Phenotypic Assay Of Hepatitis B Virus Polymerase With Extensive Site-specific Mutagenesis

A common problem during long term administration of chronic hepatitis B with Neocleot(s)ide analogues(NAs)is drug resistance.NAs resistance has been attributed to a line of mutations at specific sites in the reverse transcriptase domain of HBV polymerase.In the drug-resistance related research,phenotype assay of HBV with extensive mutagenesis at mutation hot spots is a useful means to understand the relationship between genotype and phenotype.Currently used extensive mutagenesis assay based on plasmid transfection method has some disadvantages.Here,we developed an alternative method for extensive mutagenesis phenotype assay of HBV polymerase.This method combined a randomized mutation method and the polymerase transcomplement strategy developed previously.Using rt M204,the well-known mutation site related to LAM resistance,as an example target of analysis,we demonstrated the feasibility of this method.Objective : To develop an alternative strategy for extensive mutagenesis phenotype assay of HBV polymerase using randomized mutation method combined with the polymerase transcomplement strategy.MethodExtensive site-specific mutations were obtained through the randomized mutation method and FSR(Fragment Substitution Reaction);The plasmid mixture was then used to package lentiviruses which were subsequently used to infect 293HBV-pol-cells and saved HBV replication through transcomplement strategy.After antibiotic selection and genomic DNA sequencing,Monoclonal cells were used to phenotype assay;Using rt M204,the well-known mutation site related to LAM resistance,as an example target of analysis,we constructed randomized mutation of rt204,packaged lentiviruses to infect 293HBV-pol-cell line,obtained Monoclonal cells harboring specific mutation,displayed drug sensitivity phenotype assay in vitro.ResultsRandomized mutations allowed us to construct extensive mutagenesis at specific site of RT region effectively.The reverse primer Rpol204 was used to introduce two randomized nucleotides(NNT)of the rt204 codon(ATG).The fragment was inserted at the corresponding region on p Lentipol-D by fragment substitution reaction.We obtained the wild-type and fourteen kinds of mutations of rt204 through a PCR step;Six kinds of Monoclonal cell lines were obtained and used to phenotype assay after antibiotic selection and genomic DNA sequencing;Compared with wild type,those mutations displayed a functional deficiency.The rt204 I mutant showed 3.039% the replication capacity of the wild type,which was similar to that of the rt204 N or rt204 K mutant.However the rt204 T and rt204 R mutants presented much lower replication capacities: 0.102% and 0.005% respectively;In the presence of lamivudine,the replication level of the wild type was reduced by about 7 fold(6.842 ± 0.983),whereas the rt204I/T mutants reduced by about 1 fold(1.201 ± 0.031,1.174 ± 0.146 respectively).The mutant rt204 N showed a significantly decrease of 10 fold,indicating a higher sensitivity to lamivudine.The rt204K/R mutants showed moderate sensitivities to lamivudine.Conclusion : We developed a novel mutagenesis phenotypic assay with the following characteristics:1.acquisition of extensive site-specific mutations through a PCR step and FSR;2.production of a lentivirus pool expressing different polymerase mutants via one-time lentivirus packaging;3.generation of stable cell lines that replicate HBV DNA by integrated polymerase mutants,which can help to establish an experimental system with lower variation and higher convenience.Using this strategy,we successfully constructed multiple mutants of rt204 and testified its' replication deficiency.In the presence of lamivudine,the rt204I/T mutants showed a replication advantage which was corresponding with the previous reports and clinical drug resistance demonstrating the feasibility this method.This randomized phenotypic assay of HBV polymerase provided a useful tool for assessing the replication fitness and drug susceptibility of HBV variants and quasi-species,which may offer new insight for the NAs therapy and the prognosis of patients with chronic hepatitis B.