Study of the expression of hepatitis B surface antigen (HBsAg) by different African genotypes of hepatitis B virus (HBV) using subcellular fractionation
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Date
2019
Authors
Pillay, Thanusha
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Abstract
Hepatitis B Virus (HBV) is hyperendemic in sub-Saharan Africa, and of the 292 million people chronically infected with HBV worldwide, approximately 30% reside in Africa – an approximation that is likely underestimated because of poor surveillance. Chronic HBV infection is a significant risk factor for the development of hepatocellular carcinoma (HCC), and correspondingly, the predominant strain circulating in South Africa – subgenotype A1 – is associated with hepatocarcinogenic potential.
Studies have demonstrated that HBV genotypes vary in their clinical manifestation and disease progression, accompanied by differences in expression of HBV viral proteins. Hepatitis B surface antigen (HBsAg) is one of the first virological biomarkers to appear upon the onset of HBV infection, and plays an indispensable role both in the viral life cycle as the major HBV surface protein, and in the clinical setting as an indicator of HBV infection, disease progression and treatment response. HBsAg is the term used to collectively refer to small- (S-HBs), middle- (M-HBs) and large (L-HBs) surface antigens, and distinct differences in the proportion of these antigens have been observed extracellularly and intracellularly. Additionally, variations in the subcellular localisation of HBV viral proteins have been shown to influence the severity of HBV infection and its associated hepatocarcinogenesis. However, no studies have comparatively analysed the differences in S-HBs, M-HBs and L-HBs expression or their subcellular localisation, within the HBV strains predominantly circulating in South Africa. Thus, the aim of the study was to determine the subcellular localisation of HBsAg in HCC cells transfected with wild type subgenotypes A1, A2 and D3, and further characterise the relative proportions of S-HBs, M-HBs and L-HBs expressed intracellularly and extracellularly.
To achieve this, human HCC (Huh7) cells were transiently transfected with 1.28 mer replication competent plasmids containing HBV wild type subgenotypes A1, A2 or D3. Supernatants were collected on days 1, 3 and 5 post-transfection and transfected Huh7 cells were harvested on day 5 post-transfection. Using the Thermo Scientific Subcellular Protein Fractionation Kit for Cultured Cells, Huh7 cell pellets were then fractionated into cytoplasmic, membrane, soluble-nuclear, chromatin-bound nuclear and cytoskeletal fractions. The identity of each fraction was confirmed with fraction-
specific housekeeping proteins and the subcellular fractionation protocol was optimised to provide pure, enriched fractions suitable for downstream Western blotting analysis. HBsAg was detected using a mouse monoclonal antibody targeted towards the S-domain of HBsAg, thus all forms of HBsAg were successfully detected. HBsAg was found to localise exclusively within the membrane fraction with subgenotype A1 exhibiting the lowest expression and subgenotype D3 the highest. To determine the expression of HBsAg extracellularly, supernatants collected on days 1, 3 and 5 from the culture of transfected Huh7 cells were precipitated for HBsAg using a PEG precipitation protocol developed and optimised for this study. Extracellular HBsAg was secreted the highest from cells transfected with subgenotype A1. This result was confirmed with an HBsAg enzyme-linked immunosorbent assay (ELISA).
A novel finding in this study was differences in the proportions and glycosylation patterns of S-HBs, M-HBs and L-HBs extracellularly and intracellularly between the different subgenotypes. It was observed that S-HBs is consistently expressed in the highest proportion, with variable expression in M-HBs and L-HBs expression between the subgenotypes. Furthermore, there was generally a lower proportion of glycosylated S-HBs and biglycosylated M-HBs, with nonglycosylated and glycosylated L-HBs expressing at a ratio of approximately 1:1. The relative proportions of S-HBs to M-HBs and L-HBs as well as their rates of glycosylation have important implications for HBV viral infectivity and the associated host immune response.
An HBeAg ELISA was also performed to determine the rate of HBeAg expression over days 1, 3 and 5 post-transfection. HBeAg expression increased over each consecutive day, and subgenotype A1 was noted to have the highest expression. The comparatively high extracellular HBeAg and HBsAg expression in association with low intracellular HBsAg expression in subgenotype A1 would seem to indicate that this strain has high production and secretion efficiency of HBV viral proteins – characteristics which may allow subgenotype A1 to circumvent the immune system, establish chronic HBV infection and ultimately result in HCC.
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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.
July, 2019