ADAR1 Regulates Replication Of HBV

Objective:To investigate the effect of ADAR1 on HBVMethods:1.The protein and RNA level of ADAR1 in Huh7 cell lines treated with IFN-?,?,? were measured by Western blot and RT-qPCR.2.Huh7 and Hep G2.2.15 cell lines were transfected with siRNA which targeted ADAR1 mRNA.The effect of endogenous ADAR1 on HBV replication parameters including HBV DNA,HBc,HBs,HBe and HBV RNA were measured by Southern blot,QPCR,Western blot,ELISA and RT-qPCR.3.ADAR1 expression plasmids and HBV construct(genotype D)were co-transfected into human hepatoma cell lines Huh7 and HepG2.ADAR1 expression plasmids were transfected into stable HBV-producing cell lines HepG2.2.15 and HepAD38.The effect of over-expressed ADAR1 on HBV replication parameters including HBV DNA,HBc,HBs,HBe and HBV RNA were measured by Southern blot,QPCR,Western blot,ELISA and RT-qPCR.4.Huh7 cell lines were co-tranfected with ADAR1 expressionplasmids and HBV constructs(genotype A,B,C).HBV DNA levels were analyzed by Southern blot.5.Deaminase-deficient mutants of ADAR1 expression plasmids were constructed(P110 H910 A,P110 E912 A,P110 C966 A,P110 C1036 A,P150 H910A)prior to co-transfect HBV construct(genotype D)and ADAR1 mutants into Huh7 cell lines.HBV DNA levels were tested by Southern blot.Results:1.In Huh7 cell lines,the protein and RNA level of ADAR1 P150 were remarkably increased after the treatment of IFN-?(F=326.346,P<0.05;F=8.942,P<0.05,respectively),and IFN-?(F=263.173,P<0.05;F=91.767,P<0.05,respectively).2.Endogenous ADAR1 enhanced HBV DNA level,in Huh7(F=879.386,P<0.05)and HepG2.2.15 cell lines(P<0.05).3.Over-expressed ADAR1 enhanced HBV DNA,in human hepatoma cell lines Huh7(P<0.05),HepG2(P<0.05);stable HBV-producing cell lines HepG2.2.15(P<0.05),HepAD38(P<0.05).4.ADAR1 could facilitate HBV genotype A(F=30409.807,P<0.05),B(F=139576.050,P<0.05),C(F=33952.793,P<0.05)replication in DNA level.5.Deaminase mutants diminished facilitation of ADAR1 P110,P150 on HBV replication(F=50546.022,P<0.05;F=3168.668,P<0.05,thedifferences among the groups were statistically significant).Conclusion:ADAR1 facilitates HBV replication in DNA level,which depends on its deaminase activity.