Structure and function of influenza A virus PDZ-binding motif

描述（由申请人提供）：甲型流感病毒编码一种称为NS1的非结构蛋白，其功能是毒力决定因素，特别是在高致病性H5N1病毒中。NS1是一种多功能蛋白，可作为多种干扰素介导的抗病毒机制的拮抗剂。最近的研究表明，来自禽流感病毒（包括H5N1病毒）的NS1蛋白含有四个羧基末端残基，称为pdz结合域（PDZ-BD）或pdz结合基序（PBM），它们通过未知的机制起着毒力决定因素的作用。我们最近发现禽流感H6N6病毒的NS1 PBM允许NS1与Dlg1和Scribble结合，这两种细胞蛋白含有PDZ结构域并调节细胞极性和信号传导。NS1与Scribble之间的相互作用是一种直接的蛋白质-蛋白质相互作用。我们的初步结果还表明，NS1 PBM具有保护感染细胞免于凋亡的功能，也可能破坏上皮细胞之间的紧密连接。我们最近还确定了来自H5N1流感病毒的全长NS1蛋白的晶体结构以及与dsRNA结合的NS1 （H1N1）的冷冻电镜成像。这些研究为NS1如何隔离可变长度的dsRNA并与介导NS1功能的细胞蛋白进行特异性相互作用提供了有价值的见解。在我们的研究计划中，我们提出验证NS1与禽类病毒PBM、Scribble和Dlg1的相互作用通过保护感染细胞免于凋亡和干扰紧密连接的完整性来参与病毒复制和发病的假设。我们将使用功能和结构两种方法来测试这些和其他关于NS1 PBM功能的特定假设。我们利用反向遗传学方法将禽H6N6病毒的NS1蛋白引入到乌冬H3N2毒株的背景中。重要的是，该病毒的实验可以在BSL2的遏制条件下进行，极大地促进了对禽类病毒NS1蛋白的PBM的深入分析。该模型系统将使我们能够在两年的资助期内阐明NS1 PBM功能的相关机制，为今后对H5N1病毒的NS1进行重点研究铺平道路，这将需要更高的遏制水平。

DESCRIPTION (provided by applicant): Influenza A viruses encode a non-structural protein termed NS1 that functions as a virulence determinant, especially in highly pathogenic H5N1 viruses. NS1 is a multifunctional protein that acts as an antagonist of multiple interferon-mediated antiviral mechanisms. Recent work has shown that NS1 proteins from avian influenza viruses, including H5N1 viruses, contain four carboxyl terminal residues termed the PDZ-binding domain (PDZ-BD) or PDZ-binding motif (PBM) that act as a virulence determinant through unknown mechanisms. We have recently found that the NS1 PBM of an avian H6N6 virus allows NS1 to associate with Dlg1 and Scribble, two cellular proteins that contain PDZ domains and regulate cellular polarity and signaling. The interaction between NS1 and Scribble is a direct protein-protein interaction. Our initial results also suggest that the NS1 PBM functions to protect infected cells from apoptosis and may also disrupt tight junctions between epithelial cells. We have also recently determined the crystal structure of the full-length NS1 protein from an H5N1 influenza virus along with cryo-EM imaging of NS1 (H1N1) bound to dsRNA. These studies have provided valuable insight into how NS1 can sequester variable lengths of dsRNA and make specific interactions with cellular proteins that mediate NS1 function. In our research plan, we propose to test the hypotheses that the interaction between NS1 with an avian virus PBM and Scribble and Dlg1 contributes to viral replication and pathogenesis through protecting infected cells from apoptosis and also perturbing tight junction integrity. We will use both functional and structural approaches to test these and other specific hypotheses about the function of the NS1 PBM. We have used reverse genetics to introduce the NS1 protein of an avian H6N6 virus into the background of the Udorn H3N2 strain. Importantly, experiments with this virus can be performed under BSL2 containment conditions, greatly facilitating an in-depth analysis of the PBM from an avian virus NS1 protein. This model system will allow us to elucidate mechanisms involved in the function of the NS1 PBM in a two year funding period, paving the way for future focused studies with the NS1 from H5N1 viruses which will require higher containment levels. PUBLIC HEALTH RELEVANCE: The NS1 protein of influenza A viruses contributes to viral pathogenesis, especially in current highly pathogenic H5N1 viruses. The carboxyl terminus of NS1 proteins from most avian influenza virus isolates contain a domain termed the PDZ-binding domain (PDZ-BD) or PDZ-binding motif (PBM) that contributes to virulence by an unknown mechanism. We will study the function and structure of a NS1 protein and its PBM from a H6N6 avian influenza virus.