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.
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.
