Deletion and Functional Analysis of Hepatitis B Virus X Protein: Evidence for an Effect on Cell Cycle Regulators

Abstract

Background/Aims: The hepatitis B virus X protein (HBx) is a viral trans-activator that plays a crucial role in pathogenesis of hepatocellular carcinoma (HCC) via an unknown mechanism. The role of HBx in modulating cell proliferation and programmed cell death is replete with controversies. Thus, the goal of this study was to elucidate the effect of HBx and its deletion mutants on cell cycle progression in human hepatoma cells. Methods: Huh7 cells transfected with either full-length or truncated HBx were tested for their mitogenic potential based on their effect on the expression of key cell cycle-related proteins (p27, cyclin D1, p21, and p53) and pro-apoptotic proteins such as cleaved poly (ADP-ribose) polymerase (PARP) and Bax. Western blotting and immunofluorescence techniques were applied to detect changes in the expression levels and intracellular localization, respectively, of the investigated proteins. Also, Quantitative real-time PCR (qRT-PCR) was used to detect changes in RNA levels. Results: An increased anchorage-independent growth of cells transfected with HBx-WT and its deletion mutants was observed. The cell cycle regulatory molecules were differentially modulated by full-length HBx (1-154) and its different N- and C-terminal truncated forms (HBx (31-154), HBx (61-154), HBx (1-94), and HBx (61-124)). An enhanced modulation of p27, p21, and cyclin D1 was associated with HBx (1-154), whereas p53 expression was significantly inhibited by HBx (61-124). Similarly, the expression of cleaved PARP and Bax was efficiently suppressed by HBx (1-94) and HBx (61-154). Conclusion: The HBx-WT and its mutants play a critical role in the pathogenesis and progression of HCC by modulating cell cycle regulatory proteins.