Cell Biology of Retroviral Entry and Uncoating

逆转录病毒进入和脱壳的细胞生物学

• 批准号: 7189347
• 负责人: WALTHER H MOTHES
• 金额: $ 27.59万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-12 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7189347
• 关键词: Address; Avian Leukosis Virus; Biochemical; Biological; Biological Assay; Biological Models; Capsid; Cell membrane; Cells; Cellular biology; Cytoplasm; Dominant-Negative Mutation; Endocytosis; Event; Functional Imaging; Genome; Giant Cells; Goals; HIV; Lead; Life; Link; Mediating; Membrane Fusion; Molecular; Movement; Murine leukemia virus; Numbers; Pathway interactions; Process; Proteins; RNA; RNA Interference; Reaction; Retroviridae; Route; Subgroup; Surface; Techniques; Technology; Testing; Viral; Viral Genome; Virus; Virus Diseases; base; fluorescence imaging; inhibitor/antagonist; particle; receptor

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.

描述（由申请人提供）：逆转录病毒是有包膜病毒，通过膜融合和病毒衣壳脱壳将其RNA基因组递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）和鼠白血病病毒（MLV）为研究这些过程提供了极好的模型系统。此前，人们认为这两种病毒都是通过质膜上的 pH 无关过程进入细胞的。然而，我们最近的研究提供了令人信服的证据，表明 ALV 首先被内化到达酸性细胞内区室，在该区室中，低 pH 值会诱导融合。现在必须解决一些重要问题。首先，ALV从哪个细胞内区室进入细胞质？其次，已鉴定出 ALV A 亚组的两个表面受体。两者都允许有效的病毒进入，但其内化机制不同。两种受体如何介导 ALV 进入？两种进入途径是否会合并到达一个共同的低 pH 区室，或者是否有几种可用于病毒进入？第三，人类免疫缺陷病毒（HIV）和MLV等不依赖pH的逆转录病毒从哪里进入细胞？这些病毒是从质膜进入还是通过内吞作用内化，尽管它们不依赖于 pH 值？最后，病毒衣壳的膜融合和脱壳如何导致病毒基因组递送到细胞质中？这两个事件是同时发生还是及时解决并且在生化上有所不同？该提案的目的是确定两种病毒进入细胞质的细胞区室，并了解逆转录病毒衣壳的脱壳是否与膜融合直接相关，还是作为细胞质中的独特反应进行。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测定和生化技术相结合。具体来说，我们将：1) 确定 ALV 的内吞内化途径作为两种不同受体的功能，2) 识别 pH 依赖性病毒 HIV 和 MLV 进入细胞的细胞区室，3) 剖析病毒进入过程中的融合和脱壳反应。