Study On The Regulation Of Nuclear Export Of HBV Progenomic RNA By The Change Of Stem-Loop Structure In PRE

BackgroundHepatitis B virus(HBV)has been an infectious virus that endangers the world.Although hepatitis B is effectively prevented by the injection of hepatitis B vaccine,millions of people still have new infections every year.HBV infection is one of the major etiological factors of liver cancer.Currently clinically used antiviral drugs,such as nucleotide analogs,act on the replication process of HBV,require long-term administration,and are easily resistant.New therapeutic agents are urgently needed in the clinic.Studies that target other processes of the HBV life cycle may offer new approaches to the treatment of hepatitis B.During the life cycle,HBV DNA is transcribed to form pregenomic RNA(pg RNA).After which pg RNA is exported out of the nucleus and then used as a template for reverse transcription to synthesize new HBV.The stem-loop(SL)SL? and SL? in the posttranscriptional regulation element(PRE)of pg RNA mediate the extranuclear export of pg RNA.The alteration of sl? and SL? structure in pg RNA is promising to inhibit the extranuclear export of pgRNA,thereby reducing HBV replication.New treatments for hepatitis B are available through this approach.The purpose of this study is to confirm the role of SL? and SL? in the extranuclear export of HBV pg RNA by constructing PRE,SL? or SL? deletion mutations.The structure of SL? and SL? in PRE was then changed by single base mutation to explore the influence of the change of stem-loop structure on the extranuclear export of pg RNA.MethodsWith a plasmid containing the entire genome of HBV,plasmids with complete and partial deletion of PRE,SL? deletion or SL? deletion mutants were constructed by PCR.We changed the stem-loop structure of SL? and SL? by inducing single-base mutations.Finally,we constructed SL? open,SL? close,SL? open,SL? close mutant plasmids.The mutant plasmids were then transfected into Huh7 and Hep G2 cell lines.Real-time quantitative PCR was used to detect the level of HBV DNA replication,quantitative reverse transcription PCR(RT-q PCR)was used to detect HBV pg RNA transcription levels in the nucleus and cytoplasm,and Western blotting was used to detect hepatitis B surface antigen(HBs Ag)expression level in cells.The above methods were used to explore the effects of the complete or partial deletion of PRE,the deletion of SL? or SL?,and the structural changes of SL? and(or)SL? on the transcription level of HBV pg RNA and its nuclear output.Results1.After the complete or partial deletion of PRE,the synthesis and nuclear export of pg RNA in Huh7 and Hep G2 cell lines are reduced.2.After the deletion of the stem-loop structure SL? or SL? in PRE,the nuclear export of pg RNA in Huh7 and Hep G2 cell lines decreased.3.After SL? open,SL? close,SL? open,and SL? close mutations,HBV DNA levels significantly decreased.4.After SL? open mutation,the nuclear export of pg RNA in Hep G2 cell line decreased.5.SL? open + SL? open mutation may reduce the nuclear export of pg RNA in Huh7 and Hep G2 cell lines.Conclusions1.In Huh7 and Hep G2 cells,the SL? and SL? structures in PRE play an important role in the expression and nuclear export of HBV pg RNA.2.In Huh7 and Hep G2 cells,The SL? open,SL? close,SL? open,and SL? close mutations reduce HBV DNA replication.