title: Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses.
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.
authors: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors: Huib H
authors: HH
authors: Rabouw
authors: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors: Martijn A
authors: MA
authors: Langereis
authors: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
authors: Robert C M
authors: RC
authors: Knaap
authors: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
authors: Tim J
authors: http://orcid.org/0000-0002-5916-7548
authors: TJ
authors: Dalebout
authors: Department of Molecular and Cell Biology, National Center of Biotechnology (CNB-CSIC), Campus Universidad Autonoma de Madrid, Madrid, Spain.
authors: Javier
authors: J
authors: Canton
authors: Department of Molecular and Cell Biology, National Center of Biotechnology (CNB-CSIC), Campus Universidad Autonoma de Madrid, Madrid, Spain.
authors: Isabel
authors: I
authors: Sola
authors: Department of Molecular and Cell Biology, National Center of Biotechnology (CNB-CSIC), Campus Universidad Autonoma de Madrid, Madrid, Spain.
authors: Luis
authors: L
authors: Enjuanes
authors: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
authors: Peter J
authors: PJ
authors: Bredenbeek
authors: Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
authors: Marjolein
authors: M
authors: Kikkert
authors: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors: Raoul J
authors: RJ
authors: de Groot
authors: Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
authors: Frank J M
authors: FJ
authors: van Kuppeveld
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D018352:Coronavirus Infections / Q000276:immunology / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D055786:Gene Knockout Techniques; D006801:Humans; D057131:Immune Evasion / Q000276:immunology*; D007181:Inclusion Bodies, Viral / Q000276:immunology / Q000378:metabolism; D065207:Middle East Respiratory Syndrome Coronavirus / Q000276:immunology / Q000378:metabolism; D016133:Polymerase Chain Reaction; D054334:Viral Regulatory and Accessory Proteins / Q000276:immunology / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000276:immunology / Q000378:metabolism*
chemical_list: D054334:Viral Regulatory and Accessory Proteins; D019892:eIF-2 Kinase