Functional Characterization of the Hepatitis C Virus E1-E2 Glycoproteins

描述（由申请人提供）：丙型肝炎病毒（HCV）是一种重要的病原体，全世界约有1.7亿人感染。尽管进行了广泛的努力，但令人惊讶的是，人们对HCV引发感染的机制知之甚少。由于缺乏针对这种病毒的疫苗或有效治疗方法，因此迫切需要对丙型肝炎病毒进入宿主细胞进行研究。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释放其核衣壳，这一过程由HCV E1和E2糖蛋白促进。E1E2诱导的融合严格依赖于受体和低ph值。不仅这一过程的机制尚不清楚，甚至融合蛋白（E1和E2）的身份也尚未确定。这是由于缺乏一个易于处理的模型系统来研究HCV融合。我们的工作假设是HCV融合至少通过两个主要步骤进行-通过与细胞受体相互作用使E1E2融合的启动步骤，然后是低ph依赖性融合。这一假设预测：(1)在没有受体的情况下，低pH预处理不会诱导E1E2的不可逆构象变化导致病毒失活；(2)与可溶性或膜锚定受体的结合使E1E2能够经历构象变化，并在低ph下促进融合。这些预测将通过新型病毒灭活、病毒-细胞融合和细胞-细胞融合试验进行验证。单个病毒的时间分辨成像将用于跟踪HCV摄取和随后与核内体的融合。HCV进入将通过捕捉和描述不同的融合中间阶段来进一步检查。这些实验应该为e1e2诱导融合的机制提供新的见解，并可能有助于设计阻止HCV感染的预防策略。公共卫生相关性：丙型肝炎病毒（HCV）是一种重要的病原体，全世界有超过1.7亿人感染。在HCV被细胞内化后，感染是由病毒和细胞膜合并并释放病毒基因组引起的。为了阐明HCV糖蛋白介导的膜合并机制，我们将利用荧光显微镜实时观察单个病毒进入宿主细胞的过程。了解病毒进入的过程将提出预防感染的新策略。

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.