Molecular characterization of the hepatitis C virus core protein

Abstract: Hepatitis C virus (HCV) is an RNA virus that causes chronic infection, which can lead to hepatocellular carcinomas in humans. Besides liver diseases, the chronic HCV infection causes a broad spectrum of extrahepatic complications such as lymphoproliferative, metabolic and autoimmune disorders. Notably, HCV encoded core (C) protein is the major virion component that is involved in the oncogenesis and immune subversion. Therefore, detailed molecular characterization of the C protein provides a rational starting point for identification of novel countermeasures against pathogenic HCV infections. In this thesis we have investigated the suppressive effect of the C protein on T cell functions in immortalized cell lines and clinical samples.In paper I, we found that the expression of the C protein enhanced overall tyrosine phosphorylation in immortalized T cells. Interestingly, stable expression of the C protein specifically reduced accumulation of the tyrosine phosphatase SHP-1 mRNA. Our detailed bisulfite sequencing (BS) studies revealed that the SHP-1 P2 promoter was particularly hypermethylated at CpG1 and proximal islands in these cells. In paper II, we presented a new high-throughput next generation bisulfite sequencing (NGS-BS) protocol for the analysis of locus specific CpG methylation in HCV-infected cells using SHP-1 P2 as a model promoter. In line with our data from the BS, the NGS-BS method showed similar methylation profile at CpG1 island in immortalized cells. Strikingly, peripheral blood mononuclear cells (PBMCs) isolated from healthy controls and HCV-positive (HCV+) patients, showed much lower levels of methylation at the CpG1 island with no significant difference in DNA methylation pattern. In paper III, we investigated the mechanism of the C protein-mediated release of Ca2+ from intracellular stores. We identified two distinct regions in the N- and C-terminal parts of the protein that were essential for activation of the Ca2+/NFAT pathway. Of these, the N-terminal region was required for self-association of the C protein into nucleocapsid-like structures whereas the C-terminal part is essential for anchoring the protein to the ER-membrane. In paper IV, we presented a PCR based diagnostic method for the specific detection of positive and negative strand HCV RNA using primers with a non-viral tag. The method was evaluated by analysing the plasma and PBMC samples from chronic HCV+ patients.Taken together, our studies provide more detailed molecular characterization of the HCV C protein functions in immortalized as well as in HCV+ T cells. Importantly, specific DNA methylation pattern of the SHP-1 gene promoter may function as a potential prognostic marker for the disease progression in HCV-induced tumors. In addition, our updated PCR-based HCV diagnostic method may provide a more specific tool to monitor HCV infections in minor reservoirs.