35.    The replication of the main Hepatitis B virus subgenotypes in Argentina induces an            incomplete autophagic process in human hepatocytes

Introduction. Hepatitis B virus (HBV) causes acute and chronic liver infections. HBV genotypes and particular mutations were described to affect the natural course of HBV chronic infection. F1b and F4 subgenotypes (sgts) produce most of the infections in Argentina. Mutations at the preCore and at basal core promoter (BCPdm) regions are also associated with mechanisms of pathogenesis. Objectives. Analyze whether F1b and F4 HBV sgts replication induces autophagy in human hepatoma cells and study the effect of mutations in the modulation of this process. Methods. Huh-7 cells were transfected with full-length linear F1b and F4 HBV genomes {wild type sequence (wt), BCPdm (A1762T/G1764A) and preCore (G1896A) mutations}. Autophagy was studied by: detection of LC3 puncta by immunofluorescence, transmission electron microscopy, acridine orange staining and expression of LC3 and p62 by Western Blot. Results. Replication of both F1b and F4 HBV sgts induced autophagosome formation, demonstrated by: i) the accumulation of LC3-positive puncta, ii) conversion from the cytosolic form of LC3 (LC3-I) to the autophagosome-associated form (LC3-II), and iii) detection of the typical double membrane vesicles by electron microscopy. The induction of autophagosomes was significantly higher for BCPdm than for the wt, for both sgts, and for sgtF1b preCore mutant in relation to sgtF1b wt. When analyzing the
autophagic flux, HBV replication increased p62 levels and decreased the percentage of acidic vacuoles. These results suggest that viral replication can induce early stages of autophagy but blocks later stages. Conclusions. F1b and F4 HBV sgts replication can induce autophagy in human hepatocytes. However, the autophagic flux is blocked and results in the inhibition of autophagic protein degradation. This inhibition of autophagy flux might be due to an impairment of lysosomal acidification in hepatoma cells. BCPdm and sgtF1b preCore mutants induce higher levels of autophagy than wt virus. These results contribute to describe mechanisms of HBV pathogenesis in chronic infections.