Middle East Respiratory Syndrome Coronavirus NS4b Protein Inhibits Host RNase L Activation

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is the first highly pathogenic human coronavirus to emerge since severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002. Like many coronaviruses, MERS-CoV carries genes that encode multiple accessory proteins that are not required for replication of the genome but are likely involved in pathogenesis. Evasion of host innate immunity through interferon (IFN) antagonism is a critical component of viral pathogenesis. The IFN-inducible oligoadenylate synthetase (OAS)-RNase L pathway activates upon sensing of viral double-stranded RNA (dsRNA). Activated RNase L cleaves viral and host single-stranded RNA (ssRNA), which leads to translational arrest and subsequent cell death, preventing viral replication and spread. Here we report that MERS-CoV, a lineage C Betacoronavirus, and related bat CoV NS4b accessory proteins have phosphodiesterase (PDE) activity and antagonize OAS-RNase L by enzymatically degrading 2′,5′-oligoadenylate (2-5A), activators of RNase L. This is a novel function for NS4b, which has previously been reported to antagonize IFN signaling. NS4b proteins are distinct from lineage A Betacoronavirus PDEs and rotavirus gene-encoded PDEs, in having an amino-terminal nuclear localization signal (NLS) and are localized mostly to the nucleus. However, the expression level of cytoplasmic MERS-CoV NS4b protein is sufficient to prevent activation of RNase L. Finally, this is the first report of an RNase L antagonist expressed by a human or bat coronavirus and provides a specific mechanism by which this occurs. Our findings provide a potential mechanism for evasion of innate immunity by MERS-CoV while also identifying a potential target for therapeutic intervention.

RNA regulation of the antiviral protein 2′‐5′‐oligoadenylate synthetase

“…The OAS family of enzymes comprises a group of cytosolic dsRNA innate immune sensors whose importance in this process is underscored by the numerous ways diverse viruses have developed to evade detection. These include directly inhibiting OAS, sequestering dsRNA produced as a result of viral infection, producing 2‐5A analogs, degrading 2‐5A via viral‐encoded phosphodiesterases, expression of protein or RNA inhibitors of 2‐5A interaction with RNase L or its ribonuclease activity, or through selective pressure to remove RNase L target sites within viral RNAs (Cayley, Davies, McCullagh, & Kerr, ; Drappier et al, ; J. Q. Han & Barton, ; Keel, Jha, & Kieft, ; Silverman, ; Silverman & Weiss, ; Taguchi et al, ; Thornbrough et al, ; Townsend et al, ; Townsend, Jha, Silverman, & Barton, ; Zhang et al, ; Zhao et al, ). While there is strong evidence for critical roles for each OAS family member in at least some context, defining the protective effects of each family member is an active area of investigation.…”

Interaction of SARS and MERS Coronaviruses with the Antiviral Interferon Response

“…With respect to the antiviral OAS/RNase L system that is also activated by dsRNA, the mouse coronavirus MHV-A59 was shown to expresses an ns2 protein that antagonizes by degrading the product of the OAS enzyme, 2 0 -5 0 oligoadenylate that would activate RNase L (Zhao et al, 2012). SARS-CoV and MERS-CoV do not possess an ns2 homolog (Silverman and Weiss, 2014), but the MERS-CoV ns4b was recently demonstrated to cleave 2 0 -5 0 oligoadenylate (Thornbrough et al, 2016). Although ns4b-mutated MERS-CoV was not attenuated in cell culture, it provoked increased RNAse L activity in infected cells (Thornbrough et al, 2016).…”

“…SARS-CoV and MERS-CoV do not possess an ns2 homolog (Silverman and Weiss, 2014), but the MERS-CoV ns4b was recently demonstrated to cleave 2 0 -5 0 oligoadenylate (Thornbrough et al, 2016). Although ns4b-mutated MERS-CoV was not attenuated in cell culture, it provoked increased RNAse L activity in infected cells (Thornbrough et al, 2016). A critical factor for IFN resistance of SARS-CoV (and of the low pathogenic HCoV-229E) is the ADP-ribose-1 00 -monophosphatase (ADRP) domain that is contained within the nsp3 protein (Kuri et al, 2011).…”

Epigenetic Landscape during Coronavirus Infection

“…Several downstream open reading frames like ORF3a, ORF3b and ORF6 also antagonize sensing or signaling pathways, or block karyopherin 2 nuclear import [13,21] (Figure 1). MERS-CoV also encodes several luxury functions with interferon antagonism activities, including ORF4a, ORF4b and perhaps ORF5, noting that ORF4b antagonizes phosphodiesterase activity and RNAse L activation [26,27,28,29,30]. However, how the exact underlying mechanisms allow these antagonistic molecules to interfere with the effector molecules that establish an antiviral state, assist in wound repair, or prime and enhance an adaptive immune response, which is critical for clearance, is still under study.…”