Cell Biology of Retroviral Entry and Uncoating

描述（由申请方提供）：逆转录病毒是一种包膜病毒，通过膜融合和病毒衣壳的脱壳将其RNA基因组递送到宿主细胞质中。尽管这些事件对于建立病毒感染的重要性，但对分子机制知之甚少。禽白血病病毒（ALV）和鼠白血病病毒（MLV）为研究这些过程提供了极好的模型系统。以前，这两种病毒被认为是通过质膜上的pH非依赖性过程进入细胞的。然而，我们最近的研究提供了令人信服的证据，ALV首先内化，达到酸性细胞内室，融合是由低pH值诱导。现在必须解决一些重要的问题。首先，ALV从哪个细胞内区室进入细胞质？其次，已经鉴定了ALV A亚群的两种表面受体。两者都允许有效的病毒进入，但在其内化机制不同。两种受体如何介导ALV进入？两种进入途径是否合并以达到共同的低pH区室，或者是否有几种可用于病毒进入？第三，非pH依赖性逆转录病毒（如人类免疫缺陷病毒（HIV）和MLV）从何处进入细胞？这些病毒是从质膜进入的，还是通过内吞作用内化的，尽管它们不依赖于pH？最后，膜融合和病毒衣壳的脱壳是如何导致病毒基因组进入细胞质的？这两个事件是同时发生的，还是及时解决的和生化上不同的？该提案的目标是确定两种病毒进入细胞质的细胞隔室，并了解逆转录病毒衣壳的脱壳是否直接与膜融合有关，或作为细胞质中的独特反应进行。为此，我们将采用细胞生物学方法，将活细胞中的荧光成像与功能测定和生物化学技术相结合。具体而言，我们将：1）确定ALV的内吞内化途径作为两种不同受体的函数，2）鉴定pH非依赖性病毒HIV和MLV进入细胞的细胞区室，和3）剖析病毒进入期间的融合和去包被反应。

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.