Viral Immunology Group, Trinity Biomedical Sciences Institute, School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland.
Molecular Design Group, School of Chemical Sciences, Dublin City University, Glasnevin, Ireland.
Cell Mol Life Sci. 2024 Oct 12;81(1):433. doi: 10.1007/s00018-024-05458-y.
Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an enveloped, positive-sense RNA virus that emerged in 2012, causing sporadic cases and localized outbreaks of severe respiratory illness with high fatality rates. A characteristic feature of the immune response to MERS-CoV infection is low type I IFN induction, despite its importance in viral clearance. The non-structural proteins (nsps) of other coronaviruses have been shown to block IFN production. However, the role of nsp5 from MERS-CoV in IFN induction of human respiratory cells is unclear. In this study, we elucidated the role of MERS-CoV-nsp5, the viral main protease, in modulating the host's antiviral responses in human bronchial epithelial BEAS 2b cells. We found that overexpression of MERS-CoV-nsp5 had a dose-dependent inhibitory effect on IFN-β promoter activation and cytokine production induced by HMW-poly(I:C). It also suppressed IFN-β promoter activation triggered by overexpression of key components in the RIG-I-like receptor (RLR) pathway, including RIG-I, MAVS, IKK-ε and IRF3. Moreover, the overexpression of MERS-CoV-nsp5 did not impair expression or phosphorylation of IRF3, but suppressed the nuclear translocation of IRF3. Further investigation revealed that MERS-CoV-nsp5 specifically interacted with IRF3. Using docking and molecular dynamic (MD) simulations, we also found that amino acids on MERS-CoV-nsp5, IRF3, and KPNA4 may participate in protein-protein interactions. Additionally, we uncovered protein conformations that mask the nuclear localization signal (NLS) regions of IRF3 and KPNA4 when interacting with MERS-CoV-nsp5, suggesting a mechanism by which this viral protein blocks IRF3 nuclear translocation. Of note, the IFN-β expression was restored after administration of protease inhibitors targeting nsp5, indicating this suppression of IFN-β production was dependent on the enzyme activity of nsp5. Collectively, our findings elucidate a mechanism by which MERS-CoV-nsp5 disrupts the host's innate antiviral immunity and thus provides insights into viral pathogenesis.
中东呼吸综合征冠状病毒（MERS-CoV）是一种包膜、正链 RNA 病毒，于 2012 年出现，导致散发性和局部爆发严重呼吸道疾病，死亡率高。对 MERS-CoV 感染的免疫反应的一个特征是 I 型干扰素（IFN）诱导水平低，尽管其在清除病毒中非常重要。其他冠状病毒的非结构蛋白（nsps）已被证明可阻断 IFN 的产生。然而，MERS-CoV 的 nsp5 在诱导人呼吸道细胞 IFN 方面的作用尚不清楚。在这项研究中，我们阐明了 MERS-CoV 的主要蛋白酶 nsp5 在调节人支气管上皮 BEAS-2b 细胞宿主抗病毒反应中的作用。我们发现，MERS-CoV-nsp5 的过表达对 HMW-poly（I：C）诱导的 IFN-β 启动子激活和细胞因子产生具有剂量依赖性抑制作用。它还抑制 RIG-I 样受体（RLR）途径中关键组成部分过表达触发的 IFN-β 启动子激活，包括 RIG-I、MAVS、IKK-ε 和 IRF3。此外，MERS-CoV-nsp5 的过表达不会损害 IRF3 的表达或磷酸化，但抑制了 IRF3 的核转位。进一步的研究表明，MERS-CoV-nsp5 特异性与 IRF3 相互作用。通过对接和分子动力学（MD）模拟，我们还发现 MERS-CoV-nsp5、IRF3 和 KPNA4 上的氨基酸可能参与蛋白质-蛋白质相互作用。此外，我们发现了与 MERS-CoV-nsp5 相互作用时掩盖 IRF3 和 KPNA4 的核定位信号（NLS）区域的蛋白质构象，表明该病毒蛋白阻止 IRF3 核转位的机制。值得注意的是，在用靶向 nsp5 的蛋白酶抑制剂处理后，IFN-β 的表达得到恢复，表明 IFN-β 产生的抑制依赖于 nsp5 的酶活性。总之，我们的研究结果阐明了 MERS-CoV-nsp5 破坏宿主先天抗病毒免疫的机制，从而为病毒发病机制提供了新的见解。
