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      Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

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          Abstract

          Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infections that can be life-threatening. To establish an infection and spread, MERS-CoV, like most other viruses, must navigate through an intricate network of antiviral host responses. Besides the well-known type I interferon (IFN-α/β) response, the protein kinase R (PKR)-mediated stress response is being recognized as an important innate response pathway. Upon detecting viral dsRNA, PKR phosphorylates eIF2α, leading to the inhibition of cellular and viral translation and the formation of stress granules (SGs), which are increasingly recognized as platforms for antiviral signaling pathways. It is unknown whether cellular infection by MERS-CoV activates the stress response pathway or whether the virus has evolved strategies to suppress this infection-limiting pathway. Here, we show that cellular infection with MERS-CoV does not lead to the formation of SGs. By transiently expressing the MERS-CoV accessory proteins individually, we identified a role of protein 4a (p4a) in preventing activation of the stress response pathway. Expression of MERS-CoV p4a impeded dsRNA-mediated PKR activation, thereby rescuing translation inhibition and preventing SG formation. In contrast, p4a failed to suppress stress response pathway activation that is independent of PKR and dsRNA. MERS-CoV p4a is a dsRNA binding protein. Mutation of the dsRNA binding motif in p4a disrupted its PKR antagonistic activity. By inserting p4a in a picornavirus lacking its natural PKR antagonist, we showed that p4a exerts PKR antagonistic activity also under infection conditions. However, a recombinant MERS-CoV deficient in p4a expression still suppressed SG formation, indicating the expression of at least one other stress response antagonist. This virus also suppressed the dsRNA-independent stress response pathway. Thus, MERS-CoV interferes with antiviral stress responses using at least two different mechanisms, with p4a suppressing the PKR-dependent stress response pathway, probably by sequestering dsRNA. MERS-CoV p4a represents the first coronavirus stress response antagonist described.

          Author Summary

          Human coronaviruses generally cause relatively mild respiratory disease. In the past 15 years, the world has witnessed the emergence of two coronaviruses with high mortality rates in humans; severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, both originating from animal reservoirs. Successful infection of a host not only depends on the presence of an appropriate receptor but also on the ability of a virus to evade innate antiviral host responses, which constitute the first line of defense against invading viruses. MERS-CoV has been reported to actively suppress the IFN-α/β response, but it is unknown whether it also interferes with another important innate antiviral response, the stress response pathway. Activation of this pathway by a kinase, PKR, curtails virus infection by shutting off cellular and viral protein synthesis. To date, no coronavirus protein has been recognized to suppress the stress response pathway. Here, we show that the accessory protein 4a of MERS-CoV is a potent stress antagonist that prevents PKR activation by sequestering its ligand, dsRNA. This finding furthers our understanding of the molecular mechanism used by MERS-CoV to evade infection-limiting antiviral host responses and may provide new avenues for therapeutic intervention.

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          Most cited references33

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          Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia.

          A previously unknown coronavirus was isolated from the sputum of a 60-year-old man who presented with acute pneumonia and subsequent renal failure with a fatal outcome in Saudi Arabia. The virus (called HCoV-EMC) replicated readily in cell culture, producing cytopathic effects of rounding, detachment, and syncytium formation. The virus represents a novel betacoronavirus species. The closest known relatives are bat coronaviruses HKU4 and HKU5. Here, the clinical data, virus isolation, and molecular identification are presented. The clinical picture was remarkably similar to that of the severe acute respiratory syndrome (SARS) outbreak in 2003 and reminds us that animal coronaviruses can cause severe disease in humans.
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            MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment

