Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas, USA.
Department of Pathology, The University of Texas Medical Branch, Galveston, Texas, USA.
J Virol. 2018 Oct 12;92(21). doi: 10.1128/JVI.01157-18. Print 2018 Nov 1.
Middle East respiratory syndrome coronavirus (MERS-CoV) nsp1 suppresses host gene expression in expressed cells by inhibiting translation and inducing endonucleolytic cleavage of host mRNAs, the latter of which leads to mRNA decay. We examined the biological functions of nsp1 in infected cells and its role in virus replication by using wild-type MERS-CoV and two mutant viruses with specific mutations in the nsp1; one mutant lacked both biological functions, while the other lacked the RNA cleavage function but retained the translation inhibition function. In Vero cells, all three viruses replicated efficiently with similar replication kinetics, while wild-type virus induced stronger host translational suppression and host mRNA degradation than the mutants, demonstrating that nsp1 suppressed host gene expression in infected cells. The mutant viruses replicated less efficiently than wild-type virus in Huh-7 cells, HeLa-derived cells, and 293-derived cells, the latter two of which stably expressed a viral receptor protein. In 293-derived cells, the three viruses accumulated similar levels of nsp1 and major viral structural proteins and did not induce -β and -λ mRNAs; however, both mutants were unable to generate intracellular virus particles as efficiently as wild-type virus, leading to inefficient production of infectious viruses. These data strongly suggest that the endonucleolytic RNA cleavage function of the nsp1 promoted MERS-CoV assembly and/or budding in a 293-derived cell line. MERS-CoV nsp1 represents the first CoV gene 1 protein that plays an important role in virus assembly/budding and is the first identified viral protein whose RNA cleavage-inducing function promotes virus assembly/budding. MERS-CoV represents a high public health threat. Because CoV nsp1 is a major viral virulence factor, uncovering the biological functions of MERS-CoV nsp1 could contribute to our understanding of MERS-CoV pathogenicity and spur development of medical countermeasures. Expressed MERS-CoV nsp1 suppresses host gene expression, but its biological functions for virus replication and effects on host gene expression in infected cells are largely unexplored. We found that nsp1 suppressed host gene expression in infected cells. Our data further demonstrated that nsp1, which was not detected in virus particles, promoted virus assembly or budding in a 293-derived cell line, leading to efficient virus replication. These data suggest that nsp1 plays an important role in MERS-CoV replication and possibly affects virus-induced diseases by promoting virus particle production in infected hosts. Our data, which uncovered an unexpected novel biological function of nsp1 in virus replication, contribute to further understanding of the MERS-CoV replication strategies.
中东呼吸综合征冠状病毒（MERS-CoV）nsp1 通过抑制翻译和诱导宿主 mRNA 的内切核酸酶切割来抑制宿主基因表达，后者导致 mRNA 降解。我们通过使用野生型 MERS-CoV 和两种具有 nsp1 特定突变的突变病毒来研究 nsp1 在感染细胞中的生物学功能及其在病毒复制中的作用；一种突变体缺乏这两种生物学功能，而另一种突变体缺乏 RNA 切割功能但保留了翻译抑制功能。在 Vero 细胞中，三种病毒均以相似的复制动力学有效地复制，而野生型病毒诱导的宿主翻译抑制和宿主 mRNA 降解比突变体更强，表明 nsp1 抑制了感染细胞中的宿主基因表达。突变病毒在 Huh-7 细胞、HeLa 衍生细胞和 293 衍生细胞中的复制效率低于野生型病毒，后两者稳定表达病毒受体蛋白。在 293 衍生细胞中，三种病毒积累相似水平的 nsp1 和主要病毒结构蛋白，并且不诱导 -β 和 -λ mRNAs；然而，两种突变体都不能像野生型病毒那样有效地产生细胞内病毒颗粒，导致传染性病毒的产生效率降低。这些数据强烈表明，nsp1 的内切核酸酶 RNA 切割功能促进了 MERS-CoV 在 293 衍生细胞系中的组装和/或出芽。MERS-CoV nsp1 代表第一个在病毒组装/出芽中起重要作用的 CoV 基因 1 蛋白，也是第一个鉴定出的病毒蛋白，其 RNA 切割诱导功能促进了病毒组装/出芽。MERS-CoV 代表着高度的公共卫生威胁。由于 CoV nsp1 是主要的病毒毒力因子，揭示 MERS-CoV nsp1 的生物学功能可能有助于我们了解 MERS-CoV 的致病性，并推动医疗对策的发展。表达的 MERS-CoV nsp1 抑制宿主基因表达，但它在感染细胞中的病毒复制的生物学功能和对宿主基因表达的影响在很大程度上尚未得到探索。我们发现 nsp1 抑制了感染细胞中的宿主基因表达。我们的数据还进一步表明，在 293 衍生细胞系中，未在病毒颗粒中检测到的 nsp1 促进了病毒的组装或出芽，从而导致有效的病毒复制。这些数据表明，nsp1 在 MERS-CoV 复制中起重要作用，并可能通过促进感染宿主中病毒颗粒的产生来影响病毒诱导的疾病。我们的数据揭示了 nsp1 在病毒复制中的一个意想不到的新生物学功能，有助于进一步了解 MERS-CoV 的复制策略。
