Molecular Mechanisms of Rhabdovirus Entry

弹状病毒进入的分子机制

• 批准号: 7784767
• 负责人: Sean PJ Whelan
• 金额: $ 41.82万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7784767
• 关键词: Affect; Amino Acids; Antiviral Agents; Biochemical; Biological; Biological Models; Biological Process; Cell Culture Techniques; Cell membrane; Cell surface; Cells; Cellular Membrane; Chemicals; Chimeric Proteins; Clathrin; Coupled; Endocytic Vesicle; Flavivirus; GTP-Binding Proteins; Genetic; Genome; Glycoproteins; Growth; Image; In Vitro; Individual; Infection; Influenza Hemagglutinin; Kinetics; Lipid Bilayers; Membrane; Membrane Fusion; Modeling; Molecular; Molecular Conformation; Mutation; Nucleic Acids; Pathway interactions; Process; Proteins; Reagent; Resolution; Rhabdoviridae; Ribonucleoproteins; Role; Series; Site; Staging; Structure; Techniques; Testing; Vesicular stomatitis Indiana virus; Viral; Viral Fusion Proteins; Virion; Virus; Virus Diseases; X-Ray Crystallography; conformational conversion; drug development; env Gene Products; falls; genetic manipulation; glycoprotein G; inhibitor/antagonist; insight; interest; mutant; new technology; particle; protein function; prototype; public health relevance; research study; response; tool; virus core

DESCRIPTION (provided by applicant): Enveloped viruses must fuse viral and cellular membranes to transfer the viral nucleic acid into the host cell and initiate the infectious cycle. These viruses have evolved dedicated fusion proteins that catalyze this energetically unfavorable process. These fusion proteins fall into three classes as exemplified by influenza hemagglutinin (class I), flavivirus envelope proteins (class II) and rhabdovirus glycoproteins (class III). In response to specific triggers, these fusion proteins undergo dramatic conformational changes that bring the viral and target membranes into close proximity, lowering the energy barrier to membrane fusion. The mechanism by which class III proteins accomplish this is the least well understood. Vesicular stomatitis virus (VSV), a prototype of the Rhabdoviridae, is the ideal model to study how class III fusion machines function as the structure of its single attachment and fusion glycoprotein was recently solved by X-ray crystallography in both pre and post fusion forms. Our long term objectives are to understand how VSV delivers its 286 MDa ribonucleoprotein core into cells to initiate the process of infection. Membrane fusion is a central step of this process. Here we have capitalized on the facile genetics of VSV and its robust growth in cell culture to develop new technologies to study the process of membrane fusion and viral entry. Our underlying hypothesis is that specific pH triggered conformational transitions in G drive the initial steps of membrane fusion, but that interactions between multiple G protein trimers are required to accomplish delivery of the ribonucleoprotein core of the virus across the membrane. We will examine this hypothesis in three interrelated aims. In specific aim 1, we will use genetic and biochemical approaches to determine the requirements in G for pH triggered conformational change, membrane fusion and viral infectivity. In specific aim 2, we will use high-resolution single particle imaging approaches to probe the relationships between hemifusion, fusion pore formation and transfer of the RNP across a lipid bilayer in vitro. In specific aim 3, we will use high resolution single particle imaging to determine the site of membrane fusion and RNP release in cells. Completion of these studies will reveal how a class III fusion protein functions to accomplish delivery of the viral contents into the cell. Consequently, these studies will provide new mechanistic insights into the process of enveloped virus membrane fusion and endocytic transport. PUBLIC HEALTH RELEVANCE: Membrane fusion and endocytic transport are fundamental biological processes that are of interest to cell biologists as well as virologists. Understanding fusion is of intrinsic interest, and has significant potential to impact drug development, as fusion inhibitors represent an effective and relatively new class of antiviral drugs. Understanding how viruses are targeted to specific endocytic pathways and determining how the endocytic machinery is co opted by viruses during entry is also of intrinsic interest. Viral infection may be more sensitive to inhibition of these host pathways, which may render them targets for drug development.

描述（由申请人提供）：包膜病毒必须融合病毒和细胞膜才能将病毒核酸转移到宿主细胞中并启动感染周期。这些病毒已经进化出了专用的融合蛋白，从而催化了这种不利的过程。这些融合蛋白分为三类，如流感hemagglutinin（I类），黄病毒包膜蛋白（II类）和色夫病毒糖蛋白（III类）所示。为了响应特定的触发因素，这些融合蛋白会经历巨大的构象变化，使病毒和靶向膜近距离接近，从而降低了膜融合的能屏障。 III类蛋白质完成的机制是最不理解的。副伏氏病毒的原型是囊泡孔隙病毒（VSV），是研究III类融合机如何作为其单个附着和融合糖蛋白的结构的理想模型。我们的长期目标是了解VSV如何将其286个MDA核糖核蛋白核心传递到细胞中以启动感染过程。膜融合是此过程的核心步骤。在这里，我们利用了VSV的便捷遗传学及其在细胞培养中的强大生长，以开发新技术来研究膜融合和病毒的过程。我们的基本假设是，在G中特定的pH触发了构象转变驱动膜融合的初始步骤，但是需要多个G蛋白三聚体之间的相互作用来完成病毒核糖蛋白核心在整个膜中的递送。我们将在三个相互关联的目标中检查这一假设。在特定目标1中，我们将使用遗传和生化方法来确定G对pH触发构象变化，膜融合和病毒感染性的需求。在特定的目标2中，我们将使用高分辨率的单粒子成像方法来探测半分解，融合孔的形成和RNP在体外跨脂质双层层的转移之间的关系。在特定的目标3中，我们将使用高分辨率的单粒子成像来确定细胞中膜融合和RNP释放的位点。这些研究的完成将揭示III类融合蛋白的功能如何完成病毒含量进入细胞的功能。因此，这些研究将提供有关包膜病毒膜融合和内吞转运过程的新机械见解。 公共卫生相关性：膜融合和内吞运输是细胞生物学家以及病毒学家感兴趣的基本生物学过程。了解融合具有内在兴趣，并且具有影响药物发育的巨大潜力，因为融合抑制剂代表了一种有效且相对较新的抗病毒药物。了解如何将病毒靶向特定的内吞途径，并确定进入过程中病毒选择的内吞机械如何选择。病毒感染可能对抑制这些宿主途径更敏感，这可能使它们成为药物开发的目标。