Molecular Mechanisms of Rhabdovirus Entry

总结： 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 并引发传染循环这些病毒已经进化出专门的融合蛋白， 非常不利的过程。这些融合蛋白分为三类，如流感病毒 血凝素（I类）、黄病毒包膜蛋白（II类）和弹状病毒糖蛋白（III类）。在 响应于特定的触发，这些融合蛋白经历戏剧性的构象变化， 病毒和靶细胞膜靠近，降低了膜融合的能量屏障。的 III类蛋白质实现这一点的机制是最不清楚的。水泡性口炎病毒 (VSV)，弹状病毒科的原型，是研究III类融合机器如何作为 其单一附着和融合糖蛋白结构最近通过X射线晶体学解决， 融合前和融合后的形式。我们的长期目标是了解VSV如何提供其286 MDa 病毒通过核糖核蛋白核心进入细胞以启动感染过程。膜融合是这一过程的核心步骤 过程在这里，我们利用VSV的简单遗传学和其在细胞培养中的强劲生长来开发 研究膜融合和病毒进入过程的新技术。我们的基本假设是 特定的pH触发G中的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核糖核蛋白的递送 病毒核心穿过细胞膜我们将在三个相互关联的目标中检验这一假设。在特定 目的1，我们将使用遗传和生物化学方法来确定G对pH触发的需求 构象变化、膜融合和病毒感染性。在具体目标2中，我们将使用高分辨率 单粒子成像方法，以探测半融合，融合孔形成和 RNP在体外穿过脂质双层的转移。在具体目标3中，我们将使用高分辨率单粒子 成像以确定细胞中膜融合和RNP释放的位点。完成这些研究将 揭示了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的过程 膜融合和内吞转运。

SUMMARY: 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.