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.

描述（由申请人提供）：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并启动感染周期。这些病毒已经进化出专门的融合蛋白来催化这个能量上不利的过程。这些融合蛋白可分为三类，如流感血凝素（I类）、黄病毒包膜蛋白（II类）和横纹肌病毒糖蛋白（III类）。为了响应特定的触发因素，这些融合蛋白经历了戏剧性的构象变化，使病毒和靶膜接近，降低了膜融合的能量屏障。III类蛋白实现这一功能的机制尚不清楚。水疱性口炎病毒（VSV）是Rhabdoviridae的一种原型，是研究III类融合机如何发挥作用的理想模型，其单附着结构和融合糖蛋白最近通过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核糖核蛋白核心传递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的易遗传及其在细胞培养中的强劲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触发了G的构象转变，驱动了膜融合的初始步骤，但需要多个G蛋白三聚体之间的相互作用才能完成病毒核糖核蛋白核心的跨膜递送。我们将从三个相互关联的目标来检验这一假设。在具体目标1中，我们将使用遗传和生化方法来确定G对pH触发的构象变化、膜融合和病毒感染性的要求。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索体外半融合、融合孔形成和RNP在脂质双分子层上转移之间的关系。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释放的位置。这些研究的完成将揭示III类融合蛋白如何发挥作用以完成病毒内容物进入细胞的传递。因此，这些研究将为包膜病毒膜融合和内吞运输过程提供新的机制见解。