Mechanism of Hepatitis C Virus Core Protein Domain 3 in Hepatocyte Lipid Accum

描述(由申请人提供)： 这项提案将为我提供资源，以进一步推进我最初的K08提案中概述的研究。我们一直在研究丙型肝炎核心蛋白在细胞内脂质堆积中的作用，以确定所需的特定病毒因子。我们已经确认：结构域3是细胞内脂质积累的必要条件和充分条件，结构域3中的序列在不同基因型之间的微小变化会导致脂肪积累的显著差异，结构域3的表达会导致游离胆固醇和甘油三酯的积累。在这项提议中，我将检验这样的假设：来自丙型肝炎病毒核心蛋白的结构域3-裂解产物改变胆固醇的运输并指导胆固醇的隔离。为了验证这一假说，我提出了以下具体目标：1)确定丙型肝炎病毒核心区3从结构域1-2裂解后的转运和持久性；2)阐明丙型肝炎病毒核心区3介导的胆固醇在肝细胞中积聚的机制。为了追踪结构域3的转运，我们将使用瞬时转染的表达表位标记的结构域3的肝细胞，结合免疫荧光显微镜和亚细胞梯度离心法来确定结构域3的分布。通过脉冲标记实验，我们将确定结构域3的隔室特异性稳定性，并进行比较研究，以评估不同基因型的结构域3的稳定性差异。我们将利用荧光胆固醇前体和类似物结合化学抑制剂来系统地确定Domain3[SIC]是如何改变胆固醇运输、合成和类固醇反应调节的。我们将通过确定结构域3[SIC]的表达是否调节胆固醇合成和修饰的中心酶的表达和功能来补充这些研究。总之，这些实验合理地建立在我们最初应用的核心目标之上，并将为丙型肝炎病毒的细胞内生命周期、脂肪变性的发病机制提供新的分子洞察力，并最终可能揭示所有基因类型的结构域3导致脂肪堆积的共同途径。一旦获得这些结果，我们可以在未来朝着以下方向前进：1)确定潜在的治疗方法作为抗丙型肝炎病毒的非基因型药物，2)详细研究某些宿主条件(肥胖和糖尿病)如何改变已确定的调控途径并加剧丙型肝炎病毒诱导的脂肪变性，以及3)启动细胞内信号通路的研究，这些信号通路除了脂肪变性外，还调节纤维化和癌变的发展。 与公共卫生相关：丙型肝炎病毒(丙型肝炎病毒)是国内和世界范围内的一个众所周知的公共卫生问题。随着最近发现的在体外产生病毒的JFH克隆，我们对丙型肝炎病毒细胞内生命周期的具体细节的了解有了很大的提高。很明显，脂质的积累和病毒蛋白，特别是核心蛋白与脂滴的相互作用是丙型肝炎病毒复制、组装和释放过程中不可或缺的步骤。假设在慢性丙型肝炎病毒感染中观察到的相当频繁的脂肪变性或脂肪肝是在满足丙型肝炎病毒的生殖要求后随着时间的推移过量脂肪的积累。

DESCRIPTION (provided by applicant): This proposal will provide me with resources to further advance the research outlined in my initial K08 proposal. We have been examining the role of Hepatitis C Core protein in intracellular lipid accumulation to identify the specific viral factors that are required. We have identified that: domain 3 is necessary and sufficient for intracellular lipid accumulation, small changes in sequence within domain 3 between genotypes lead to significant differences in the lipid accumulation and domain 3 expression results in free cholesterol and triglyceride accumulation. In this proposal, I will test the hypothesis that: the domain 3- cleavage product from the HCV Core protein alters cholesterol trafficking and directs cholesterol sequestration. In order to test this hypothesis, I have laid out the following specific aims: 1) To determine the trafficking and persistence of HCV Core domain 3 after its cleavage from domains 1-2 and 2) To elucidate the mechanism of HCV core domain 3-mediated cholesterol accumulation in hepatocytes. To trace domain 3 trafficking, we will use transient transfected hepatocytes expressing epitope-tagged domain 3 constructs in conjunction with immunofluorescence microscopy and subcellular gradient centrifugation to determine the distribution of domain 3. Through pulse labeling experiments, we will determine the compartment-specific stability of domain 3 and perform comparative studies to assess stability differences between domain 3 of different HCV genotypes. We will utilize fluorescent cholesterol precursors and analogs combined with chemical inhibitors to systematically determine how domain3 [sic] changes cholesterol trafficking, synthesis and sterol-responsive regulators. We will complement these studies by determining if domain3 [sic] expression regulates the expression and function of enzymes central to cholesterol synthesis and modification. Together, these experiments rationally build on the core aims of our initial application and will provide new molecular insights into the intracellular life cycle of HCV, the pathogenesis of steatosis and may ultimately uncover the common pathway by which domain 3 constructs from all genotypes cause lipid to accumulation. Once these results are achieved, we can move in the following directions for the future: 1) identify potential therapeutics as genotype-independent agents against HCV, 2) examine in detail how certain host conditions (obesity and diabetes mellitus) alter the identified regulatory pathways and exacerbate HCV-induced steatosis and 3) initiate studies of intracellular signaling pathways that regulate the development of both fibrosis and carcinogenesis in addition to steatosis. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a well known public health problem both domestically and around the world. With the recent discovery of the JFH clone that produces virus in vitro, our understanding of the specific details of the HCV intracellular life cycle has increased substantially. It has become clear that the accumulation of lipid and the interaction of viral proteins, specifically Core, with lipid droplets is an integral step in the replication, assembly and release of HCV. It is hypothesized that steatosis, or fatty liver, that is observed with considerable frequency in chronic HCV infection is the accumulation over time of excess lipid after the reproductive requirements of HCV are met.