Structure and function of influenza A virus PDZ-binding motif

描述（由申请方提供）：甲型流感病毒编码一种称为NS 1的非结构蛋白，其作为毒力决定因子发挥作用，尤其是在高致病性H5 N1病毒中。NS 1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H5 N1病毒）的NS 1蛋白含有四个羧基末端残基，称为PDZ结合结构域（PDZ-BD）或PDZ结合基序（PBM），其通过未知机制作为毒力决定因子。 我们最近发现，禽H6 N6病毒的NS 1 PBM允许NS 1与Dlg 1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。NS 1和Scribble之间的相互作用是直接的蛋白质-蛋白质相互作用。我们的初步结果还表明，NS 1 PBM的功能，以保护感染的细胞凋亡，也可能破坏上皮细胞之间的紧密连接。我们最近还确定了全长NS 1蛋白的晶体结构，从H5 N1流感病毒沿着与冷冻电镜成像的NS 1（H1N1）结合到dsRNA。这些研究提供了有价值的见解NS 1如何可以螯合可变长度的dsRNA，并与介导NS 1功能的细胞蛋白质进行特异性相互作用。 在我们的研究计划中，我们建议测试的假设，即NS 1与禽病毒PBM和Scribble和Dlg 1之间的相互作用有助于病毒复制和发病机制，通过保护感染的细胞凋亡，也扰乱紧密连接的完整性。我们将使用功能和结构的方法来测试这些和其他具体的假设NS 1 PBM的功能。我们已经使用反向遗传学将禽类H6 N6病毒的NS 1蛋白引入Udorn H3 N2毒株的背景中。重要的是，这种病毒的实验可以在BSL 2遏制条件下进行，极大地促进了对禽病毒NS 1蛋白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NS 1 PBM功能所涉及的机制，为未来针对H5 N1病毒的NS 1的重点研究铺平道路，这将需要更高的遏制水平。 公共卫生相关性：A型流感病毒的NS 1蛋白参与病毒的发病机制，特别是在目前的高致病性H5 N1病毒中。来自大多数禽流感病毒分离株的NS 1蛋白的羧基末端含有被称为PDZ结合结构域（PDZ-BD）或PDZ结合基序（PBM）的结构域，其通过未知机制促成毒力。我们将研究来自H6 N6禽流感病毒的NS 1蛋白及其PBM的功能和结构。

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.