A single amino acid in the reverse transcriptase domain of hepatitis B virus affects virus replication efficiency

To explore functional domains in the hepatitis B virus (HBV) polymerase, tw o naturally occurring HBV isolates (56 and 2-18) with 98.7% nucleic acid se quence homology but different replication efficiencies were studied. After transfection into HepG2 cells, HBV DNA isolated from intracellular virus co re particles was much higher in 56-transfected cells than in cells transfec ted with 2-18. The structural basis for the difference in replication effic iency between these two isolates was studied by functional domain gene subs titution. The complete polymerase (P) gene and its gene segments coding for the terminal protein (TP), spacer (SP), reverse transcriptase (RT), and RN ase H in 2-18 were separately replaced with their counterparts from 56 to c onstruct full-length chimeric genomes. Cell transfection analysis revealed that substitution of the complete P gene of 2-18 with the P gene front 56 s lightly enhanced viral replication. The only chimeric genome that regained the high replication efficiency of the original 56 isolate was the one with substitution of the RT gene of 2-18 with that from 56. Within the RT regio n, amino acid differences between isolates 2-18 and 56 were located at posi tions 617 (methionine versus leucine), 652 (serine versus proline), and 682 (valine versus leucine). Point mutation Identified amino acid 652 as being responsible for the difference in replication efficiency. Homologous model ing studies of the HBV RT domain suggest that the mutation of residue 652 f rom proline to serine might affect the conformation of HBV RT which interac ts with the template-primer, leading to impaired polymerase activity.