ISG15 conjugation system targets the viral NS1 protein in influenza A virus-infected cells
ISG15 conjugation system targets the viral NS1 protein in influenza A virus-infected cells
复制标题
DOI：
10.1073/pnas.0909144107
复制
发表时间：
2010-02-02
影响因子：
11.1
通讯作者：
Krug, Robert M.
中科院分区：
文献类型：
--
作者：
Zhao, Chen;Hsiang, Tien-Ying;Krug, Robert M.
ISG15是一种ifn - α / β诱导的泛素样蛋白，通过三种也由ifn - α / β诱导的偶联酶的连续作用结合到多种细胞蛋白上。最近的研究表明，ISG15和/或其偶联物在保护细胞免受包括甲型流感病毒在内的多种病毒感染方面发挥着重要作用。然而，ISG15修饰发挥抗病毒活性的机制尚未确定。在这里，我们通过证明甲型流感病毒的NS1蛋白（NS1A蛋白）是一种重要的多功能蛋白，在病毒感染的细胞中被ISG15修饰，将ISG15靶点扩展到病毒蛋白。我们证明了感染细胞中NS1A蛋白的主要ISG15受体位点是n端rna结合域（RBD）中的关键赖氨酸残基（K41）。K41的ISG15修饰破坏了NS1A RBD结构域与importin- α（介导NS1A蛋白核输入的蛋白）的关联，而RBD保留了其双链rna结合活性。最重要的是，我们发现K41的ISG15修饰抑制了甲型流感病毒的复制，从而有助于ifn - α的抗病毒作用。我们还发现NS1A蛋白直接特异性结合Herc5， Herc5是人类细胞中ISG15结合的主要E3连接酶。这些结果建立了ifn诱导的ISG15偶联系统抗病毒活性的“功能丧失”机制，即通过将ISG15偶联到特定的病毒蛋白上抑制病毒复制，从而抑制其功能。
ISG15 is an IFN-alpha/beta-induced, ubiquitin-like protein that is conjugated to a wide array of cellular proteins through the sequential action of three conjugation enzymes that are also induced by IFN-alpha/beta. Recent studies showed that ISG15 and/or its conjugates play an important role in protecting cells from infection by several viruses, including influenza A virus. However, the mechanism by which ISG15 modification exerts antiviral activity has not been established. Here we extend the repertoire of ISG15 targets to a viral protein by demonstrating that the NS1 protein of influenza A virus (NS1A protein), an essential, multifunctional protein, is ISG15 modified in virus-infected cells. We demonstrate that the major ISG15 acceptor site in the NS1A protein in infected cells is a critical lysine residue (K41) in the N-terminal RNA-binding domain (RBD). ISG15 modification of K41 disrupts the association of the NS1A RBD domain with importin-alpha, the protein that mediates nuclear import of the NS1A protein, whereas the RBD retains its double-stranded RNA-binding activity. Most significantly, we show that ISG15 modification of K41 inhibits influenza A virus replication and thus contributes to the antiviral action of IFN-alpha. We also show that the NS1A protein directly and specifically binds to Herc5, the major E3 ligase for ISG15 conjugation in human cells. These results establish a "loss of function" mechanism for the antiviral activity of the IFN-induced ISG15 conjugation system, namely, that it inhibits viral replication by conjugating ISG15 to a specific viral protein, thereby inhibiting its function.