A New System For HBV Phenotypic Assay Based On Recombinant Lentivirus

Objective To build a new in vitro HBV phenotypic assay system usingHBV polymerase trans-complementation technology based on HBVpolymerase recombinant lentivirus.Methods1. HBV pol gene was cloned into a lentiviral vector, generating theplasmid pLenti-pol. The HBV pol recombinant lentivirus, named Lenti-pol,was obtained by co-transfection of pLenti-pol and other vectors forlentivirus packaging.2. The codon aa130of the polymerase gene in PCH9/3091was mutatedto a stop codon to delete the expression of the nomal polymerase, and aneomycin resistant gene expression fragment was cloned down stream fromthe HBV DNA, leading to the plasmid HBV1.1P--neo. After the transfectionof HBV1.1P--neo into HEK293cells and two months of selection with G418,a stably transduced cell line, named HBV P-293, was obtained.3. The recombinant lentivirus was used to infect HBV P-293cells.After10days of Blasticidin selection, intracellular viral core DNA was extracted and assayed by southern blot.4. A line of recombinant lentiviruses expressing the typicaldrug-resistant HBV polymerase were constructed. These lentiviruses wereused to generating several cell lines which can replicate HBV DNA.Results1. The HBV pol gene recombinant lentivirus was produced.2. A cell line that stably transduced with the polymerase deleted1.1×HBV DNA was established.3. After being infected by the HBV pol lentivirus, HBV P-293cells canreplicate HBV DNA, which was demonstrated by the HBV replicationintermediates detected by Southern blotting.4. Several cell lines which replicate HBV DNA using the typicaldrug-resitant HBV polymerase was obtained.5. Conclusion A new system for in vitro HBV phenotypic assay basedon the recombinant lentivirus was established. It can be helpful to performmore effectively phenotypic assay of HBV isolates for their susceptibilitiesto specific drugs.