Mechanistic Studies of HCV E2

描述（由申请人提供）：丙型肝炎病毒（HCV）感染是人类慢性肝病的病原体，困扰着全球超过1.23亿人（约占人口的2%）。在许多情况下，HCV感染变成慢性，可进展为肝硬化和肝细胞癌。目前还没有针对HCV的疫苗，唯一的治疗方法是聚乙二醇干扰素？与利巴韦林联合使用，只有50%的患者有持续反应。高感染率，缺乏HCV特异性抑制剂，以及对当前治疗的低应答率强调了新疗法的重要性。HCV病毒粒子由膜包膜的核衣壳和两个糖蛋白（E1和E2）组成，这两个糖蛋白对于识别和进入靶宿主细胞是重要的。与其他包膜病毒如瘟病毒、甲病毒和其他黄病毒的情况一样，HCV感染需要在病毒基因组释放到宿主细胞胞质溶胶中之前在病毒和宿主细胞膜之间融合。这一关键步骤被认为发生在内体区室中，由低pH触发，并且可能需要糖蛋白的结构重排。除了其在膜融合中的作用之外，E2被认为负责靶向，因为已经显示其结合宿主细胞受体CD 81和I型B类清道夫受体（SR-BI）。尽管已经取得了进展，在理解E2的功能，使用病毒中和抗体和遗传方法，更深入的机制解剖E2需要互补的生物化学，生物物理和分子病毒学技术。由于E2蛋白质的重糖基化和分子内二硫键的存在，E2蛋白质的生产具有挑战性，因此无法制备足够量的正确折叠的蛋白质阻碍了进展。最近，我们的实验室设计并发表了一种新的表达系统，在哺乳动物细胞中产生eE 2（E2胞外域）蛋白，保留了病毒粒子上的E2功能。结合我们对eE 2的全面生物化学和生物物理学表征，我们为更好地确定E2在HCV感染中的功能作用奠定了基础。在这项提案中，我们将重点评估E2在触发低pH值依赖性膜融合中的作用，并探索HCV感染这一关键步骤所需的可能的寡聚体和结构重排。一个整合了生物化学、生物物理学、遗传学和分子病毒学技术的多学科实验计划将用于：（1）定义与HCV感染相关的E2的生理寡聚体形式;（2）表征E2在感知pH降低中的作用，如病毒在内体酸化过程中遇到的;和（3）确定E2的结构域组织和pH变化时可能的重排。从拟议的研究结果将大大有助于我们的理解负责病毒进入的机制，并有重要的意义，新的治疗方法和疫苗的发展，以控制HCV感染。对HIV糖蛋白的类似研究已经导致开发出一种新的抗逆转录病毒药物，可以阻止HIV进入。 公共卫生相关性：目前，2%的人口-全世界约1.23亿人-慢性感染丙型肝炎病毒（HCV），使病毒传播成为一个主要的公共卫生问题。没有针对HCV的疫苗，目前的药物对许多人无效。拟议拨款的目标将提供更好地了解HCV如何识别和进入细胞。这些信息对于设计新的治疗方法或抗HCV疫苗将是有价值的。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) infection is a causal agent of chronic liver disease in humans, afflicting more than 123 million people worldwide (approximately 2% of the human population). In many cases HCV infection becomes chronic, which can progress to liver cirrhosis and hepatocellular carcinoma. There is no vaccine available for HCV and the only treatment is pegylated interferon-? in combination with ribavirin, which leads to a sustained response in only 50% of patients. The high prevalence of infection, lack of HCV specific inhibitors, and poor response rate to the current treatment underscore the importance of new therapies. The HCV virion consists of a membrane enveloped nucleocapsid and two glycoproteins (E1 and E2), which are important for recognizing and entering a target host cell. As in the case of other enveloped viruses such as pestiviruses, alphaviruses, and other flaviviruses, HCV infection requires fusion between viral and host cell membrane before the viral genome can be released into the host cell cytosol. This critical step is thought to occur in the endosomal compartment, is triggered by low pH, and likely requires structural rearrangement of the glycoproteins. In addition to its role in membrane fusion, E2 is thought to be responsible for targeting since it has been shown to bind host cellular receptors CD81 and scavenger receptor class B type I (SR-BI). Despite the progress that has been made in understanding E2 function using virus-neutralizing antibodies and genetic approaches, more in-depth mechanistic dissection of E2 requires complementary biochemical, biophysical and molecular virology techniques. Progress has been hindered by the inability to make sufficient quantities of properly folded protein as E2 protein production is challenging due to its heavy glycosylation and presence of intramolecular disulfide bonds. Recently, our laboratory has devised and published a novel expression system in mammalian cells to produce eE2 (E2 ectodomain) protein that retains the functionality of E2 present on virions. Together with our comprehensive biochemical and biophysical characterization of eE2, we have established a foundation to better define the functional role of E2 in HCV infection. In this proposal, we will focus on assessing the role of E2 in triggering low-pH dependent membrane fusion and explore possible oligomeric and structural rearrangements necessary for this critical step in HCV infection. A multidisciplinary experimental plan that integrates biochemical, biophysical, genetic and molecular virology techniques will be used to: (1) define the physiological oligomeric form of E2 relevant to HCV infection; (2) characterize the role of E2 in sensing a decrease in pH, as encountered by the virus during endosomal acidification; and (3) determine the domain organization of E2 and possible rearrangements upon pH change. Results from the proposed studies will contribute substantially to our understanding of the mechanisms responsible for viral entry and have important implications for the development of novel therapies and vaccines to control HCV infection. Similar studies on the HIV glycoproteins have lead to the development of a new class of antiretroviral that block HIV entry. PUBLIC HEALTH RELEVANCE: Currently, 2% of the human population - approximately 123 million people worldwide - is chronically infected with hepatitis C virus (HCV), making virus transmission a major public health concern. There is no vaccine against HCV and the current drugs are not affective for many people. The goal of the proposed grant will provide a better understanding of how HCV recognizes and enter cells. This information will be valuable for designing new therapies or a vaccine against HCV.