Mechanistic Studies of HCV E2

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)感染是人类慢性肝病的原因之一，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，丙型肝炎病毒感染变为慢性，可发展为肝硬变和肝细胞癌。目前还没有丙型肝炎病毒疫苗，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%的患者有持续反应。感染的高流行率，缺乏丙型肝炎病毒特异性抑制剂，以及对当前治疗的低应答率，突出了新疗法的重要性。丙型肝炎病毒粒子由一个被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目标宿主细胞是重要的。与其他被包膜的病毒如鼠疫病毒、甲型病毒和其他黄病毒一样，丙型肝炎病毒感染需要病毒和宿主细胞膜之间的融合，然后病毒基因组才能释放到宿主细胞胞浆中。这一关键步骤被认为发生在内体隔室，由低pH值触发，可能需要糖蛋白的结构重排。除了在膜融合中的作用外，E2还被认为是负责靶向的，因为它已经被证明与宿主细胞受体CD81和清道夫受体BI型(SR-BI)结合。尽管使用病毒中和抗体和遗传方法了解E2的功能已经取得了进展，但更深入地剖析E2的机制需要补充生化、生物物理和分子病毒学技术。由于E2蛋白的糖基化和分子内二硫键的存在，生产E2蛋白具有挑战性，因此无法制造足够数量的正确折叠的蛋白质阻碍了进展。最近，我们实验室设计并发表了一种在哺乳动物细胞中表达EE2(E2胞外区)蛋白的新系统，该蛋白保留了病毒粒子上存在的E2的功能。结合我们对EE2的全面生化和生物物理特征，我们已经为更好地确定E2在丙型肝炎病毒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依赖的膜融合中的作用，并探索在丙型肝炎病毒感染的这一关键步骤中可能需要的寡聚和结构重排。综合生物化学、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)确定与丙型肝炎病毒感染相关的E2的生理寡聚体形式；(2)表征E2在感知pH下降中的作用，如病毒在内体酸化过程中遇到的；以及(3)确定E2的结构域组织和在pH变化时可能的重排。这项研究的结果将有助于我们理解病毒进入的机制，并对开发控制丙型肝炎病毒感染的新疗法和疫苗具有重要意义。对HIV糖蛋白的类似研究已经导致了一类新的抗逆转录病毒药物的开发，这种药物可以阻止HIV病毒进入。 公共卫生相关性：目前，2%的人类人口--全球约1.23亿人--慢性感染丙型肝炎病毒，使病毒传播成为一个主要的公共卫生问题。目前还没有针对丙型肝炎病毒的疫苗，而且目前的药物对许多人都没有效果。拟议拨款的目标将使人们更好地了解丙型肝炎病毒如何识别和进入细胞。这些信息将对设计新的治疗方法或抗丙型肝炎病毒疫苗很有价值。

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.