Variant species of HBV with reduced susceptibility to lamivudine emerge in chronically infected patients on long-term lamivudine therapy (1
). In these HBV species, the affinity of the viral polymerase for lamivudine triphosphate is decreased by the substitution of either valine or isoleucine for the methionine at position 552 in the conserved YMDD region of the polymerase. In many clinical isolates, an upstream methionine substitution at L528 also emerges, presumably as a compensatory mutation (15
). While these substitutions result in decreased viral susceptibility to lamivudine, HBV appears to pay a replicative price, as previous studies have indicated that HBV strains with YMDD-mutant polymerase replicate with lower efficiency than wt strains (1
). Based on a computer model of the HBV polymerase, these amino acid changes were shown to be in the catalytic domain of the enzyme and could possibly influence interactions with the normal dNTP substrates as well as interactions with lamivudine triphosphate (1
). One study indicated that not only were amino acid changes in the polymerase involved in decreased replication efficiency but also the concentrations of dNTPs within HBV-producing cells may play a role in diminishing replication efficiency (28
). To examine the influence of dNTP concentrations on replication fitness of YMDD-mutant HBV strains, the levels and relative amounts of dNTPs were determined for cells that both supported HBV replication and exhibited differences in replication efficiency between wt and YMDD-mutants. The enzyme kinetics of the HBV polymerase reaction was measured in order to investigate the molecular interactions between the dNTPs and HBV polymerase, both wt and mutant.
The system chosen for kinetic analysis was one in which the HBV polymerase activity measured is in the native state. During replication the polymerase is enclosed within the HBV core particle, using the encapsidated HBV pregenomic RNA as a template for the RNA-directed DNA polymerase activity and the minus-strand DNA as a template for the DNA-directed DNA polymerase activity. We assayed the polymerase activity found within partially replicated subviral core particles isolated from liver-derived cells which support HBV replication. This assay system is in contrast to other systems which have used recombinant HBV polymerases to obtain kinetic data (44
). In highlighting the fact that the Ki
values reported in their assay did not match the values that had been reported in a study using a cell-based system, Xiong et al. (52
) noted that the enzymes studied were partially purified recombinant protein preparations isolated from insect cells and that the primer template was activated calf thymus DNA, rather than the normal HBV template (K. P. Fischer, Letter, Hepatology 29:
996, 1999; X. Xiong, Letter, Hepatology 29:
996, 1999). While the Ki
values determined from the core polymerase assay show as much as a 1,000-fold difference between wt and YMDD-mutant polymerases (Table ), the assay using isolated polymerase preparations derived from insect cells showed a maximum difference of only 20-fold between the polymerases (52
). The core polymerase assay also discriminates between the two single M552 substitutions by demonstrating that MV is much less sensitive to lamivudine triphosphate than is MI, consistent with results previously reported in cell-based systems (1
). However, the opposite conclusion was reached with the recombinant polymerase assay, suggesting that recombinant HBV polymerases produced in heterologous systems cannot discriminate between natural substrates or lamivudine triphosphate as well as core polymerases in the native state. Moreover, the kinetic data were derived by using linear regression analysis (44
), which is not as precise as the nonlinear regression analysis used in this study (described at http://www.curvefit.com/avoid_linearizing.htm
Determination of enzyme kinetic constants for an enzymatic reaction gives insight into the nature of the reaction, especially the interaction between substrate and enzyme. In a simple enzymatic reaction with one substrate, the Km
value is the concentration of substrate at which 50% of the enzyme is bound to the substrate (43
). When the substrate concentration is equal to the Km
value, the rate of the enzymatic reaction is 50% of the maximal rate, Vmax
. During replication the HBV polymerase utilizes five substrates: four dNTPs and a template primer. The steady-state velocities for wt and mutant polymerases are the result of the complex interplay of all reactants and depend on the Km
values of each dNTP, the concentrations of each dNTP, and the nucleotide sequence of the template primer. Consequently, the kinetic equation describing the velocity of the polymerase reaction is quite complex. To simplify the interpretation of the data presented here, an apparent Km
value for each naturally occurring dNTP substrate has been defined as the concentration of the selected dNTP yielding 50% of the Vmax
value when the other three dNTPs and template primer are at saturating concentrations.
