Identification Of Host Factors Regulating Anti-HBV Function Of APOBEC3B And Investigation Its Related Mechnisms

Objectives: Hepatitis B virus(HBV)infection has been a seriously public problem.There are about 257 million patients with chronic HBV infection,these patients can develop HBV-related diseases,such as chronic hepatitis B(CHB),liver cirrhosis(LC)and primary hepatocellular carcinoma(HCC).Our previous reports also indicated that host factor APOBEC3 B can significantly inhibit HBV replication during reverse transcription which is dependent on its deaminase activity.However,much less is known about how host factors are involved in this process.Thus,it is of great interest to identify host factors that are involved in the regulation of the anti-HBV function of APOBEC3 B.In this research,we performed co-immunoprecipitation(co-IP)coupled with mass spectrometry(MS)to systematically analyze APOBEC3B-interacting proteins,and clarified the effect of HSP70 or DHX9 on the anti-HBV function of APOBEC3 B.This research not only provides a full understanding of the host factors involved in the regulation of interaction between APOBEC3 B and virus,which contribute to clarify the mechanism of virus persistent infection,but also helps to refine and distinguish whether the host factors regulates deaminase activity of APOBEC3 B or the combination between APOBEC3 B and HBV core protein/RNAs,which provides theoretical basis for new antiviral strategy.Methods:PART1: Screening of host factors interacting with APOBEC3 B and regulating anti-HBV function of APOBEC3B: HEK293 T cells were transfected with HBV expression plasmid or/and APOBEC3 B expression plasmid,co-IP was conduct to enrich proteins that potentially interacted with APOBEC3 B,MS was used to identify APOBEC3B-interacting proteins.Protein-protein interaction(PPI)networks were carried out based on GO analysis by bioinformatics methods.Then APOBEC3B-interacting proteins with the higher scores were selected for further research: heat shock protein(HSP)members and DEAD-box RNA helicases(DDX)members.The potential factors of HSP70 and DHX9 involved in the anti-HBV functions of APOBEC3 B were selected by silencing endogenous HSP family proteins(including HSP70,HSP90 and GRP78)and DDX family proteins(including DDX3,DDX5 and DHX9),respectively.PART2: Research on molecular mechanism of HSP70 contributing to the anti-HBV function of APOBEC3B: Firstly,the core-associated viral DNA was detected by Southern blot in the presence of overexpression of HSP70;HSP70 mutants of ATPase activity(D10N K71 A,K77A and E175S)were constructed by site-directed mutagenesis and investigated their effect on the anti-HBV effect of APOBEC3B;VER155008,a HSP70 inhibitor,was performed to study its role in the anti-HBV function of APOBEC3B;effect of HSP70 on APOBEC3 B deamination was explored by using fluorescent-labeled substrate probe in vitro;effect of HSP70 on HBV DNA deamination edited by APOBEC3 B was verified by 3D-PCR and signle cloning sequencing;the direct interaction and binding region between HSP70 and APOBEC3 B were studied by co-IP and GST Pulldown.PART3: Research on molecular mechanism of DHX9 attenuating the anti-HBV function of APOBEC3B: Firstly,the binding of APOBEC3 B to DHX9 protein was detected by co-IP and GST Pulldown;effect of DHX9 on HBV DNA or the anti-HBV effect of APOBEC3 B was investigated by silencing the expression of endogenous DHX9 or overexpression of DHX9 in defferent cell modles including LPP-APOBEC3B-Huh7 cell model and NTCP-HepG2 HBV infection model;effect of DHX9 on APOBEC3 B edited substrates was detected by using fluorescent-labeled substrate probe in vitro and 3D-PCR;effect of DHX9 on the interaction between APOBEC3 B and HBc was performed by co-IP;three deletion mutants of DHX9,including DHX9-?N(?3-252 aa),DHX9-?C(?1173-1260 aa)and DHX9-? N ? C(? 3-252 aa,? 1173-1260 aa),were built by site-directed mutagenesis for investigating the key interaction region between DHX9 and APOBEC3 B by co-IP,and whether DHX9 attenuating the anti-HBV function of APOBEC3 B depends on their interaction was explored further;DHX9 attenuating the interaction between APOBEC3 B with HBV pgRNA was detected by silencing and overexpression of DHX9 in HEK293 T transfection-replication model and HepAD38 stable HBV replication model,respectively.Results:PART1: Screening of host factors interacting with APOBEC3 B and regulating anti-HBV function of APOBEC3B: MS analysis indicated that 126 proteins exclusively interact with APOBEC3 B in the presence of HBV.PPI network analysis revealed that the HA-APOBEC3 B interacting proteins were mainly involved in specific mRNA metabolism,protein folding and viral process.The Cancer Genome Atlas(TCGA)database analysis showed that there was a certain positive correlation between APOBEC3 B and HSP70 mRNA expression in HBV infected HCC samples(r=0.13103,p=0.0075),but there was no correlation between GRP78(r=-0.1757,p=0.1371)and HSP90(r=0.0212,p=0.8585);the inhibitory effect of APOBEC3 B on HBV replication was weakened by specifically interfering with HSP70 expression;the inhibitory effect of APOBEC3 B on HBV replication was enhanced by specifically interfering with DHX9 expression.PART2: Research on molecular mechanism of HSP70 contributing to anti-HBV function of APOBEC3B: The level of HBV DNA decreased in a concentration-dependent manner after overexpression of HSP70,but not dependent on the HSP70 ATPase activity;The anti-HBV function of APOBEC3 B decreased gradually with the increasing of VER155008 treatment;HSP70 enhanced the deaminase activity of APOBEC3 B measured by the fluorescent-labeled assay in vitro or the viral DNAs editing ability of APOBEC3 B detected by 3D-PCR and cloning sequencing,by means of overexpression of HSP70 or VER155008 treatment;co-IP and GST Pulldown experiments confirmed that HSP70 had a direct binding with APOBEC3 B.PART3: Research on molecular mechanism of DHX9 attenuating the anti-HBV function of APOBEC3B: DHX9 specifically interacted with APOBEC3 B,and the levels of DHX9 bound to APOBEC3 B were significantly enhanced in the presence of HBV.The promotion of HBV replication by DHX9 was decreased after silencing the expression of endogenous APOBEC3B;conversly,the inhibition of HBV replication by APOBEC3 B was enhanced by silencing the expression of DHX9 in LPP-A3B-Huh7 cell model and NTCP-HepG2 cells that support HBV infection;silencing or ectopic expression of DHX9 had no effect on the APOBEC3 B editing of viral DNA or the interaction of APOBEC3 B with HBc.Both the dsRBDs and RGG region of DHX9 served as the main binding region for APOBEC3 B,and DHX9 attenuating the anti-HBV function of APOBEC3 B was dependent on their interaction.DHX9 attenuated the binding between APOBEC3 B and 3.5 kb HBV RNA by silencing the expression or overexpression of DHX9;the 3.5kb HBV RNA interacted with APOBEC3 B was identified as HBV pgRNA by anchored PCR.Conclusions: When HBV infects hepatocytes,APOBEC3 B restricits viral DNA replication.Meanwhile,host factor HSP70 positively contributes to the antiviral activity of APOBEC3 B by enhancing the deaminase activity of APOBEC3B;while HBV induced upregulation of DHX9 binds to APOBEC3 B dependent on the dsRBDs domain at the N-terminal and RGG domain at the C-terminal of DHX9,and antagonizes the binding of APOBEC3 B to HBV pgRNA in the nucleus of hepatocytes,thus contributing to viral DNA replication by partially decreasing the antiviral function of APOBEC3 B.