MERS-CoV orf4a -an antagonist of antiviral signaling

As mentioned in a previous entry MERS-CoV is capable to inhibit antiviral signaling by antagonizing interferon signaling pathways which are induced by the recognition of dsRNA by intracellular pathogen-associated molecular pattern (PAMP) receptors. Although having a ssRNA genome, dsRNA is produced during Coronavirus replication as an intermediate and thus can be recognized by Toll-like receptors -3 and -9 (TLR-3/TLR-9). Imaging of newly synthesized RNA in cells infected with MHV revealed the formation of spherules tethered to the cellular membrane shielding these intermediates from the recognition by TLR-3 and/or TLR-9. If however these spherules also exist in MERS-CoV remains to be investigated.

In addition to TLR mediated signaling, following the infection of cells with a Coronavirus, cytoplasmic viral RNAs are recognised by two RIG-1 like proteins, RIG-1 and MDA-5. Both RIG-1 and MDA-5 require PACT, which binds to RIG-1 and MDA-5, followed by intracellular signaling involving the mitochondrial adaptor protein MAVS and two protein kinases (TBK1 and IKKε).  This in turn allows the nuclear translocation of two transcription factors, IRF3 and IRF7 and subsequent induction of recruited to the IFN promoters to activate transcription of Interferon-β.

In the case of MERS-CoV, the 4a protein was shown previously to be capable of not only binding dsRNA but also to inhibit MDA5 mediated signaling - and thus to block antiviral signaling. A result from another group however favors a different mechanism. They observed that MERS-CoV 4a fails to suppress MDA5 mediated activation of the IFN-β promoter and does not form a complex with RIG-1or MDA5 but forms a complex with PACT in a RNA-dependent manner. Both groups however agree that MERS-CoV 4a inhibits the expression of IFN-β. If and how 4b contributes to the antiviral signaling is still debated. Importantly, BtCoV-HKU-4 4a and a point mutation of MERS-CoV 4a incapable of binding dsRNA prevented PACT mediated signaling thus emphasizing the role of PACT - in contrast to BtCoV HKU-5 4a. 

As always , these results need to be confirmed in other cell lines, besides the Human Airway Epithelia (Calu3) cell line used, such as monocyte derived cell lines (THP-1); the only camel derived cell line I am aware of are Dubca cells (skin fibroblast).

If confirmed, MERS-CoV 4a will be not the only viral protein inhibiting PACT signaling. Other examples of viral proteins inhibiting the activation of RIG-1 by binding to PACT include Influenza NS1, Herpes Virus Us11 and Ebola VP35 and the list might be growing once new viruses are discovered and old ones are revisited.

Finally it remains to be seen if the inhibition of PACT by MERS-CoV also has an impact on the activation of Dicer, which can cleave viral RNA. In cells infected with Influenza A, the inactivation of Dicer has been associated with a modest increase in virus production. Ebola virus VP30 and VP35 proteins have also been postulated to prevent the formation of siRNA by inhibition of the activation of Dicer or inhibiting the contact of Dicer with PACT.

From the present data it is not clear if the viral ssRNA or dsRNA intermediates are responsible for the activation of the antiviral response in absence of orf 4a. Indeed the present data only indicate that 4a is capable of inhibiting experimentally, Poly (I:C), induced dsRNA antiviral response, but the use of the reverse genetic system available should allow to characterize the role of MERS-CoV orf4a in the context of viral replication.

Last but not least a few words about MERS-CoV 4b protein. In a recent paper it was shown that the protein encoded by MERS-CoV orf4b as well as BtCoV-HKU-4 orf4b and BtCoV HKU-5 orf 4b inhibit the IFN type 1 response by localizing to the nucleus. In this case, the protein inhibits RIG-1 mediated IFN-β signaling while only having a moderate effect on NF-κB signalling. 

Further reading

Siu KL, Yeung ML, Kok KH, Yuen KS, Kew C, Lui PY, Chan CP, Tse H, Woo PC, Yuen KY, & Jin DY (2014). Middle East respiratory syndrome coronavirus 4a protein is a double-stranded RNA-binding protein that suppresses PACT-induced activation of RIG-I and MDA5 in innate antiviral response. Journal of virology PMID: 24522921

Hagemeijer MC, Vonk AM, Monastyrska I, Rottier PJ, & de Haan CA (2012). Visualizing coronavirus RNA synthesis in time by using click chemistry. Journal of virology, 86 (10), 5808-16 PMID: 22438542

Kok KH, Lui PY, Ng MH, Siu KL, Au SW, & Jin DY (2011). The double-stranded RNA-binding protein PACT functions as a cellular activator of RIG-I to facilitate innate antiviral response. Cell host & microbe, 9 (4), 299-309 PMID: 21501829

Niemeyer D, Zillinger T, Muth D, Zielecki F, Horvath G, Suliman T, Barchet W, Weber F, Drosten C, & Müller MA (2013). Middle East respiratory syndrome coronavirus accessory protein 4a is a type I interferon antagonist. Journal of virology, 87 (22), 12489-95 PMID: 24027320

Yang Y, Zhang L, Geng H, Deng Y, Huang B, Guo Y, Zhao Z, & Tan W (2013). The structural and accessory proteins M, ORF 4a, ORF 4b, and ORF 5 of Middle East respiratory syndrome coronavirus (MERS-CoV) are potent interferon antagonists. Protein & cell, 4 (12), 951-61 PMID: 24318862

Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, Taira K, Akira S, & Fujita T (2004). The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nature immunology, 5 (7), 730-7 PMID: 15208624

Kok KH, Ng MH, Ching YP, & Jin DY (2007). Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA. The Journal of biological chemistry, 282 (24), 17649-57 PMID: 17452327

Matskevich AA, & Moelling K (2007). Dicer is involved in protection against influenza A virus infection. The Journal of general virology, 88 (Pt 10), 2627-35 PMID: 17872512

Fabozzi G, Nabel CS, Dolan MA, & Sullivan NJ (2011). Ebolavirus proteins suppress the effects of small interfering RNA by direct interaction with the mammalian RNA interference pathway. Journal of virology, 85 (6), 2512-23 PMID: 21228243

Matthews KL, Coleman CM, van der Meer Y, Snijder EJ, & Frieman MB (2014). The ORF4b-encoded accessory proteins of Middle East respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling. The Journal of general virology, 95 (Pt 4), 874-82 PMID: 24443473