Molecular Mechanisms of Rhabdovirus Entry

弹状病毒进入的分子机制

• 批准号: 8007418
• 负责人: Sean PJ Whelan
• 金额: $ 41.39万
• 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/8007418
• 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.

性状（由申请方提供）：包膜病毒必须融合病毒和细胞膜，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专门的融合蛋白，催化这种能量上不利的过程。这些融合蛋白分为三类，例如流感血凝素（I类）、黄病毒包膜蛋白（II类）和弹状病毒糖蛋白（III类）。为了响应特定的触发，这些融合蛋白经历了巨大的构象变化，使病毒和靶膜紧密靠近，降低了膜融合的能量屏障。III类蛋白实现这一点的机制是最不清楚的。水泡性口炎病毒（VSV）是弹状病毒科的一种原型病毒，是研究III类融合器如何发挥作用的理想模型，其单个附着和融合糖蛋白的结构最近通过X射线晶体学在融合前和融合后形式中得到了解决。我们的长期目标是了解VSV如何将其286 MDa核糖核蛋白核心递送到细胞中以启动感染过程。膜融合是这一过程的核心步骤。在这里，我们利用VSV的简单遗传学和其在细胞培养中的稳健生长来开发新技术来研究膜融合和病毒进入的过程。我们的基本假设是，特定的pH值引发的构象转换G驱动膜融合的初始步骤，但需要多个G蛋白三聚体之间的相互作用，以完成交付的核糖核蛋白核心的病毒跨膜。我们将在三个相互关联的目标中检验这一假设。在具体目标1中，我们将使用遗传和生物化学方法来确定G对pH触发的构象变化、膜融合和病毒感染性的需求。在具体目标2中，我们将使用高分辨率单粒子成像方法来探测半融合、融合孔形成和RNP在体外穿过脂质双层的转移之间的关系。在具体目标3中，我们将使用高分辨率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类融合蛋白如何发挥功能以实现将病毒内容物递送到细胞中。因此，这些研究将提供新的机制的见解包膜病毒膜融合和内吞转运的过程。 公共卫生相关性：膜融合和内吞转运是细胞生物学家和病毒学家感兴趣的基本生物学过程。了解融合是一个内在的兴趣，并具有显着的潜力，影响药物开发，融合抑制剂代表了一种有效的和相对较新的一类抗病毒药物。了解病毒如何靶向特定的内吞途径，并确定内吞机制如何被病毒在进入过程中吸收也是内在的兴趣。病毒感染可能对这些宿主途径的抑制更敏感，这可能使它们成为药物开发的靶点。