Functional characterisation of pre S1/pre S2 deletion mutants of hepatitis B virus isolated from Southern Africans

Abstract

Both HBV and HIV are hyperendemic in sub-Saharan Africa, and there is a correspondingly high incidence of hepatocellular carcinoma (HCC) in this region, contributing to a high burden of disease. In regions where HBV genotypes B and C prevail, a strong relationship exists between the rapid and more likely development of HCC and infection with HBV containing deletions in the preS region. Similar preS deletion mutants have been detected in southern African and Indian HCC patients infected with subgenotype A1. Disturbingly in a cohort study conducted in Mpumalanga, South Africa by our team, equivalent deletion-mutants were detected in 5 treatmentnaïve HBV-HIV coinfected patients. Thus, the aim of this study was to characterize the quasispecies of HBV in these patients and to construct plasmids containing preS deletion-mutants in a subgenotype A1 backbone, in order to functionally characterize them in vitro. Such studies may determine how these preS deletions contribute to the working model of hepatocarcinogenesis and explain the high hepatocarcinogenic potential of subgenotype A1. The quasispecies populations of HBV deletion mutants isolated from four HIV-positive patients were analysed phylogenetically, using both Neighbor-Joining and Bayesian methods. It was found that the preS deletion mutants represented the majority population, as 70% or more of clones in each of the four patients were sequentially highly similar to the respective parental strains. In addition to the major populations in each patient, minor populations were identified in the quasispecies. The overlength subgenotype A1 wild-type replication competent plasmid, was successfully altered to remove an unwanted XbaI site. This altered construct served as a positive control to test whether the removal of this restriction site would affect replication and viral protein expression of this construct compared to the original plasmid. This plasmid was then successfully used as the backbone to construct three overlength deletion mutant constructs. Using a new strategy a 784 bp fragment flanking preS deletions from each of 4 patients, was inserted into the subgenotype A1 backbone. Three fragments were derived from isolates with preS deletion mutants and one fragment, without the deletion, was isolated from a patient with occult HBV infection. The resulting plasmids together with the appropriate control plasmids were used to transfect Huh7 cells in culture. Viral replication was followed at days 1, 3 and 5 using enzyme linked immunosorbent assay (ELISA) for hepatitis B surface antigen (HBsAg) and Hepatitis B e antigen (HBeAg). All deletion mutants were shown to express HBsAg and HBeAg at levels comparable to the controls, with the highest levels on day 3. There were no significant differences in the expression of HBeAg in Huh7 transfected cells between the deletion-mutant constructs and the positive controls. The HBV viral loads (VL) were measured at the same time points, using real-time quantitative PCR (qPCR). Both the extracellular (supernatant) and intracellular (lysate) compartment of the Huh7 cells were tested. Furthermore, to clarify whether encapsidated virus was being secreted, an immunocapture technique on the supernatant was employed prior to DNA extraction and qPCR. In a similar manner to the ELISA experiments, qPCR measurement of VL showed that despite the replacement of the 784 bp fragment with a fragment containing deletion mutations, all constructs were producing detectable amounts of HBV DNA suggesting that viral replication was occurring. Analogous to the HBsAg results, the highest VL was seen in all constructs on day 3 post transfection in both the supernatant and lysate experiments. Cell-associated (lysate compartment) VL was not particularly increased when compared to the VL measured in the supernatant, which does not indicate an inability to secrete the mature virions, nor an accumulation of viral DNA within the cell. Negligible HBsAg expression was observed on days 1, 3 and 5 following transfection with the overlength construct that contained the 784 bp fragment derived from a patient with occult HBV infection. The VL following transfection with this construct was higher than both positive controls at all three time points, in both the supernatant and lysate compartment. This is again consistent with the clinical characteristics of the patient, which also had high viral loads. This finding is novel, since the in vitro experiments mimicked the phenotype of occult infection seen in the patient in vivo. As far as we are aware, we are the first group to have constructed plasmids with deletion mutants and an occult mutant in a subgenotype A1 backbone and to show that they express viral proteins and viral DNA following transfection into HuH7 cells. These constructs will be important resource for further research into the high hepatocarcinogenic potential of subgenotype A1.