Hepatitis B virus X protein (HBx), human voltage-dependent anion channel 3 (HVDAC3) and mitochondria.

摘要

Hepatitis B virus (HBV) causes acute/chronic hepatitis. HBV infection is strongly associated with hepatocellular carcinoma (HCC). Hepatitis B viral X protein (HBx) is a regulatory protein encoded by HBV genome. The mechanism by which HBx exerts its effects on many cellular functions is not clearly understood. A partial cDNA of HVDAC3, a novel member of the human voltage-dependent anion channel (HVDAC) family, has been shown to interact with HBx in a yeast two hybrid assay by a previous postdoctoral fellow in this laboratory. My thesis work focused on the characterization of HBx's association with VDAC and mitochondria.; GST pull down assays and in vivo coimmunoprecipitation analyses established that HBx and HVDAC3 directly interact in vitro and in vivo. Double immunofluorescence, confocal microscopic analysis, and subcellular fractionation showed that HBx and HVDAC3 colocalized to mitochondria in transiently transfected human hepatoma cells. In addition, HBx expression in Huh7 cells led to a decrease in mitochondrial membrane potential and an increase in cellular reactive oxygen species. These results suggest that HBx not only localizes to mitochondria but also significantly affects its functions.; Various truncation and substitution mutations of HBx cDNA were generated to map regions necessary for mitochondrial association. Double immunofluorescence and confocal microscopic analyses of those mutants indicated that a putative transmembrane region (aa 54–70) is necessary for mitochondrial association and additional amino acids in putative alpha helical regions (aa 75–88 and as 109–131) also potentially contribute to this a association. The majority of HBx localizes to the outer mitochondrial membrane as determined by trypsin sensitivity assay and alkaline treatment.; Mitochondria is a key organelle that controls various cellular events including energy metabolism and apoptosis. The subcellular localization of HBx to mitochondria and its effects on the organelle may explain previously reported pleiotropic functions such as transcription transactivation, apoptosis and activation of signal transduction pathways. The observations described in this thesis, raise the possibility that HBx may regulate the mitochondrial physiology in the HBV-infected liver. These activities may potentially contribute to the liver disease pathogenesis associated with the HBV infection.