The higher apparent Km values for the mutant polymerases determined by the core polymerase assay indicate that higher concentrations of dNTPs are required for activity equivalent to that of wt polymerase. One indication that the Km differences are involved in replication efficiency is the fact that the differences in apparent Kms between wt and mutant polymerases correlated with the change in yields and not the changes in lamivudine sensitivity. Because of the complexity of the actual polymerase reaction, as noted above, both the actual concentration of each dNTP and their relative amounts will have a profound effect on the observed velocity. Thus, it was important to determine the levels and relative amounts of dNTPs in cells that both supported HBV replication and exhibited differences in replication efficiency between wt and mutants.
dNTP pools were determined in HepG2 cells that had been grown under a variety of conditions: (i) mock production of extracellular virus, (ii) mock production of intracellular particles, and (iii) normal passage. Pools of dNTPs were also measured in the HepG2-derived 2.2.15 producer cell line under conditions that induce overexpression of extracellular virus. For the most part, maximum levels of dNTP concentrations were in the low micromolar range (<5 μM), with the highest concentrations observed at very low cell density or under conditions used for mock intracellular harvest. In most cases at high cell densities the concentrations of individual dNTPs were ≤1 μM. The levels of dCTP in HepG2 cells reported in this paper are similar to those reported recently (22
). A review of the literature indicates that similarly low concentrations of dNTPs are found in liver or liver-derived cells from the rat or mouse (41
). Nakamura et al. (29
) measured dNTP levels in mouse fetal liver and in mouse adult liver; the dNTP levels for mouse fetal liver reported by these investigators are similar in ratio and concentration to those measured in low-density, rapidly growing HepG2 cells after 3 days of growth (Table ). By contrast, dNTP levels in the liver of the adult dam were lower (<0.4 μM) than those found in dense HepG2 monolayers. These data indicate that as HepG2 cells increase in density, intracellular dNTP levels decrease to levels that may approximate those in normal adult liver (2
). One caveat to these calculations is that assessment of the dNTP pools was based upon a cellular average and does not reflect possible intracellular compartmentation. If there are two distinct dNTP pools, with cytoplasmic dNTP concentrations lower than those in the nucleus (3
), then actual concentrations available for HBV replication may be lower than our estimates indicate.
A comparison of the catalytic specificities, kcat/Km, of wt and mutant polymerases indicated that at low levels of dNTPs the relative velocity of HBV polymerase would be lower for the mutants than for the wild type (Table ). Polymerase assays were performed to see whether the levels of dNTPs measured in HepG2 cells were sufficiently low to detect differences between wt and mutant polymerases. As predicted by the enzyme kinetic data, there were marked differences between wt and mutant polymerases at low levels of dNTPs (Tables and ). The results were similar whether the nonlabeled dNTPs were equimolar, with the labeled dNTP at Km, or whether the concentrations of the nonlabeled dNTPs varied and the labeled dNTP was present in high concentration. The differences in polymerase activity between wt and mutant polymerases based on kcat/Km calculations are almost identical to the differences observed in polymerase assays performed at low dNTP levels.
The results of the kinetic experiments and polymerase assays at limiting levels of dNTP substrates indicated that the elongation rate of polymerases containing YMDD mutations would be slower than the elongation rate of wt polymerase in HepG2 cells. In order to test this hypothesis, the extent of DNA polymerization within core particles of wt and YMDD-mutant HBV grown in HepG2 cells was measured. The results showed that in a population of core particles that had polymerized at least the first 200 nt of DNA, a much greater number of wt core particles than of YMDD-mutant core particles had extended polymerization by at least 5,000 nt, which is twice as many wt core particles as LMMV or LMMI core particles and three times as many wt core particles as MI core particles.
In summary, the results presented in this paper confirm the lower replication efficiency of YMDD-mutant HBV strains and show that they have reduced fitness. The YMDD-mutant polymerases have a lowered affinity for the natural dNTP substrates used for viral DNA replication, and cells capable of supporting HBV replication have intracellular concentrations of dNTPs that are low enough to influence the differences in the enzymatic rates between YMDD-mutant polymerases and wt polymerase.