Cross-resistance testing of antihepadnaviral compounds using novel recombinant baculoviruses which encode drug-resistant strains of hepatitis B virus

Abstract

Long-term nucleoside analog therapy for hepatitis B virus (HBV)-related disease frequently results in the selection of mutant HBV strains that are resistant to therapy. Molecular studies of such drug-resistant variants are clearly warranted but have been difficult to do because of the lack of convenient and reliable in vitro culture systems for HBV. We previously developed a novel in vitro system for studying HBV replication that relies on the use of recombinant baculoviruses to deliver greater than unit length copies of the HBV genome to HepG2 cells. High levels of HBV replication can be achieved in this system, which has recently been used to assess the effects of lamivudine on HBV replication and covalently closed circular DNA accumulation. The further development of this novel system and its application to determine the cross-resistance profiles of drug-resistant HBV strains are described here. For these studies, novel recombinant HBV baculoviruses which encoded the L526M, M550I, and L526M M550V drug resistance mutations were generated and used to examine the effects of these substitutions on viral sensitivity to lamivudine, penciclovir (the active form of famciclovir), and adefovir, three compounds of clinical importance. The following observations were made: (i) the L526M mutation confers resistance to penciclovir and partial resistance to lamivudine, (ii) the YMDD mutations M550I and L526M M550V confer high levels of resistance to lamivudine and penciclovir, and (iii) adefovir is active against each of these mutants. These findings are supported by the limited amount of clinical data currently available and confirm the utility of the HBV-baculovirus system as an in vitro tool for the molecular characterization of clinically significant HBV strains.

FIG. 1

Diagrammatic representation of the HBV construct used to generate HBV-baculovirus recombinants. HBV-baculovirus recombinants were constructed from a 1.28 times unit length HBV genome of genotype A, serotype adw2. This HBV clone has been fully sequenced and contains no nucleotides which lie outside a published wt consensus sequence (3). Note that there is only one copy each of the precore, core, pre-S1–pre-S2–S, polymerase, and X open reading frames within the construct. The positions of the core (C), pre-S, S, and X promoters, enhancer I (EI), and enhancer II (EII), as well as the baculovirus polyhedron promoter (PH), are indicated.

FIG. 2

Polymerase mutations in drug-resistant HBV. Drug resistance mutations in the 1.28 times unit length wt HBV construct (Fig. 1) were generated by site-directed mutagenesis. The following amino acid changes in the polymerase gene product resulted: (i) an L-to-M amino acid substitution at position 526 (L526M) in domain B of the polymerase, (ii) an M-to-I amino acid substitution at position 550 (M550I) in domain C of the polymerase, and (iii) a double mutation with the L526M amino acid substitution and a second, M-to-V, amino acid substitution at position 550 (L526M M550V). The M550I and M550V mutations also resulted in changes to the deduced amino acid sequence of the surface antigen gene, as indicated. TP, terminal protein; R., reverse; ORF, open reading frame; HBsAg, hepatitis B surface antigen.

FIG. 3

Inhibition of wt HBV replication by lamivudine, adefovir, or penciclovir. Double-stranded HBV replicative intermediates were quantified by densitometry of appropriately exposed autoradiographs of Southern blots (see Fig. 4 to 6). Image densities were expressed as percentages of the mean density of untreated controls (defined as 100%). The resulting data, which describe the dose-dependent effects of drugs on wt HBV replication, were fitted to logistic dose-response equations with the aid of TableCurve2D software. The 95% confidence limits appear on either side of the fitted dose-response curves; the equation y = a/(1 + [x/b]^c) accurately describes the data. With the exceptions noted in footnote d to Table 1 and the legend to Fig. 5, all data sets were also accurately described by this equation. As shown, the lowest concentrations of lamivudine and adefovir (0.001 and 0.01 μM, respectively) appeared to stimulate wt HBV replication. This is also reflected by >100 values for the a (amplitude) parameter listed in Table 1. The cause(s) of this phenomenon is not known but may include (i) allosteric effects of (d)NTP analogs on intracellular nucleotide pools and/or (ii) interference with virus secretion, which might result in accumulation of intracellular replicative intermediates.

FIG. 4

Effects of lamivudine on wt and drug-resistant HBV. HepG2 cells were transduced at 50 PFU/cell with HBV-baculovirus which encoded either wt or mutant (L526M, M550I, or L526M M550V) polymerase. Immediately after transduction, cultures were treated with the indicated concentrations of lamivudine (3TC). After 7 days of treatment, HBV replicative intermediates were extracted from cell lysates and analyzed by Southern blotting and autoradiography. (A) autoradiographs of Southern blots. After equivalent exposure times, image densities in (drug-free) control lanes were such as to suggest that replication fitness decreased in the order wt > L526M > L526M M550V > M550I, assuming that transduction efficiencies were equal and that the polymerase changes did not differentially affect virus secretion. The autoradiographs shown were exposed for different times so that the image density in each control lane is approximately the same. (B) results of graphical analysis of data from panel A. rc, relaxed circular.

FIG. 5

Effects of penciclovir on wt and drug-resistant HBV. HepG2 cells were transduced at 50 PFU/cell with HBV-baculovirus which encoded either wt or mutant (L526M, M550I, or L526M M550V) polymerase. Immediately after transduction, cultures were treated with the indicated concentrations of penciclovir (PCV). After 7 days of treatment, HBV replicative intermediates were extracted from cell lysates and analyzed by Southern blotting and autoradiography. (A) autoradiographs of Southern blots (exposures were chosen to give approximately equal image densities, as noted in the legend to Fig. 4). (B) results of graphical analysis of data from panel A. As noted in Table 1, footnote d, it was not possible to fit logistic dose-response curves to data for the M550I single and L526M M550V dual mutants. rc, relaxed circular.

FIG. 6

Effects of adefovir on wt and drug-resistant HBV. HepG2 cells were transduced at 50 PFU/cell with HBV-baculovirus which encoded either wt or mutant (L526M, M550I, or L526M M550V) polymerase. Immediately after transduction, cultures were treated with the indicated concentrations of adefovir (PMEA). After 7 days of treatment, HBV replicative intermediates were extracted from cell lysates and analyzed by Southern blotting and autoradiography. (A) autoradiographs of Southern blots (exposures were chosen to give approximately equal image densities, as in Fig. 4 and 5). (B) results of graphical analysis of data from panel A. rc, relaxed circular.