Mechanistic Studies of HCV E2

描述（由申请人提供）：丙型肝炎病毒（HCV）感染是人类慢性肝脏疾病的致病因子，全世界有超过1.23亿人（约占人口的2%）受到影响。在许多情况下，丙型肝炎病毒感染是慢性的，可发展为肝硬化和肝细胞癌。目前还没有针对丙肝病毒的疫苗，唯一的治疗方法是聚乙二醇化干扰素。与利巴韦林联合使用，只有50%的患者有持续的反应。感染的高流行率，缺乏HCV特异性抑制剂，以及对当前治疗的不良反应率强调了新疗法的重要性。HCV病毒粒子由一个膜包裹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很重要。与其他包膜病毒（如鼠疫病毒、甲病毒和其他黄病毒）一样，HCV感染需要病毒与宿主细胞膜融合，然后才能将病毒基因组释放到宿主细胞质中。这一关键步骤被认为发生在内体室，由低pH触发，可能需要糖蛋白的结构重排。除了在膜融合中的作用，E2被认为是负责靶向，因为它已被证明结合宿主细胞受体CD81和清道夫受体B类I型（SR-BI）。尽管利用病毒中和抗体和遗传方法了解E2的功能取得了进展，但更深入的E2机制解剖需要补充的生化、生物物理和分子病毒学技术。由于E2蛋白的重糖基化和分子内二硫键的存在，E2蛋白的生产具有挑战性，因此无法制造足够数量的适当折叠蛋白阻碍了进展。最近，我们的实验室设计并发表了一种在哺乳动物细胞中产生eE2 （E2外畴）蛋白的新表达系统，该蛋白保留了病毒粒子上E2的功能。结合我们对E2的全面生化和生物物理表征，我们为更好地确定E2在HCV感染中的功能作用奠定了基础。在本研究中，我们将重点评估E2在触发低ph依赖性膜融合中的作用，并探索HCV感染中这一关键步骤所需的可能的寡聚物和结构重排。一个整合生化、生物物理、遗传和分子病毒学技术的多学科实验计划将用于：(1)确定与HCV感染相关的E2的生理寡聚形式；(2)表征E2在感知pH值下降中的作用，正如病毒在内体酸化过程中遇到的那样；(3)确定E2的结构域组织和pH变化时可能的重排。拟议研究的结果将大大有助于我们了解病毒进入的机制，并对开发控制HCV感染的新疗法和疫苗具有重要意义。对HIV糖蛋白的类似研究导致了一种新的抗逆转录病毒药物的开发，这种药物可以阻止HIV进入。

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.