Molecular Mechanisms of Rhabdovirus Entry

描述（由申请人提供）：有包膜病毒必须融合病毒和细胞膜，以将病毒核酸转移到宿主细胞中并启动感染周期。这些病毒进化出了专用的融合蛋白，可以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因素，这些融合蛋白会发生巨大的构象变化，使病毒和目标膜非常接近，从而降低膜融合的能量障碍。 III 类蛋白实现这一目标的机制是最不为人所知的。水疱性口炎病毒 (VSV) 是弹状病毒科的原型，是研究 III 类融合机如何发挥作用的理想模型，因为其单一附着物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得到解决。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。膜融合是该过程的核心步骤。在这里，我们利用 VSV 的简单遗传学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 值触发的 G 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跨膜的传递。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讨半融合、融合孔形成和 RNP 体外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作用，将病毒内容物递送到细胞中。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。

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.