Hepatitis B Virus X Protein Sensitizes Cells to Starvation-Induced Autophagy via Up-regulation of Beclin 1 Expression

Human beclin 1 is the first identified mammalian gene to induce autophagy. It is commonly expressed at reduced levels in breast tumors; however, it is overexpressed in hepatitis B virus (HBV)-infected cancerous liver tissues. To expose the possible mechanism and biological significance of this up-regulation of beclin 1, we investigated the regulation of beclin I expression by HBV x protein (HBx) in hepatic or hepatoma cell lines. Here, we showed that enforced expression of HBx by transfection technology results in the up-regulation of the endogenous messenger RNA (mRNA) and protein levels of Beclin I in the tested cells. Using a luciferase- reporter assay, we demonstrated that HBx transactivates beclin I promoter activity in a dose-dependent manner. The promoter region of the beclin I gene identified in this study is located at nt -277/ +197 and has the maximum transcriptional activity. HBx-mediated up-regulation of beclin I expression might be direct, that is, via its promoter. Furthermore, the cells that transiently or stably expressed HBx showed an enhanced accumulation of vacuoles carrying the autophagy marker LC3 as compared with the control cells, which was induced by nutrient starvation, indicating HBx-enhanced autophagy. Moreover, this enhanced autophagy occurred in HepG2.2.15 cells that replicate HBV and in cells transfected with HBV genomic DNA, suggesting that HBV infection also causes increased levels of autophagy under starvation conditions. Treatment of cells with beclin I small interfering RNA (siRNA) blocked HBx-enhanced autophagy, demonstrating that the function of HBx in influencing autophagy is Beclin I dependent. Conclusion: Our findings suggest a novel function of HBx in increasing autophagy through the up-regulation of beclin] expression, and this may provide an important mechanism in HBV-infected hepatocytes growing under nutrient-deficient conditions. (HEPATOLOGY 2009;49:60-71.)