Molecular Mechanisms of Rhabdovirus Entry

描述(由申请人提供)：被包裹的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，并启动感染循环。这些病毒进化出了专门的融合蛋白，催化了这一不利的能量过程。这些融合蛋白分为三类，如流感血凝素(I类)、黄病毒囊膜蛋白(II类)和横纹病毒糖蛋白(III类)。作为对特定触发因素的反应，这些融合蛋白经历了戏剧性的构象变化，使病毒和靶膜更接近，降低了膜融合的能量障碍。III类蛋白完成这一任务的机制是最不为人所知的。水泡性口炎病毒(VSV)是横纹病毒科的一个原型，是研究III类融合机如何发挥作用的理想模型，因为它的单附着和融合糖蛋白的结构最近通过X射线结晶学在融合前和融合后被解决。我们的长期目标是了解VSV如何将其286丙二醛核糖核蛋白核心运送到细胞内，以启动感染过程。膜融合是这一过程的中心步骤。在这里，我们利用VSV简单的遗传学和它在细胞培养中的强劲生长来开发新的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G的构象转变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引发的构象变化、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索体外半融合、融合孔形成和RNP跨脂质双层转移之间的关系。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内膜融合和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，完成病毒内容物进入细胞的输送。因此，这些研究将为包膜病毒膜融合和胞内转运过程提供新的机制见解。

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.