Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, University of Texas at Austin, TX 78712, USA.
Proc Natl Acad Sci U S A. 2010 Feb 2;107(5):2253-8. doi: 10.1073/pnas.0909144107. Epub 2010 Jan 19.
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-beta. 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.
ISG15 是一种 IFN-α/β诱导的、泛素样蛋白，通过三种连续作用的连接酶将其连接到广泛的细胞蛋白上，这三种连接酶也被 IFN-α/β诱导。最近的研究表明，ISG15 和/或其缀合物在保护细胞免受多种病毒感染方面发挥着重要作用，包括流感 A 病毒。然而，ISG15 修饰发挥抗病毒活性的机制尚未建立。在这里，我们通过证明流感 A 病毒（NS1A 蛋白）的 NS1 蛋白是一种必需的多功能蛋白，在病毒感染的细胞中被 ISG15 修饰，将 ISG15 的靶标扩展到一种病毒蛋白。我们证明，感染细胞中 NS1A 蛋白的主要 ISG15 接受位点是 N 端 RNA 结合域（RBD）中的关键赖氨酸残基（K41）。ISG15 修饰 K41 破坏了 NS1A RBD 域与导入蛋白-α的结合，导入蛋白-α是介导 NS1A 蛋白核输入的蛋白，而 RBD 保留其双链 RNA 结合活性。最重要的是，我们表明 ISG15 修饰 K41 抑制了流感 A 病毒的复制，从而有助于 IFN-β 的抗病毒作用。我们还表明，NS1A 蛋白直接且特异性地与 Herc5 结合，Herc5 是人类细胞中 ISG15 缀合的主要 E3 连接酶。这些结果建立了 IFN 诱导的 ISG15 缀合系统抗病毒活性的“失活功能”机制，即通过将 ISG15 缀合到特定的病毒蛋白上抑制其功能，从而抑制病毒复制。
