Hepatitis B virus X protein (HBx)-induced apoptosis in HuH-7 cells: influence of HBV genotype and basal core promoter mutations.
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.
Kanda
T
T
Dept. of Medicine and Clinical Oncology, Graduate School of Medicine, Health Sciences Center, Chiba University, Chiba, Japan. kandat-cib@umin.ac.jp
Yokosuka
O
O
Imazeki
F
F
Yamada
Y
Y
Imamura
T
T
Fukai
K
K
Nagao
K
K
Saisho
H
H
D017209:Apoptosis / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival; D005838:Genotype; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D008113:Liver Neoplasms / Q000235:genetics* / Q000473:pathology; D009154:Mutation; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics* / Q000502:physiology*; D014162:Transfection; D054334:Viral Regulatory and Accessory Proteins
C105465:KLF6 protein, human; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D011518:Proto-Oncogene Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein