Investigation of the Oncogenic Potential of HBV Drug Resistant Mutants

Hepatitis B virus (HBV) is an oncogenic DNA virus. Several viral proteins, including X protein, pre-S deletion mutants, and pre-S/S truncation mutants, have been proposed to have significant oncogenic potential. Because of rapid advance and successful development of new antiviral agents followed by extensive clinical usage of these drugs, novel drug resistant mutants have emerged. Furthermore, our recent study showed that other antiviral agent-related, but not drug resistant mutants could also develop (Hsu and Yeh et al., Gastroenterology 2007). Although antiviral therapy could reduce the risk of hepatocellular carcinoma (HCC) through long-term suppression of HBV replication, lines of evidence suggested that such effect could be compromised in patients harboring drug resistant mutants. Since it is the drug-related HBV mutants but not the wild type HBV that constitute major viral population in patients receiving long-term antiviral therapy, it is pivotal to understand the oncogenic potential of the drug-related HBV mutants. This issue is of particular importance in Taiwan where HBV antiviral agents are widely used. This proposal aimed at investigating the oncogenic potential of antiviral agents-related HBV mutants. Specific aims: 1. Identification of antiviral agent-related HBV mutants developed in HCC patients. Serum and tissue samples obtained from patients who received oral antiviral therapy against HBV but the treatment failed to prevent development of HCC will be collected. Sequence analysis will be performed for HBV X and pre-S/S regions to identify novel HBV mutants. Our preliminary experiments identified several pre-S/S truncation mutants. 2. Evaluation of the oncogenic potential of the aforementioned HBV X and pre-S/S mutants. 2-1. Site-directed mutagenesis experiment will be performed to generate target antiviral drug related HBV mutations in a controlled wild type construct. 2-2. Stable transformants expressing these mutants will be established for characterization of cell behavior including proliferation, ER stress, invasion, and apoptosis. 2-3. Transactivation of oncogene promoters by these mutants will be evaluated. 2-4. Transformation and tumorigenicity assays will be performed using immortalized cells. 3. Establishment of transgenic mice expressing newly identified oncogenic HBV X or pre-S/S mutants. In our preliminary data, we have identified a potentially oncogenic mutant, rtA181T/sW172*, in lamivudine treated, HCC patients. Unless other more potent oncogenic mutants are identified, this mutant will be used to generate transgenic mice. In the first 2 years, pre-S/S truncation mutants were found to enhance oncogenesis in NIH3T3 cells (published in Antivir Ther 2008;13:875; Antivir Ther 2009;14:249 and Antivir Ther 2010;15:471[review]). In the 3rd year, we will examine the oncogenic potential of the rtA181T/sW172* mutant in transgenic mice.

Project ID:PC10006-0026
External Project ID:NSC100-3112-B182-003