The study of the expression of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) by pres deletion mutants and an occult strain of subgenotype A1 hepatitis B virus (HBV) using subcellular fractionation

Abstract

Hepatitis B surface antigen (HBsAg) expression is a vital diagnostic marker in the course of HBV infection. Its presence in the serum for longer than six months heralds chronic infection. The large, middle and small hepatitis B surface protein (LHBs, MHBs and SHBs, respectively), are three types of HBsAg that are expressed from the preS/S opening reading frame (ORF), with varying lengths since they are translated from separate in-frame initiation codons. LHBs comprises the preS1, the preS2, and the S domains; MHBs comprises the preS2 and the S domains whereas the SHBs contains the S domain. Strains of HBV, with deletions in the preS1 and/or preS2 regions, have been identified in HIV-infected individuals and are also frequent in HBV strains from hepatocellular carcinoma (HCC) patients. These deletions have been shown to be a risk factor for the development HCC. The objective of the study was to functionally characterize deletion mutants and an occult strain isolated from South African HIV-infected adults. In this study, previously constructed plasmids shown to express HBV DNA and proteins for wild-type (A1 WT SW), occult strain (SHH193A), and with different HBsAg mutations: preS1 & preS2 deletion mutant (SHH011A), preS2 deletion mutants (SHH045A and SHH167A) of subgenotype A1 were transfected in Huh7 cells. Intracellular protein expression was determined following subcellular fractionation and subsequent immunoblotting. Extracellular protein expression was determined by ELISA. The subcellular localization, extracellular expression and the ratios of the different types of HBsAg was determined for the occult and preS deletion mutant strains relative to the wild-type strain. This study demonstrated that the ratio and subcellular localization of the occult and preS deletion mutant strains were affected relative to the A1 wild-type. Furthermore, we found that HBsAg from the occult strain aggregated primarily in the membrane-bound fraction, suggesting that these proteins were retained in this cellular compartment. The retention of HBsAgs in the ER, can result in ER stress and lead to reactive oxygen species accumulation, which can trigger oxidative DNA damage and lead to hepatocarcinogenesis. These findings may provide a further explanation as to why the occult and preS deletion mutants may contribute to an increased hepatocarcinogenic potential and/or the HBsAg-negative phenotype.