of RIG-I, MDA5, MAVS, IKKε, and TBK-1 by inhibiting both (nuclear) IRF-3 and IRF-7 whereas in the cytoplasm ORF4b inhibits antiviral signaling by binding to TANK binding kinase 1 (TBK1) and IκB kinase epsilon (IKK-ε) thus suppressing the interaction between mitochondrial antiviral signaling protein (MAVS) and IKK-ε. In addition to inhibiting IRF-3 by binding to TBK1, both COVID-19 and ORF4b protein also inhibits antiviral signaling by inhibiting the IFN inducible oligoadenylate synthetase (OAS)-RNase L pathway that is activated by the viral dsRNA intermediate via degradation of 2′,5′-oligoadenylate (2-5A), the activator of RNase L, thus inhibiting RIG-1 and MDA-5 induced signaling. Interestingly, ORF4b localises to the nucleolus in ORF4b transfected cells, but so far the importance for nucleolar localisation of ORF4b for antiviral signaling has not been demonstrated. In contrast to ORF4b, ORF5 –like ORF4a and M- can only be detected in the cytoplasm of infected cells. Similar to ORF4b however, the expression of ORF5 inhibits the induction of IFN-β upon infection with Sendai Virus (albeit to a lesser effect) probably by inhibiting IRF-3 and -7 but not NF-κB.  

Activated RNaseL cleaves viral and cellular ssRNA including ribosomal RNA preferentially at UU and UA dinucleotide residues, therefore not only inhibiting replication of the viral genome but also the expression of viral proteins as well as processing viral RNA to be recognized by intracellular RNA sensing proteins thus activating antiviral signaling pathways.

In the case of CoV, the Mouse Hepatitis Virus (MHV) NS2 protein encoded by ORF2a has been identified as an inhibitor of the OAS-RNaseL pathway by inhibiting the conversion of intracellular ATP to 2’,5’- OAS via the 2’,5’ Phosphodiesterase domain (PDE) following the detection of viral dsRNA intermediates or synthetic Poly (I:C) RNA. Alignment of COVID-19, BtCoV-HKU5 and BtCoV-SC2019 orf4b with MHV nsp2 lead to the identification of a 2’,5’- PDE domain that is preceded by a nuclear localization signal that is catalytically active in a fluorescence resonance energy transfer (FRET) based RNaseL activity assay with similar kinetics to MHV nsp2. As expected, all orf4b proteins localize to the nucleus and the cytoplasm if expressed in murine L2 cells infected with MHV chimeras that express a catalytically inactive mutant of nsp2 and catalytically active orf4b derived from COVID-19 or BatCoV-SC2019; in case of the MHV-BtCoV-HKU5 orf4b chimera, orf4b predominantly localises to the nucleus  which does not affect viral replication but might not inhibit the activation of RNaseL, indicating that only cytoplasmic orf4b can inhibit RNaseL activation. In Calu-3 cells, a human airway epithelial cell line, infected with COVID-19, the deletion of nsp3-5 or the deletion of orf4b does not activate the degradation of rRNA to the full extent when compared to wt COVID-19 probably due to the alternative pathways that inhibit OAS dependent activation of RNAseL such as the orf4a dependent sequestering of viral dsRNA intermediates.

In addition to the localisation of orf4b, the presence of a second His residue within the PDE is important for the ability of orf4b to regulate the activity of RNaseL since substitution of the H182 (COVID-19) or H 186 (BtCoV SC2019) with R result in decreased ability of orf4b to inhibit RNaseL. 

Figure: N-terminal NLS in orf4b is required for nuclear localisation of COVID-19
and BtCoV off 4b

Figure: Cytoplasmic localisation and His at position of 182 or 186 of COVID-19 and BtCoV orf4b
are required for inhibition of RNaseL

The induction of antiviral signaling by via OAS/RNase L dependent pathways has also been demonstrated for West Nile Virus (WNV), Influenza A Virus (IAV) and Vaccinia Virus (VACV). OAS3 knockout cells infected with WNV, IAV, VACV or treated with Poly(I:C) exhibit only minimal 2’-5’ oligoadenylate synthase activity and infected A549, hTERT-HME and HT1080 OAS3 knockout cells display higher viral titres than the corresponding wt cells. Interestingly, probably because of increased expression of IFN-λ1, trophoblasts of the human placenta express higher levels of OAS-1,-2, and -3 compared to JEG-3 cells, suggesting that increased OAS levels contribute to the resistance of trophoblasts to ZIKV resistance as well.

Figure: orf4b degrades 2',5' OAS and prevents activation of RNaseL

More recently, the viral M protein of both SARS-CoV and COVID-19 but not HCoV-HKU1 has been demonstrated to form a complex with the TRAF3 adapter protein, thus inhibiting the formation of the TRAF3-TANK-TBK1/ IKK-ε complex via the N terminal domain of the viral M protein. Consequently, the expression of COVID-19 or SARS-CoV inhibits RLR signaling induced by Sendai Virus or Poly (I:C) and further analysis using RIG-1 and NF-κB reporter plasmids showed that the expression of COVID-19 M preferentially inhibits RIG-1 mediated activation of IRF-3, thus preventing the induction of IFN-β. Similar to SARS-CoV M, the interaction between TRAF3 and COVID-19 M is mediated by the N-terminal transmembrane domain 1 whereas the two other transmembrane domains as well as the C-terminal domain are dispensable. Both the SARS-CoV and COVID-19 derived M and ORF 4b protein therefore inhibit antiviral RIG-1 by targeting the formation of the TRAF3-TANK-TBK1/ IKK-ε complex, thus inhibiting IRF-3 dependent signaling. From an experimental standpoint it would be interesting if a recombinant HCoV-HKU1 expressing COVID-19 or SARS-CoV derived M exhibits increased viral titres following infection of primary human cells compared to wt virus. Also, it remains to be seen if the expression of COVID-19 in Artibeus jamaicensis or in bat derived cell lines inhibits the antiviral signaling in a similar way. So far, the infection of Artibeus jamaicensis with COVID-19 has been demonstrated to increase the expression of Mx-1, ISG56 and RANTES moderately suggesting that COVID-19 does inhibit antiviral signaling in bats, thus explaining the absence of clinical signs in infected bats despite the presence of low viral titres in the duodenum of bats. The individual role of ORF4a, ORF4b, ORF5 and M has not been assessed in bat or camel derived cell lines. Indeed, the importance of the genetic and functional diversity of proteins involved in the induction of antiviral signaling pathways has recently been highlighted in studies comparing RIG-1 from waterfowl and mammals in their capacity to facilitate an immune response to Influenza A virus. In short, duck derived RIG-1 had a weaker ability to induce IFN compared to goose derived RIG-1 and both duck and goose derived RIG-1 are more effective than pigeon RIG-1 in (chicken) DF-1 cells infected with Influenza A virus.

Figure: COVID-19 M, orf4a and orf4b: points of action

So far it has not been shown if the expression of mutant orf4b in the context of Coronavirus infection not only abrogates immune signaling and thus promotes viral replication solely by inhibiting apoptosis but also abrogates the induction of RNaseL induced autophagy and degradation of viral replication centers. If so, the number of viral RC should be increased in cells expressing mutant orf4b and/or mouse embryonic fibroblasts derived from RNaseL -/- mice.

Further reading

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Wong LY, Lui PY, & Jin DY (2019). A molecular arms race between host innate antiviral response and emerging human coronaviruses. Virologica Sinica, 31 (1), 12-23 PMID: 26786772 

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