Mechanism Study Of TTPA And Rab7A Affect HBV Expression By Activating MTORCl Signals

ObjectiveHepatitis B virus(HBV)infection has always been a major global health problem.Despite the establishment of HBV vaccine programs since the early 1990s,and the use of effective viral suppression drugs since 1998,there are still more than 70 million people still suffer from chronic hepatitis B infection in China.HBV can maintain the stability of ccc DNA and promote the replication of HBV through the interaction between viral proteins and host factors,leading to chronic infection of HBV,which makes the complete cure of HBV extremely difficult.Therefore,the study of HBV replication mechanism and its therapeutic pathway is of great significance.The previous work in this laboratory showed that ADAR1 down-regulated the expression of TTPA through the RNA editing function to promote the replication of HBV,and initially revealed the anti-HBV effect of TTPA in vitro experiments;in addition,the modified biotin switch assay combined with mass spectrometry analysis were used to screen out the occurrence of sulfhydrated protein,namely Rab7A.Therefore,this study further explores the effects of TTPA and Rab7A on downstream effector molecules,and provides new targets for hepatitis B therapy.MethodsIn this study,full-HBV tg mice were injected with AAV-TTPA and AAV-NC,respectively.The expression levels of serum and intracellular HBV markers after the injection were detected by ELISA,qPCR,IHC.After overexpression of TTPA in HepG2.2.15 cells,western blot method detected the change of phosphorylation level of S6K1-mTORC1 downstream effector molecule.The core promoter region was determined by the dual-luciferase reporter gene experiment,and the possible mechanism affecting TTPA promoter transcription was further verified by ChIP,CoIP and other experiments.The functional sulfhydrylation sites in Rab7A were determined by biotin switch method.After overexpression of Rab7A and mutant plasmids,the expression level of HBV markers in the supernatant and in the cells were detected by ELISA and qPCR,and the GTP binding ability experiment was used to verify the change in the binding ability of Rab7A and mutants.The Western Blot was used to detect the change in phosphorylation level of S6K1,and the CoIP experiment was used to detect whether the binding of Rab7A and mTORC1 was affected by sulfhydrylation.Results1.The values of HBV surface antigen(HBsAg),HBV DNA,AST,ALT in the serum and HBV total RNA and 3.5kb RNA of liver tissues in the transgenic mice were significantly lower than those in the control group(p<0.05).2.Overexpression of TTPA in HepG2.2.15 cells promoted the expression of p-S6K1 and p-AKT(p<0.05),while TTPA mutation(R59W)had no significant effect on p-S6K1,p-AKT and p-ERK.TTPA R59W or RAP treatment had no effect on HBsAg expression.However,mTORC1 inhibitor rapamycin treated or knocked down TTPA significantly reduced the promoting effect of insulin on p-S6K1(p<0.05).3.The core promoter fragment TTPA200 with high activity was initially screened by the double luciferase reporter gene assay,and the transcription complex SP1/HBXIP/HBx was verified to reduce TTPA promoter activity by the bioinformatics software prediction combined with ChIP,CoIP and double luciferase reporter assay(p<0.05).4.The sulfhydration of Rab7A(?)protein was weakened most obviously in the biotin switch assay.5.After NaHS treatment,the supernatant and intracellular HBV marker expression levels in Rab7A group were significantly lower than those in the control group(p<0.05),while there was no significant difference in Rab7A(?)group.6.Rab7A promoted phosphorylation of S6K1.The binding capacity of GTP was Q67L>Rab7A>Rab7A(?)>N125I(p<0.05).The effect of Rab7A and its mutant on downstream effector molec?Les of mTORC1 was detected.Q67L significantly promoted phosphorylation of S6K1,and Rab7A(?)and N125I inhibited expression of p-S6K1(p<0.05).7.COIP experiment verified that Rab7A could bind to mTOR,and the binding of Rab7A and mTORC1 was weakened after treating immunoprecipitation complex with 0.5%tx-100.Rab7A interacted with mTOR in a GTP-dependent manner.ConclusionsTTPA inhibits HBV replication by activating mTORC1 signaling pathway,and HBx inhibits TTPA promoter transcription by transcription complex SP1/HBXIP/HBx;Rab7A binds to mTOR in a GTP-dependent manner,thus regulating mTORC1 signaling pathway,and sulfhydrated Rab7A inhibits HBV replication in HepG2.2.15 cells...