The Impact Of The Polymorphic Sites Of The Hepatitis B Virus Reverse Transcriptase On HBV Replication

Objective Phenotypic assays to investigate the relationship betweenrtN118H polymorphic sites found in some resistant strains and ADVprimary resistanceMethods The plasmid PCH9-EGFP containing rtN118H mutant fromHBV sequence derived from a ADV drug-resistant patient was constuctedby the FSR (Fragment substitutation reaction) method；On the other hand,the rtN118H mutant were introduced into the plasmid PCH9-EGFPcontaining the wild-type HBV genotype D sequence by site-directedmutagenesis method；and the two constructed plasmids were transfectedinto hepatoma cell line(HepG2) to accomplish in vitro ADVsusceptibility test. Compared with control plasmid PCH9-EGFP containingwild-type HBV sequence, phenotypic assays were made on the tworeconstructed plasmids. Results The two mutant plasmids constructed by different methods wereable to successfully transfected into HepG2cells, and further supported invitro expression of HBsAg and HBeAg. In vitro, IC50of plasmidcontaining the wild-type HBV sequence to ADV were0.109±0.004μM;IC50of plasmid containing rtN118H mutant constructed by FSR to ADVwere0.112±0.001μM (P>0.05); IC50of rtN118H mutant plasmid builtby site-directed mutagenesis to ADV were0.108±0.002μM (P>0.05);Compared with the wild type, there were no statistically significantdifference.Conclusion There was no relationship between rtN118H mutation siteand the ADV primary resistance. In other words, strains were stillsensitive to ADV in the presence of rtN118H mutation.