The effect of the accumulation of Hepatitus B virus e-antigen precursor on cell viability

[摘要] The G1862T mutation in the bulge of the RNA encapsidation signal, in the precoreregion of hepatitis B virus, results in reduced expression of HBeAg and accumulation ofthe HBeAg precursor in the endoplasmic reticulum (ER)/Golgi apparatus of the cell.This accumulation can disturb the functioning of the ER and lead to the ER stressresponse that can affect various cellular pathways, in turn affecting cell viability. The aimof this study was to determine whether apoptosis or necrosis occurred when culturedHuh7 cells were transfected with a plasmid expressing the G1862T mutation. Plasmidconstructs, with and without the G1862T mutation, were used to transfect cells. Todifferentiate between necrosis and apoptosis cells were stained with propidium iodide orYO-PRO-1®, respectively. These were analyzed quantitatively using flow cytometry andqualitatively using confocal microscopy. Confocal microscopy, using monoclonal anti-HBe and the Hoechst stain, was performed to ensure that apoptosis was present as a resultof the accumulation of the G1862T mutant HBeAg precursor. Caspase profiling wascarried out using a fluorogenic-based assay. When cells were transfected with wild-typeplasmid, necrosis predominated over apoptosis. Apoptosis predominated when the cellswere transfected with the G1862T mutant plasmid. The highest levels of apoptosisoccurred at 72 hours post-transfection. Confocal microscopy revealed the co-localizationof aggregates of mutant HBeAg precursor with apoptotic nuclei. Transfection withG1862T mutant plasmids resulted in significant differences in the expression of caspase3, 8, and 9 relative to the wild-type, at 48 and 72 hours post-transfection. Theaccumulation of the G1862T mutant HBeAg precursor, in the ER/ Golgi compartment,leads to apoptosis and affects the levels of caspase expression.

[发布机构] University of the Witwatersrand