Cell Biology of Retroviral Entry and Uncoating

描述（由申请人提供）：逆转录病毒是一种包膜病毒，通过膜融合和病毒衣壳的剥离将其RNA基因组传递到宿主的细胞质中。尽管这些事件对病毒感染的建立具有重要意义，但对其分子机制知之甚少。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很好的模型系统。以前，这两种病毒都被认为是通过一个不依赖于ph值的质膜过程进入细胞的。然而，我们最近的研究提供了令人信服的证据，表明ALV首先被内化到一个酸性的细胞内室，在那里低ph诱导融合。现在必须解决许多重要的问题。首先，ALV从哪个细胞内腔室进入细胞质？其次，ALV A亚群的两个表面受体已被确定。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？是两种进入途径合并到一个共同的低pH区，还是有几种途径可以用于病毒进入？第三，不依赖ph的逆转录病毒，如人类免疫缺陷病毒（HIV）和MLV，从哪里进入细胞？这些病毒是通过质膜进入还是通过内吞作用被内化，尽管它们不依赖于ph ？最后，膜融合和病毒衣壳的剥离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，并且在生物化学上是不同的？这一建议的目的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剥离是否与膜融合直接相关，还是作为细胞质中的一个独特反应进行。为此，我们将应用细胞生物学方法，将活细胞荧光成像与功能分析和生化技术相结合。具体来说，我们将：1)确定ALV作为两种不同受体的内吞内化途径；2)鉴定ph无关的HIV和MLV进入细胞的细胞室；3)解剖病毒进入细胞时的融合和剥膜反应。

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.