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Structural basis for HIV-1 Gag interactions with cellular constituents

HIV-1 Gag 与细胞成分相互作用的结构基础

基本信息

批准号：
8638883
负责人：
Jamil Subhi Saad
金额：
$ 36.26万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-15 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8638883
关键词：
Affect Affinity Antiviral Agents Binding Biochemical Biological Assay Calcium Calmodulin Cell membrane Cell physiology Cells Complex Cytokine Inducible SH2-Containing Protein Cytoplasm Data Development Drug Design Electrostatics Gagging HIV HIV Infections HIV-1 Histidine Human Infection Integration Host Factors Knowledge Lead Lecithin Life Lipids Location Membrane Membrane Lipids Methods Micelles Modeling Molecular Mutagenesis Nuclear Magnetic Resonance Nucleocapsid Pathway interactions Peptides Phase Phosphatidylethanolamine Phosphatidylinositol 4,5-Diphosphate Phosphatidylserines Phospholipids Property Protein Binding Proteins Public Health Research Resolution Role Safety Signal Pathway Signal Transduction Specificity Staging Structure Techniques Therapeutic Agents Virus Replication base design gag Gene Products in vivo membrane assembly membrane model novel strategies particle pathogen protein complex public health relevance structural biology three dimensional structure tool trafficking

项目摘要

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.

描述(申请人提供)：在细胞内，蛋白质通过与蛋白质和磷脂等细胞成分的特定相互作用而定位于明确的位置。在HIV-1感染的晚期，Gag多聚蛋白被运输到质膜(PM)进行组装。利用核磁共振技术，我们已经确定了针对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还与细胞脂质和蛋白质形成生物上重要的相互作用，这些作用与有效的颗粒组装有关。我们发现MA与磷脂酰丝氨酸(PtdSer)、磷脂酰胆碱(PtdCho)和磷脂酰乙醇胺(PtdEtn)直接相互作用，提示GAG靶向和组装在PM上是通过MA结构域与PI(4，5)P2和主要膜脂的双重结合进行的。已经提出了一些细胞蛋白来促进GAG在细胞内的转运和靶向PMHIV-1 Gag与胞浆中的钙调蛋白(CaM)相互作用和共定位。此外，最近的研究发现，细胞因子信号转导抑制因子1(SOCS1)是HIV感染过程中的一个可诱导宿主因子，它通过与Gag蛋白直接相互作用来调节病毒复制途径的晚期。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与CaM以钙依赖的方式直接相互作用，提示HIV-1劫持CaM细胞信号通路以促进Gag转运。此外，我们还获得了SOCS1和MA蛋白之间直接相互作用的证据。在这项提案中，我们将使用生化、生物物理和结构生物学工具来确定关键的蛋白质-蛋白质和蛋白质-脂质相互作用，这些相互作用涉及调控HIV-1 Gag在细胞内运输和组装的分子机制。我们的三个主要目标是：(I)确定GAG与PM结合的确切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SOCS1-Gag相互作用的结构要求。这项研究产生的结果将使人们更好地了解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新的抗病毒治疗剂的新方法，以抑制Gag的贩运和组装。