A valine to phenylalanine mutation in the precore region of hepatitis B virus causes intracellular retention and impaired secretion of HBe-antigen.
OBJECTIVE
Hepatitis B virus (HBV) e antigen (HBeAg) is translated from precore mRNA as a precore/core protein, which is post-translationally modified to give rise to the protein that is secreted into the serum. The G1862T mutation in HBV occurs in the bulge of the encapsidation signal within the pregenomic RNA. When the precore mRNA is translated, this mutation results in a valine to phenylalanine substitution at the -3 position to the signal peptide cleavage site at the amino end of the precursor protein. The aim of this study was to determine whether this mutation could affect HBV replication and/or HBeAg expression.


METHODS
Following transfection of Huh 7 cells, HBV replication was followed using real time polymerase reaction (PCR) and expression of HBeAg expression was monitored using confocal microscopy.


RESULTS
HBV replication was reduced when this mutation was introduced into genotype D but not into genotype A replication-competent constructs. Using mutant HBeAg-expressing plasmids, we demonstrated a 54% reduction in HBeAg secretion relative to the wild type. Confocal microscopy demonstrated that the mutant HBeAg accumulated in the endoplasmic reticulum, endoplasmic reticulum intermediate compartment and Golgi. These aggregates of mutant protein increased in size following treatment of the cells with a proteasome inhibitor, MG132, and had the hallmark features of aggresomes. They attracted ubiquitin, heat shock proteins and proteasomes and were isolated from the cytosol by the intermediate filaments, vimentin and cytokeratin.


CONCLUSIONS
The formation of aggresomes, as a result of the G1862T mutation, may play a contributory role in HBV-induced liver disease.
Chen
Chien Yu
CY
MRC/University Molecular Hepatology Research Unit, Department of Medicine, University of the Witwatersrand, Johannesburg, South Africa.
Crowther
Carol
C
Kew
Michael C
MC
Kramvis
Anna
A