Expression of envelope proteins by pre-s1/pre-s2 deletion mutants of HBV isolated from Southern African HIV-positive patients
Date
2018
Authors
Simelane, Daniel
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Abstract
Hepatitis B Virus (HBV) pre-S deletion mutants of genotypes B and C have been shown to predispose to hepatocellular carcinoma (HCC). Studies have reported that envelope protein expression can be affected by the deletion mutants, leading to endoplasmic reticulum (ER) stress, apoptosis and hepatic injury. In South Africa, genotype A/subgenotype A1 prevails and it also has a relatively high hepatocarcinogenic potential compared to non-A genotypes. The aim of the study was to determine whether strains of HBV subgenotype A1, isolated from HIV-infected patients, affect envelope protein expression in vitro. The strains included pre-S deletion mutants (also frequently found in HCC patients) and the strain isolated from HBsAg negative HIV positive individual. Different 1.28 mer replication-competent constructs, generated previously, including two wild type subgenotype A1 strains from an HBsAg-positive infection (A12C15; A12C15 ALT9.3), a strain from an occult infection (SHH193A) and 3 different deletion mutants: SHH011A (with deletions in the pre-S1/pre-S2 regions), SHH045A and SHH167A (with deletions in the pre-S2 region) were used.
The plasmids were transfected into Huh7.5 cells and envelope protein expression followed by immunofluorescence using anti-HBs antibody and confocal microscopy on days 1, 3 and 5 post-transfection as well as quantification co-localization of the HBsAg using Zen software (Zeiss). Mean of fluorescence was quantified by Image J software.
All the constructs expressed HBsAg and the HBsAg was located mainly in the cytoplasm for 100% of the transfected cells with a diffuse staining for wild-type (A12C15_OL and A12C15 ALT 9.3) and the 3 deletion mutants (SHH011A, SHH045A & SHH167A). The strain from an occult infection (SHH193A) showed aggregates of HBsAg in the cytoplasm with more extensive accumulation at the perinuclear region of the cells, a finding that is suggestive of envelope protein retention in the cellular compartments,
which prevented secretion leading to the HBsAg-negative phenotype of the patient. In general, the deletion mutants (SHH011A, SHH045A & SHH167A), expressed envelope proteins at comparable amounts to that shown by the wild-type constructs (A12C15_OL and A12C15 ALT 9.3).
Although other groups have shown the expression of the envelope proteins by pre-S deletion mutants of different HBV genotypes, this study is the first to show the expression of the envelope proteins for subgenotype A1 constructs. Immunocytochemistry and high resolution confocal microscopy was successfully used to follow the expression of HBsAg in the secretory pathway. The accumulation of HBsAg in the perinuclear region following transfection with the strain from the occult infection could account for the HBsAg-negative phenotype seen in the patient.
Description
A Dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine, Johannesburg 2018
Keywords
Deletion Mutants, Envelope proteins