The Potential Role Of Autophagy In The Pathogenesis Of Hepatitis B Virus Infection

Objective Autophagy serves as an important part of innate immunity to defeat against pathogen invasion.However,hepatitis B virus(HBV)infection could induce autophagosomes formation without the following lysosomal degradation.The study aims to investigate the underlying mechanism by which HBV escapes from the autophagic degradation.Methods Expression of autophagy-related protein 5(Atg5)and microtubule-associated protein light chain 3(LC3)in HepG2 cells,starvation-treated HepG2 cells,HBV plasmid p1.3×HBV(pHBV)transfected HepG2 cells(pHepG2)and HepG2.2.15 cells were measured by western blot,respectively.Autophagosome accumulation in pHepG2 cells and HepG2 cells was observed by transmission electron microscopy.Mice model of HBV infection was established by high-pressure intravenous injection of pHBV,while control group was injected with empty vector.All mice were sacrificed at 96 h after the injection,then liver tissues and serums were collected.Serum HBsAg level was determined using the ARCHITECT i2000 SR HBsAg QT assay,and serum HBV DNA level was detected by Roche COBAS HBV Amplicor MonitorTM assay.In terms of liver tissue,histological features were observed by hematoxylin-eosin(H&E)staining,and expressions of Atg5 and LC3-II were detected by western blot.Moreover,fluorescence confocal microscopy was performed to analyze autophagic flux in pHepG2 cells and HepG2 cells.In addition,HepG2 cells,starvation-treated HepG2 cells,pHepG2 cells and HepG2.2.15 cells were subjected for western blot and semi-quantitative PCR to assess expressions of Ras-related protein 7(Rab7)in protein level and mRNA level,respectively.Results Compared to HepG2 cells,HepG2.2.15 cells,pHepG2 cells and starvation treated HepG2 cells showed markedly elevated LC3-II and Atg5 levels.Intracellular autopahgic vacuoles were observed directly in pHepG2 cells,whereas there were no double membrane vacuoles in HepG2 cells.Furthermore,pHBV injection caused obvious inflammation in mice liver with increases in LC3-II and Atg5 protein levels.Autophagic flux analysis indicated that autophagosome-lysosome fusion was blocked in pHepG2 cells.Meanwhile,expression of Rab7,both in protein and mRNA levels,was dramatically reduced in pHepG2 cells and HepG2.2.15 cells compared with that in HepG2 cells.Whereas,starvation treatment increased the expression of Rab7 in HepG2 cells.Conclusions Our study suggests that HBV could induce autophagosome formation but block their fusion with lysosome to escape virus removal.Functionally,downregulation of Rab7 might be responsible for the autolysosome-lysosome fusion deficiency.In this study,it might help to clarify Rab7 occupies an indispensable place in HBV-induced incomplete autophagic degradation,which provides a therapeutic approach to further manipulation of autophagy in HBV infection therapy.