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

描述（由申请人提供）： 这份提案将为我提供资源，以进一步推进我最初的K 08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积累中的作用，以确定所需的特定病毒因子。我们已经确定：结构域3是细胞内脂质积累所必需和充分的，基因型之间结构域3内序列的微小变化导致脂质积累的显著差异，并且结构域3表达导致游离胆固醇和甘油三酯积累。在这个建议中，我将测试的假设：从HCV核心蛋白的结构域3切割产物改变胆固醇运输和指导胆固醇螯合。为了验证这一假设，我提出了以下具体目标：1）确定HCV核心结构域3从结构域1-2切割后的运输和持久性; 2）阐明HCV核心结构域3介导的肝细胞中胆固醇积累的机制。为了追踪结构域3的运输，我们将使用瞬时转染的肝细胞表达表位标记的结构域3构建体，结合免疫荧光显微镜和亚细胞梯度离心来确定结构域3的分布。通过脉冲标记实验，我们将确定结构域3的区室特异性稳定性，并进行比较研究，以评估不同HCV基因型结构域3之间的稳定性差异。我们将利用荧光胆固醇前体和类似物与化学抑制剂相结合，系统地确定结构域3 [原文如此]如何改变胆固醇运输，合成和甾醇响应调节剂。我们将通过确定结构域3表达是否调节胆固醇合成和修饰的核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HCV的细胞内生命周期、脂肪变性的发病机制提供新的分子见解，并可能最终揭示所有基因型的结构域3构建体导致脂质积累的共同途径。一旦取得这些成果，我们可以朝着以下方向前进：1）鉴定作为针对HCV的基因型非依赖性试剂的潜在治疗剂，2）详细检查某些主机条件（肥胖和糖尿病）改变已确定的调节途径并加剧HCV诱导的脂肪变性，以及3）启动细胞内信号传导途径的研究，这些信号传导途径调节除了脂肪变性之外的纤维化和癌发生的发展。 公共卫生相关性：丙型肝炎病毒（HCV）是国内和世界各地众所周知的公共卫生问题。随着最近发现的JFH克隆，产生病毒在体外，我们的理解的具体细节的HCV细胞内的生命周期大大增加。已经清楚的是，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HCV复制、组装和释放中不可或缺的步骤。据推测，在慢性HCV感染中观察到的相当频繁的脂肪变性或脂肪肝是在满足HCV的生殖要求后过量脂质随时间的积累。

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.