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

Abstract

To explore functional domains in the hepatitis B virus (HBV) polymerase, two naturally occurring HBV isolates (56 and 2-18) with 98.7% nucleic acid sequence homology but different replication efficiencies were studied. After transfection into HepG2 cells, HBV DNA isolated from intracellular virus core particles was much higher in 56-transfected cells than in cells transfected with 2-18. The structural basis for the difference in replication efficiency between these two isolates was studied by functional domain gene substitution. The complete polymerase (P) gene and its gene segments coding for the terminal protein (TP), spacer (SP), reverse transcriptase (RT), and RNase H in 2-18 were separately replaced with their counterparts from 56 to construct full-length chimeric genomes. Cell transfection analysis revealed that substitution of the complete P gene of 2-18 with the P gene from 56 slightly 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 region, amino acid differences between isolates 2-18 and 56 were located at positions 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 modeling studies of the HBV RT domain suggest that the mutation of residue 652 from proline to serine might affect the conformation of HBV RT which interacts with the template-primer, leading to impaired polymerase activity.

FIG. 1

Functional region of the 2-18 polymerase gene (open bars), HBV region flanking the polymerase gene of 2-18 (checkered bars), functional region of the 56 polymerase gene (shaded bars), and HBV region flanking the polymerase gene of 56 (stippled bars). 2-18P⁵⁶, 2-18SP⁵⁶, 2-18TP⁵⁶, 2-18RNaseH⁵⁶, and 2-18RT⁵⁶ are 2-18 chimeric genomes in which the complete P gene or each of the functional regions of the P gene, namely, spacer, TP, RNase H, and RT, was replaced with either the complete P gene or each of its counterparts from 56. 56RT^2-18 is a 56 HBV chimeric genome in which the RT region of the P gene was replaced with its 2-18 counterpart.

FIG. 2

p2-18 and p56 are pUC18 recombinant plasmids containing either full-length HBV strain 2-18 or 56 cloned at the SstI site. The polymerase gene, ranging from nt 2309 to 1625, consists of four functional regions, TP (terminal protein), spacer, RT/DNA polymerase (DNAP), and RNase H. The 5′ border of the TP region, 3′ border of TP/5′ border of spacer, 3′ border of spacer/5′ border of RT/DNA polymerase, 3′ border of RT/DNA polymerase/5′ border of RNase H, and 3′ border of the RNase H region are located at nt 2309, 2843, 135, 1168, and 1625, respectively, and are indicated by vertical bars. Gray boxes indicate consensus regions flanking the borders of functional regions, and the nucleotide positions are shown. P1 is the pUC18 reverse universal primer, and P2 to P8 are primers located in the consensus regions. P1, P2, P5, and P6 were used for construction of the TP chimera of the P gene, whereas P3, P4, P7, and P8 were used for the construction of the RT/DNA polymerase and RNase H chimeras. The sequences of these primers are given in the text.

FIG. 3

2-18 and 56 are original isolates with low and high replicative efficiencies, respectively. 2-18P⁵⁶, 2-18SP⁵⁶, 2-18TP⁵⁶, 2-18RNaseH⁵⁶, and 2-18RT⁵⁶ are 2-18 HBV chimeric genomes in which the full-length P gene and functional regions of the P gene (spacer, TP, RNase H, and RT) were replaced with their counterparts from 56. Intensity of hybridization signal was measured by densitometry, and the relative intensity of these samples is indicated at the bottom. Lane M, molecular size standards.

FIG. 4

56RT^2-18 is an HBV chimeric genome in which the RT region of the P gene was replaced with its 2-18 counterpart. Intensity of hybridization signal was measured by densitometry, and the relative intensity of samples is indicated at the bottom. Lane M, molecular size standards.

FIG. 5

2-18^617Met→Leu, 2-18^652Ser→Pro, and 2-18^682Val→Leu are 2-18 HBV chimeric genomes in which the nucleotide sequences coding for amino acids at positions 617, 652, and 682 within the RT region were each mutated to change the amino acid to the corresponding amino acid of isolate 56. Intensity of hybridization signal was measured by densitometry. The intensity of signal of 2-18RT^652Ser→Pro was similar to that of 2-18RT⁵⁶, in which the RT region of 2-18 was replaced with its 56 counterpart. Lane M, molecular size standards.

FIG. 6

Ribbon diagram showing the three-dimensional model of the RT domain of HBV polymerase (residues 319 to 700). HBV RT is composed of three subdomains, fingers (319 to 400 and 451 to 515, in blue), palm (401 to 450 and 516 to 610, in red), and thumb (611 to 700, in green). A docked template-primer double-stranded DNA is shown as a ribbon, with the template in magenta and the primer in cyan. The three catalytically essential aspartic acid residues, Asp429, Asp551, and Asp552, are shown with side chains. The locations of the mutations are indicated with gold balls.