Abstract
The Hepatitis B Virus (HBV) is a primary cause of chronic liver disease in humans. An aftermath of infection may be hepatocellular carcinoma (HCC). The time required for development of carcinoma varies, but there is a significant correlation with the chronic carrier state. The Woodchuck Hepatitis Virus (WHV) is a virus similar to the HBV, and is an important model for studying HBV pathogenesis. In the woodchuck model, infection of neonate woodchucks with WHV leads to development of HCC in 100% of chronic carriers. These hepadnaviruses are related to the retrovirus family, which use reverse transcriptase for replication, integrate into the host genome and encode four structural proteins and one transcriptional activator (X-ag) on the coding strand. The X-ag is actively expressed in the replication cycle of the virus. However, in the antisense orientation, an ORF6 overlaps the X-ag and contains a splice site that if integrated into the host DNA, expression of a protein may ensue. The ORF6 protein is approximately 22kD in size and positive in charge. The objective of this project is to express the ORF6 gene, referred to as WHAsi, in eukaryotic cells. This will be accomplished by constructing a fusion protein of the green fluorescent protein (N-term) and WHAsi (C-term). This construct will be transfected into Chinese hamster ovary (CHO) cells and localization of the fusion protein will be tracked using fluorescence microscopy. The protein is hypothesized to localize to the nucleus which would support its role in development of HCC by interacting with host DNA. Subsequent research may examine the influence of the gene during embryonic development.
