Research On The Mechanism Of Host Restrictive Factor MOV10 On HBV Replication

Objective: Hepatitis B virus is a hepadnavirus that replicates in a reverse transcription manner.MOV10 is an important host antiviral factor in cells.MOV10 not only inhibits various viral replication,including human immunodeficiency virus type 1,hepatitis C virus,influenza A virus and vesicular stomatitis virus,but also limits retrotransposon activity such as LINE-1,Alu,SVA.However,whether and how MOV10 inhibit HBV replication are less reported.Therefore,this study focuses on the effect of MOV10 on HBV replication and investigated the mechanism of MOV10 inhibits HBV replicaiton.Methods:(1)Phenotype of MOV10 inhibiting HBV replication: We first investigated the relationship of MOV10 expression level and HBV replication in clinical CHB samples and HBV stable replcation cells: the expression level of MOV10 in peripheral blood mononuclear cells of healthy people and patients with chronic hepatitis B infection were compared,the expression level of MOV10 were measured aftertetracycline was withdraw in HepAD38 cells.Whether MOV10 regulated HBV replication was explored by gene lost and gain of function experiment;the viral DNA and RNA were extracted and measured by qPCR and Northern blot,the core particle was determined by native gel after knocking down the endoexpression of MOV10 in Huh7 or HepG2-NTCP;the viral DNA and RNA were extracted and measured by Southern blot and Northern blot after overexpressing MOV10 in Huh7 or HepG2-NTCP.(2)Mechanism of MOV10 inhibit HBV replication: the HBV replication plasmid which contain HBV self-promoter were cotransfected with MOV10 plasmid,and whether MOV10 inhibited the viral DNA replication by regulating the viral promoter;Huh7 cells were stimulated by IFN-? and IFN-?,then cellular RNA and protein were extracted to detect the expression level of MOV10,then we investigated werther MOV10 inhibited HBV replication as interferon induced gens;After IRF3 and STAT1 in the interferon pathway were knocked down,HBV and MOV10 were cotransfected into Huh7 cell,viral DNA levels were measured by qPCR;We constructed MOV10 conserved domain II enzyme active mutants(D645A,E646 Q and G648A),and investigated the antiviral effect of different MOV10 enzyme mutants;we exploring whether MOV10 exerts antiviral effects by binding to viral proteins or viral RNAs: the HBV replication plasmid and MOV10 were cotransfected into 293 cell to investigate whether MOV10 interact with HBc byImmunoprecipitation;the HBV replication plasmid and MOV10 were co-transfected into 293 cell to investigate whether MOV10 can bind to HBV RNA.Results: The expression level of MOV10 in peripheral blood mononuclear cells of patients with chronic hepatitis B infection was lower than that of healthy people,MOV10 protein decreased after tetracycline was withdraw from HepAD38,indicated that MOV10 is associated with HBV replication.After knocking down endogenous MOV10 by siRNA in Huh7 cells,HBV DNA replication increased about 1.5-fold comparded with control group,while viral RNA and core particle have no significant changes.The HBV DNA levels also increased 1.5-fold after knocking down the expression of MOV10 in HepG2-NTCP cell;the viral DNA levels significantly decreased when overexpressed MOV10 in Huh7 cells,while the RNA has no change,which indicated that MOV10 inhibits viral replication mainly at DNA replication step,overexpression of MOV10 in HepG2-NTCP cells also showed significant inhibitory effect;MOV10 did not act through the HBV promoter;MOV10 was not induced by IFN-?and IFN-?;the antiviral effect of MOV10 do not disappeared after knocking down interferon upstream gene IRF3 or interferon downstream gene STAT1,suggested that MOV10 do not exert antiviral effects through interferon pathway.;MOV10 domain II mutants also significantly inhibited HBV replication,which indicated that MOV10 inhibit HBVreplication independent on its enzymatic activity t;MOV10 can bind to HBV RNAs.Conclusion: In hepatoma cells,the expression of the host restriction factor MOV10 is associated with HBV replication,which inhibits HBV replication not through the promoter,and is independent of its helicase activity,and not acting as an interferon-stimulated gene or promoting interferon-stimulated gene expression for inhibiting viral replication.MOV10 can bind to HBV RNAs,which may be important for its inhibition of viral replication.