Structural basis for HIV-1 Gag interactions with cellular constituents

描述（由申请方提供）：在细胞内，蛋白质通过与细胞成分（如蛋白质和磷脂）的特异性相互作用靶向明确的位置。在HIV-1感染的晚期，Gag多聚蛋白被转运到质膜（PM）进行组装。使用核磁共振（NMR），我们先前已经定义了靶向PM的Gag的一个组分的分子基础，即基质（MA）结构域与磷脂酰肌醇-4，5-二磷酸（PI（4，5）P2）的相互作用。我们的初步数据表明，Gag还与涉及有效颗粒组装的细胞脂质和蛋白质形成生物学上重要的相互作用。我们发现MA直接与磷脂酰丝氨酸（PtdSer），磷脂酰胆碱（PtdCho）和磷脂酰乙醇胺（PtdEtn）相互作用，表明Gag靶向和PM上的组装通过MA结构域与PI（4，5）P2和主要膜脂质的双重参与进行。已经提出了许多细胞蛋白以促进Gag细胞内运输和靶向PM。HIV-1 Gag与钙调蛋白（CaM）在细胞质中相互作用并共定位。此外，最近的研究已经将细胞因子信号转导抑制因子1（SOCS 1）鉴定为HIV感染期间的诱导性宿主因子，其通过与Gag蛋白的直接相互作用来调节病毒复制途径的晚期阶段。然而，Gag与这些因子相互作用的分子机制尚未建立。初步研究表明，HIV-1 MA以钙依赖性方式与CaM直接相互作用，表明HIV-1劫持CaM细胞信号传导途径以促进Gag运输。此外，我们已经获得了SOCS 1和MA蛋白之间直接相互作用的证据。在这项提案中，我们将采用生物化学，生物物理学和结构生物学工具，以确定关键的蛋白质-蛋白质和蛋白质-脂质相互作用参与的分子机制，管理HIV-1 Gag细胞内运输和组装。我们的三个主要目标是：（i）确定Gag与PM结合的精确分子机制。我们将在结构水平上确定各种脂质成分如何与MA蛋白相互作用，并将确定疏水插入，特异性和非特异性静电相互作用的功能重要性，（ii）确定CaM在Gag运输和组装中的功能作用，以及（iii）阐明SOCS 1-Gag相互作用的结构要求。这项研究产生的结果将更好地了解HIV-1 Gag如何在原子水平上与细胞成分相互作用，这可能导致合理设计抑制Gag运输和组装的新型抗病毒治疗剂的新方法。

DESCRIPTION (provided by applicant): Within cells proteins are targeted to well-defined locations by specific interactions with cellular constituents such as proteins and phospholipids. During the late phase of HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM) for assembly. Using nuclear magnetic resonance (NMR) we have previously defined the molecular basis of one component of Gag targeting to the PM, the interaction of the matrix (MA) domain with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Our preliminary data indicate that Gag also forms biologically important interactions with cellular lipids and proteins implicated in efficient particle assembly. We show that MA interacts directly with phosphatidylserine (PtdSer), phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn), suggesting that Gag targeting and assembly on the PM proceed via a dual engagement of the MA domain with PI(4,5)P2 and major membrane lipids. A number of cellular proteins have been proposed to facilitate Gag intracellular trafficking and targeting to the PM. HIV-1 Gag was shown to interact and co-localize with calmodulin (CaM) in the cytoplasm. In addition, more recent studies have identified the suppressor of cytokine signaling 1 (SOCS1) as an inducible host factor during HIV infection, which regulates the late stages of the virus replication pathway through direct interactions with the Gag protein. However, the molecular mechanism by which Gag interacts with these factors has yet to be established. Preliminary studies demonstrate that HIV-1 MA interacts directly with CaM in a calcium- dependent manner, suggesting that HIV-1 hijacks CaM cell-signaling pathway to facilitate Gag trafficking. In addition, we have obtained evidence for direct interactions between SOCS1 and the MA protein. In this proposal, we will employ biochemical, biophysical and structural biology tools to identify key protein-protein and protein-lipid interactions involved in the molecular mechanism governing HIV-1 Gag intracellular trafficking and assembly. Our three main aims are: (i) to determine the precise molecular mechanism for Gag binding to the PM. We will determine at the structural level how various lipid constituents interact with the MA protein and will identify the functional importance of the hydrophobic insertion, specific and non- specific electrostatic interactions, (ii) to identify the functional role of CaM in Gag trafficking and assembly, and (iii) to elucidate the structural requirements for SOCS1-Gag interactions. The results generated by this research will offer a better understanding of how HIV-1 Gag interacts with cellular constituents at the atomic level, which could lead to new approaches to rational design of new antiviral therapeutic agents that inhibit Gag trafficking and assembly.