Effect of hepatitis B virus rtA181T mutation on autophagy of Huh7 hepatoma cells

Objective To investigate the effect of reverse transcriptase A181T( rtA181T) mutation in hepatitis B virus( HBV) on autophagy of Huh7 hepatoma cells. Methods Huh7 hepatoma cells were transfected with wild- type( WT) 1. 3mer HBV plasmids( WT group),1. 3mer HBV rt A181 T mutant plasmids( 181 T group),or WT and rt A181 T mutant plasmids at a ratio of 1∶ 1( WT + 181 T group). Western blot and immunofluorescence assay were performed to measure the expression of intracellular HBV surface protein( HBs),and ELISA was used to measure the expression of extracellular HBs Ag. Western blot was used to measure the expression of endogenous autophagy- related proteins microtubule- associated light chain 3- II( LC3- Ⅱ) and P62,and Real time PCR was used to measure the mRNA expression of autophagy- related genes Atg5 and Beclin1. After transfection with green fluorescent protein- LC3,immunofluorescence assay was used to measure autophagy particles. An analysis of variance was used for comparison of continuous data between multiple groups and the LSD- t test was used for further comparison between any two groups. Results Immunofluorescence assay showed that there was expression of HBs in the 181 T group,WT group,and WT + 181 T group,and the 181 T group had significantly higher expression of intracellular HBs than the WT group and WT + 181 T group. The results of ELISA showed that there were significant differences in the level of HBs Ag in cell culture supernatant between the three groups( F = 66. 739,P < 0. 001),and the 181 T group had a significantly lower level of extracellular HBs Ag than the WT group and WT + 181 T group( 0. 111 ± 0. 056 vs 3. 157 ± 0. 490 /2. 240 ± 0. 797,both P < 0. 001). The WT group,181 T group,and WT + 181 T group all showed the aggregation of LC3- II autophagy particles; compared with the WT group and WT + 181 T group,the181 T group had significantly higher expression of LC3- II and significantly lower expression of P62( both P < 0. 05). The 181 T group had significantly higher mRNA expression of autophagy- related genes Atg5 and Beclin1 than the WT group and WT + 181 T group( P < 0. 05).