Effect of Phosphorylation of CM2 Protein on Influenza C Virus Replication
Effect of Phosphorylation of CM2 Protein on Influenza C Virus Replication
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DOI：
10.1128/jvi.00773-17
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发表时间：
2017-11-01
影响因子：
5.4
通讯作者：
Hongo, Seiji
中科院分区：
文献类型：
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作者：
Goto, Takanari;Shimotai, Yoshitaka;Hongo, Seiji
CM 2是C型流感病毒的第二个膜蛋白，已被证明在C型流感病毒复制的脱壳和基因组包装过程中发挥作用。虽然已经分析了CM 2的N-连接的糖基化、二硫键连接的寡聚化和棕榈酰化对病毒复制的影响，但CM 2的磷酸化对病毒复制的影响仍有待确定。在该研究中，CM 2的残基78和/或103处的磷酸化位点被丙氨酸残基取代，并分析了磷酸化丧失对丙型流感病毒复制的影响。在由表达野生型CM 2的293 T细胞产生的C型流感病毒样颗粒（VLP）和由表达缺乏磷酸化位点的CM 2突变体的细胞产生的VLP之间，在报告基因的包装中没有观察到显著差异。与野生型VLP感染的细胞相比，含有CM 2突变体的VLP感染的HMV-II细胞中的报告基因表达被抑制。具有在残基78处含有丝氨酸至丙氨酸变化的CM 2突变体的重组丙型流感病毒的病毒产量显著低于野生型重组丙型流感病毒。此外，具有CM 2的重组病毒的病毒生长与野生型病毒几乎相同，CM 2在第78位具有丝氨酸至天冬氨酸的变化，以模拟组成性磷酸化。这些结果表明，磷酸化的CM 2在有效的病毒复制中发挥作用，可能通过添加一个负电荷的Ser 78磷酸化site.IMPORTANCE它是众所周知的，许多主机和病毒蛋白质的posteraditionally修改磷酸化，这起着重要的作用，这些蛋白质的功能。在甲型和B型流感病毒中，未整合到膜中的病毒蛋白NP、M1、NS 1和核输出蛋白（NEP）的磷酸化影响这些蛋白的功能，从而影响病毒复制。然而，据报道，整合到膜中的甲型流感病毒M2离子通道蛋白的磷酸化在体外或体内对病毒复制没有影响。我们以前证明，C型流感病毒CM 2离子通道蛋白的N-糖基化，寡聚化，棕榈酰化和磷酸化修饰，并分析了这些修饰的影响，除了磷酸化，对病毒复制。这是第一份证明丙型流感病毒CM 2离子通道蛋白的磷酸化与甲型流感病毒M2蛋白的磷酸化不同，在病毒复制中发挥作用的报告。
CM2 is the second membrane protein of the influenza C virus and has been demonstrated to play a role in the uncoating and genome packaging processes in influenza C virus replication. Although the effects of N-linked glycosylation, disulfide-linked oligomerization, and palmitoylation of CM2 on virus replication have been analyzed, the effect of the phosphorylation of CM2 on virus replication remains to be determined. In this study, a phosphorylation site(s) at residue 78 and/or 103 of CM2 was replaced with an alanine residue(s), and the effects of the loss of phosphorylation on influenza C virus replication were analyzed. No significant differences were observed in the packaging of the reporter gene between influenza C virus-like particles (VLPs) produced from 293T cells expressing wild-type CM2 and those from the cells expressing the CM2 mutants lacking the phosphorylation site(s). Reporter gene expression in HMV-II cells infected with VLPs containing the CM2 mutants was inhibited in comparison with that in cells infected with wild-type VLPs. The virus production of the recombinant influenza C virus possessing CM2 mutants containing a serine-to-alanine change at residue 78 was significantly lower than that of wild-type recombinant influenza C virus. Furthermore, the virus growth of the recombinant viruses possessing CM2 with a serine-to-aspartic acid change at position 78, to mimic constitutive phosphorylation, was virtually identical to that of the wild type virus. These results suggest that phosphorylation of CM2 plays a role in efficient virus replication, probably through the addition of a negative charge to the Ser78 phosphorylation site.IMPORTANCE It is well-known that many host and viral proteins are posttranslationally modified by phosphorylation, which plays a role in the functions of these proteins. In influenza A and B viruses, phosphorylation of viral proteins NP, M1, NS1, and the nuclear export protein (NEP), which are not integrated into the membranes, affects the functions of these proteins, thereby affecting virus replication. However, it was reported that phosphorylation of the influenza A virus M2 ion channel protein, which is integrated into the membrane, has no effect on virus replication in vitro or in vivo. We previously demonstrated that the influenza C virus CM2 ion channel protein is modified by N-glycosylation, oligomerization, palmitoylation, and phosphorylation and have analyzed the effects of these modifications, except phosphorylation, on virus replication. This is the first report demonstrating that phosphorylation of the influenza C virus CM2 ion channel protein, unlike that of the influenza A virus M2 protein, plays a role in virus replication.