Rabouw Huib H, Langereis Martijn A, Knaap Robert C M, Dalebout Tim J, Canton Javier, Sola Isabel, Enjuanes Luis, Bredenbeek Peter J, Kikkert Marjolein, de Groot Raoul J, van Kuppeveld Frank J M
Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands.
PLoS Pathog. 2016 Oct 26;12(10):e1005982. doi: 10.1371/journal.ppat.1005982. eCollection 2016 Oct.
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.
中东呼吸综合征冠状病毒（MERS-CoV）可引发严重的呼吸道感染，甚至可能危及生命。为了实现感染与传播，MERS-CoV与大多数其他病毒一样，必须在错综复杂的抗病毒宿主反应网络中“穿行”。除了众所周知的I型干扰素（IFN-α/β）反应外，蛋白激酶R（PKR）介导的应激反应正被视为一条重要的固有反应途径。一旦检测到病毒双链RNA（dsRNA），PKR就会使真核翻译起始因子2α（eIF2α）磷酸化，从而抑制细胞和病毒的翻译，并导致应激颗粒（SGs）的形成，而应激颗粒越来越被认为是抗病毒信号通路的平台。目前尚不清楚MERS-CoV的细胞感染是否会激活应激反应途径，也不清楚该病毒是否已进化出抑制这种限制感染途径的策略。在此，我们表明MERS-CoV的细胞感染不会导致应激颗粒的形成。通过单独瞬时表达MERS-CoV的辅助蛋白，我们确定了蛋白4a（p4a）在防止应激反应途径激活中的作用。MERS-CoV p4a的表达阻碍了dsRNA介导的PKR激活，从而挽救了翻译抑制并防止了应激颗粒的形成。相比之下，p4a未能抑制独立于PKR和dsRNA的应激反应途径激活。MERS-CoV p4a是一种dsRNA结合蛋白。p4a中dsRNA结合基序的突变破坏了其PKR拮抗活性。通过将p4a插入缺乏天然PKR拮抗剂的小RNA病毒中，我们表明p4a在感染条件下也发挥PKR拮抗活性。然而，一种缺乏p4a表达的重组MERS-CoV仍然抑制了应激颗粒的形成，这表明至少还有一种其他应激反应拮抗剂的表达。这种病毒还抑制了不依赖dsRNA的应激反应途径。因此，MERS-CoV至少利用两种不同机制干扰抗病毒应激反应，其中p4a可能通过隔离dsRNA来抑制依赖PKR的应激反应途径。MERS-CoV p4a是所描述首个冠状病毒应激反应拮抗剂。
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