Cell Biology of Retroviral Entry and Uncoating

描述(申请人提供)：逆转录病毒是有包膜的病毒，通过膜融合和病毒衣壳的剥离将其RNA基因组运送到宿主的细胞质中。尽管这些事件对于病毒感染的建立很重要，但对其分子机制知之甚少。禽白血病病毒(ALV)和小鼠白血病病毒(MLV)为研究这些过程提供了很好的模型系统。此前，这两种病毒都被认为是通过质膜上不依赖于pH的过程进入细胞的。然而，我们最近的研究提供了令人信服的证据，证明ALV首先内化到酸性的细胞内，在那里融合是由低pH诱导的。现在必须解决一些重要的问题。首先，ALV是从哪个胞内室进入细胞质的？其次，已鉴定出ALV A亚群的两种表面受体。两者都允许有效的病毒进入，但它们的内化机制不同。这两种受体是如何介导ALV进入的？这两条进入途径是合并到一个共同的低pH值区间，还是有几条途径可用于病毒进入？第三，人类免疫缺陷病毒(HIV)和MLV等不依赖于pH的逆转录病毒是从哪里进入细胞的？这些病毒是进入质膜还是被内吞作用内化，尽管它们不依赖于pH？最后，病毒衣壳的膜融合和剥离如何导致病毒基因组进入细胞质？这两个事件是同时进行的，还是它们是及时解决的，并且在生化上是截然不同的？这项提议的目标是确定两种病毒进入细胞质的细胞室，并了解逆转录病毒衣壳的剥离是直接与膜融合有关，还是作为细胞质中的不同反应进行。为此，我们将应用一种细胞生物学方法，将活细胞中的荧光成像与功能分析和生化技术相结合。具体地说，我们将：1)确定ALV的内吞途径作为两个不同受体的功能，2)确定与pH无关的HIV和MLV进入细胞的细胞室，以及3)剖析病毒进入过程中的融合和脱壳反应。

DESCRIPTION (provided by applicant): Retroviruses are enveloped viruses that deliver their RNA genome into the cytoplasm of the host by membrane fusion and uncoating of the viral capsid. Despite the importance of these events for the establishment of viral infection, little is known about the molecular mechanism. The Avian Leukosis virus (ALV) and Murine Leukemia viruses (MLV) provide excellent model systems to study these processes. Previously, both viruses were believed to enter cells by a pH-independent process at the plasma membrane. However, our recent studies provide compelling evidence that ALV is first internalized to reach an acidic intracellular compartment where fusion is induced by low pH. A number of important questions must now be addressed. First, from which intracellular compartment does ALV enter the cytoplasm? Secondly, two surface receptors for subgroup A of ALV have been identified. Both allow efficient viral entry but differ in their internalization mechanisms. How do both receptors mediate ALV entry? Do both entry pathways merge to reach a common low pH compartment or are there several that can be utilized for viral entry? Third, where do pH-independent retroviruses such as the human immunodeficiency virus (HIV) and MLV enter cells? Do these viruses enter at the plasma membrane or are they internalized by endocytosis despite their pH-independence? Finally, how do membrane fusion and uncoating of the viral capsid lead to the delivery of the viral genome into the cytoplasm? Do both events proceed simultaneously or are they timely resolved and biochemically distinct? The goal of this proposal is to identify the cellular compartments through which both viruses enter the cytoplasm and to understand whether uncoating of retroviral capsids is directly linked to membrane fusion or proceeds as a distinct reaction in the cytoplasm. Toward this end we will apply a cell biological approach that integrates fluorescence imaging in living cells with functional assays and biochemical techniques. Specifically, we will: 1) determine endocytic internalization routes of ALV as a function of two different receptors, 2) identify cellular compartments through which the pH-independent viruses HIV and MLV enter cells, and 3) dissect fusion and uncoating reactions during viral entry.