            Coronavirus (CoV) infections are commonly associated with respiratory and enteric disease in humans and animals. The 2003 outbreak of severe acute respiratory syndrome (SARS) highlighted the potentially lethal consequences of CoV-induced disease in humans. In 2012, a novel CoV (Middle East Respiratory Syndrome coronavirus; MERS-CoV) emerged, causing 49 human cases thus far, of which 23 had a fatal outcome. In this study, we characterized MERS-CoV replication and cytotoxicity in human and monkey cell lines. Electron microscopy of infected Vero cells revealed extensive membrane rearrangements, including the formation of double-membrane vesicles and convoluted membranes, which have been implicated previously in the RNA synthesis of SARS-CoV and other CoVs. Following infection, we observed rapidly increasing viral RNA synthesis and release of high titres of infectious progeny, followed by a pronounced cytopathology. These characteristics were used to develop an assay for antiviral compound screening in 96-well format, which was used to identify cyclosporin A as an inhibitor of MERS-CoV replication in cell culture. Furthermore, MERS-CoV was found to be 50–100 times more sensitive to alpha interferon (IFN-α) treatment than SARS-CoV, an observation that may have important implications for the treatment of MERS-CoV-infected patients. MERS-CoV infection did not prevent the IFN-induced nuclear translocation of phosphorylated STAT1, in contrast to infection with SARS-CoV where this block inhibits the expression of antiviral genes. These findings highlight relevant differences between these distantly related zoonotic CoVs in terms of their interaction with and evasion of the cellular innate immune response.
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              Molecular diversity of coronaviruses in bats

              The existence of coronaviruses in bats is unknown until the recent discovery of bat-SARS-CoV in Chinese horseshoe bats and a novel group 1 coronavirus in other bat species. Among 309 bats of 13 species captured from 20 different locations in rural areas of Hong Kong over a 16-month period, coronaviruses were amplified from anal swabs of 37 (12%) bats by RT-PCR. Phylogenetic analysis of RNA-dependent-RNA-polymerase (pol) and helicase genes revealed six novel coronaviruses from six different bat species, in addition to the two previously described coronaviruses. Among the six novel coronaviruses, four were group 1 coronaviruses (bat-CoV HKU2 from Chinese horseshoe bat, bat-CoV HKU6 from rickett's big-footed bat, bat-CoV HKU7 from greater bent-winged bat and bat-CoV HKU8 from lesser bent-winged bat) and two were group 2 coronaviruses (bat-CoV HKU4 from lesser bamboo bats and bat-CoV HKU5 from Japanese pipistrelles). An astonishing diversity of coronaviruses was observed in bats.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                26 October 2016
                October 2016
                : 12
                : 10
                Affiliations
                [1 ]Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
                [2 ]Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands
                [3 ]Department of Molecular and Cell Biology, National Center of Biotechnology (CNB-CSIC), Campus Universidad Autonoma de Madrid, Madrid, Spain
                University of Maryland School of Medicine, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                • Conceptualization: HHR MAL RJdG FJMvK.

                • Formal analysis: HHR.

                • Funding acquisition: MAL FJMvK.

                • Investigation: HHR MAL TJD RCMK PJB JC.

                • Methodology: HHR MAL RJdG FJMvK.

                • Project administration: FJMvK.

                • Resources: IS LE PJB MK.

                • Supervision: LE IS MK RJdG FJMvK.

                • Validation: HHR MAL RJdG FJMvK.

                • Visualization: HHR MAL FJMvK.

                • Writing – original draft: HHR MAL RJdG FJMvK.

                • Writing – review & editing: HHR MAL RJdG FJMvK.

                Article
                PPATHOGENS-D-16-01222
                10.1371/journal.ppat.1005982
                5081173
                27783669
                fa6672ec-0806-4807-8240-3c52b8bff023
                © 2016 Rabouw et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 9, Tables: 0, Pages: 26
                Product
                Funding
                Funded by: Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NL)
                Award ID: NWO-918.12.628
                Award Recipient :
                Funded by: Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NL)
                Award ID: NWO-863.13.008
                Award Recipient :
                The work is supported by a Vici grant (NWO-918.12.628) from the Netherlands Organization for Scientific Research. MAL is supported by a Veni grant (NWO-863.13.008) from the Netherlands Organization for Scientific Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
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                Biology and life sciences
                Organisms
                Viruses
                RNA viruses
                Coronaviruses
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                Coronaviruses
                Biology and Life Sciences
                Organisms
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                Cell Biology
                Cell Processes
                Cellular Stress Responses
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                All relevant data are within the paper and its Supporting Information files.

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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