Functional Characterization of the Hepatitis C Virus E1-E2 Glycoproteins

丙型肝炎病毒 E1-E2 糖蛋白的功能表征

基本信息

批准号：
8116923
负责人：
Gregory B Melikian
金额：
$ 15.55万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-22 至 2011-12-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8116923
关键词：
Acids Binding Biological Assay Biological Models Cell fusion Cell membrane Cell surface Cells Cellular Membrane Chimeric Proteins Clathrin Endocytosis Endosomes Failure Fluorescence Microscopy Genus Alpharetrovirus Glycoproteins HIV Hepatitis C Hepatitis C virus Image Incubated Individual Infection Infection prevention Kinetics Light Measures Mediating Membrane Methodology Modeling Monitor Nucleocapsid Pathway interactions Prevention strategy Process Retroviral Vector Site Staging Stress Temperature Testing Time Vaccines Viral Viral Fusion Proteins Viral Genome Virus Virus Inactivation Virus Receptors Work base beta-Lactamase design effective therapy insight novel virus particle pathogen public health relevance receptor receptor binding research study trafficking uptake

Acids Binding Biological Assay Biological Models Cell fusion Cell membrane Cell surface Cells Cellular Membrane Chimeric Proteins Clathrin Endocytosis Endosomes Failure Fluorescence Microscopy Genus Alpharetrovirus Glycoproteins HIV Hepatitis C Hepatitis C virus Image Incubated Individual Infection Infection prevention Kinetics Light Measures Mediating Membrane Methodology Modeling Monitor Nucleocapsid Pathway interactions Prevention strategy Process Retroviral Vector Site Staging Stress Temperature Testing Time Vaccines Viral Viral Fusion Proteins Viral Genome Virus Virus Inactivation Virus Receptors Work base beta-Lactamase design effective therapy insight novel virus particle pathogen public health relevance receptor receptor binding research study trafficking uptake

展开

项目摘要

DESCRIPTION (provided by applicant): The hepatitis C virus (HCV) is an important pathogen that has infected approximately 170 million people worldwide. In spite of extensive efforts, surprisingly little is known about the mechanism by which HCV initiates infection. The lack of vaccine or effective therapy against this virus stresses the urgent need for studies of HCV entry into host cells. HCV is an enveloped virus that enters cells via a clathrin-mediated pathway and releases its nucleocapsid by fusing the viral membrane with an endosomal membrane a process that is promoted by HCV E1 and E2 glycoproteins. The fusion induced by E1E2 is strictly receptor- and low pH-dependent. Not only is the mechanism of this process poorly understood, but even the identity of the fusion protein (E1 vs. E2) has not been established. This is due to the lack of a tractable model system to study HCV fusion. Our working hypothesis is that HCV fusion precedes through at least two major steps - a priming step through interactions with cellular receptors which render E1E2 fusogenic, followed by low pH-dependent fusion. This hypothesis predicts that: (1) low pH pre-treatment in the absence of receptors does not induce irreversible conformational changes in E1E2 leading to virus inactivation; (2) binding to soluble or to membrane-anchored receptors renders E1E2 competent to undergo conformational changes and promote fusion at low pH. These predictions will be tested using novel virus inactivation, virus-cell fusion and cell-cell fusion assays. Time-resolved imaging of single virus will be implemented for tracking HCV uptake and subsequent fusion with an endosome. HCV entry will be further examined by capturing and characterizing distinct intermediate stages of fusion. These experiments should provide new insights into the mechanism of E1E2-induced fusion and might help to design preventive strategies to block HCV infection. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is an important pathogen that has infected over 170 million people worldwide. Following HCV internalization by a cell, infection is initiated by merging of viral and cellular membranes that releases the viral genome. To elucidate the mechanism of membrane merger mediated by HCV glycoproteins, single virus entry into a host cell will be visualized by real-time by fluorescence microscopy. Understanding the virus entry process will suggest new strategies to prevent infection.

描述（由申请人提供）：丙型肝炎病毒（HCV）是一种重要的病原体，已感染了全球约1.7亿人。尽管努力进行了广泛的努力，但令人惊讶的是，HCV启动感染的机制知之甚少。缺乏疫苗或针对该病毒的有效疗法强调，迫切需要研究HCV进入宿主细胞。 HCV是一种包膜病毒，通过网格蛋白介导的途径进入细胞，并通过将病毒膜与内体膜融合来释放其核蛋白酶，该过程由HCV E1和E2糖蛋白促进的过程。 E1E2诱导的融合是严格依赖受体和低pH值的。该过程的机制不仅鲜为人知，而且即使是融合蛋白的身份（E1 vs. E2）也没有建立。这是由于缺乏研究HCV融合的模型系统。我们的工作假设是，HCV融合至少两个主要步骤 - 启动步骤通过与细胞受体相互作用，而细胞受体的相互作用使E1E2融合，然后是低pH依赖性融合。该假设预测：（1）在没有受体的情况下，pH值低的预处理不会引起E1E2的不可逆构象变化，从而导致病毒失活；（2）与可溶性或与膜锚定受体结合的结合使E1E2具有符合构象变化并促进低pH值融合的E1E2。这些预测将使用新型病毒灭活，病毒细胞融合和细胞融合测定测试。将实施单个病毒的时间分辨成像，以跟踪HCV摄取并随后与内体融合。通过捕获和表征融合的不同中间阶段，将进一步检查HCV进入。这些实验应提供有关E1E2诱导的融合机制的新见解，并可能有助于设计预防性策略以阻止HCV感染。公共卫生相关性：丙型肝炎病毒（HCV）是一种重要的病原体，在全球范围内感染了超过1.7亿人。通过细胞进行HCV内在化后，感染是通过释放病毒基因组的病毒和细胞膜合并而引发的。为了阐明HCV糖蛋白介导的膜合并的机理，将通过荧光显微镜实时可视化单个病毒进入宿主细胞。了解病毒进入过程将提出防止感染的新策略。