Hepatitis B virus X protein (HBx)-induced apoptosis in HuH-7 cells: influence of HBV genotype and basal core promoter mutations

• Published in: Scandinavian journal of gastroenterology 2004-05, Vol.39 (5), p.478-485
• Main Authors: Kanda, T., Yokosuka, O., Imazeki, F., Yamada, Y., Imamura, T., Fukai, K., Nagao, K., Saisho, H.
• Format: Article
• Language: English
• Subjects: Apoptosis; Apoptosis - genetics; Biological and medical sciences; carcinogenesis; Carcinoma, Hepatocellular - genetics; Carcinoma, Hepatocellular - pathology; Cell Line, Tumor; Cell Survival; Gastroenterology. Liver. Pancreas. Abdomen; Genotype; HBV genotype; HBx; Hepatitis B virus - genetics; Human viral diseases; Humans; Infectious diseases; Kruppel-Like Factor 6; Kruppel-Like Transcription Factors; Liver Neoplasms - genetics; Liver Neoplasms - pathology; Medical sciences; mutagenesis; Mutation; Proto-Oncogene Proteins - genetics; Trans-Activators - genetics; Trans-Activators - physiology; Transfection; Viral diseases; Viral hepatitis
• ISSN: 0036-5521; 1502-7708
• DOI: 10.1080/00365520310008719

Background: Hepatitis B virus (HBV) infection is a serious, world-wide problem. HBV genotype and basal core promoter (BCP) mutations affect the clinical course of HBV-infected patients. BCP mutations also lead to mutations at HBV X protein (HBx) codons 130 131. The functional significance of naturally occurring variants of human HBx remains largely unknown. The purpose of the study was to investigate whether HBV genotypes or double mutations affect HBx-induced apoptosis. Methods: We constructed genotype A, B, C, and D HBx expression vectors and HBx expression vectors with double mutations at HBx codons 130K and 131V or positions 130M and 131I using site-directed mutagenesis. A transient expression system in HuH-7 cells was established and this model was utilized to address the effect of HBx on cell viability. Results: HBx-transfected cells showed a dose-dependent decrease in cell viability by MTS assay. A subset of cells expressing HBx underwent apoptosis according to terminal transferase enzyme-mediated end labeling of DNA and caspase-3 activity. This study demonstrated that HBx can induce cell death by apoptosis in a dose-dependent manner and that HBV genotypes and double mutations did not affect HBx-induced apoptosis. Conclusions: HBV genotypes and mutation of two amino acids directly adjacent to the conserved Kunitz domain essential for transcription activating activity of HBx did not change the pro-apoptotic activity of HBx. Further study is needed to determine whether HBV genotypes and double mutations have any effect on the function of HBx.