A valine to phenylalanine mutation in the precore region of hepatitis B virus causes intracellular r

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http://www.blackwell-synergy.com/doi/abs/10.1111/j.1872-034X.2007.00315.x

Hepatology Research 38 (6) , 580–592 doi:10.1111/j.1872-034X.2007.00315.x

Abstract

Original Article

A valine to phenylalanine mutation in the precore region of hepatitis B virus

causes intracellular retention and impaired secretion of HBe-antigen

Chien Yu Chen,11MRC/University Molecular Hepatology Research Unit, Department of

Medicine and Carol Crowther,22Hepatitis B Virus Research Unit, Department of

Molecular Medicine and Haematology, University of the Witwatersrand,

Johannesburg, South Africa C. Kew11MRC/University Molecular Hepatology

Research Unit, Department of Medicine and and Kramvis11MRC/University

Molecular Hepatology Research Unit, Department of Medicine andProfessor

Kramvis, Department of Internal Medicine, University of the Witwatersrand, 7

York Road, Parktown, Johannesburg 2193, South Africa. Email:

.Kramvis@... 1MRC/University Molecular Hepatology Research Unit,

Department of Medicine and 2Hepatitis B Virus Research Unit, Department of

Molecular Medicine and Haematology, University of the Witwatersrand,

Johannesburg, South Africa

Professor Kramvis, Department of Internal Medicine, University of the

Witwatersrand, 7 York Road, Parktown, Johannesburg 2193, South Africa. Email:

.Kramvis@...

Abstract

Aim: 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.

Conclusion: The formation of aggresomes, as a result of the G1862T mutation, may

play a contributory role in HBV-induced liver disease.

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