Mutations of the woodchuck hepatitis virus polymerase gene that confer resistance to lamivudine and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil

Abstract

Administration of either lamivudine (2'-deoxy-3'-thiacytidine) or L-FMAU (2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil) to woodchucks chronically infected with woodchuck hepatitis virus (WHV) induces a transient decline in virus titers. However, within 6 to 12 months, virus titers begin to increase towards pretreatment levels. This is associated with the emergence of virus strains with mutations of the B and C regions of the viral DNA polymerase (T. Zhou et al., Antimicrob. Agents Chemother. 43:1947-1954, 1999; Y. Zhu et al., J. Virol. 75:311-322, 2001). The present study was carried out to determine which of the mutants that we have identified conferred resistance to lamivudine and/or to L-FMAU. When inserted into a laboratory strain of WHV, each of the mutations, or combinations of mutations, of regions B and C produced a DNA replication-competent virus and typically conferred resistance to both nucleoside analogs in cell culture. Sequencing of the polymerase active site also occasionally revealed other mutations, but these did not appear to contribute to drug resistance. Moreover, in transfected cells, most of the mutants synthesized viral DNA nearly as efficiently as wild-type WHV. Computational models suggested that persistence of several of the WHV mutants as prevalent species in the serum and, by inference, liver for up to 6 months following drug withdrawal required a replication efficiency of at least 10 to 30% of that of the wild type. However, their delayed emergence during therapy suggested replication efficiency in the presence of the drug that was still well below that of wild-type WHV in the absence of the drug.

FIG. 1.

Mutations in the polymerase active site. (A) Eleven WHV variants (types I to XI) with mutations in the polymerase active site were detected in the sera of lamivudine- and/or l-FMAU-treated woodchucks. Two (VIII and XI) have mutations in the C region; the remainder have mutations only in the B region. The amino acid sequence of a cloned wild-type WHV polymerase (12, 27) is shown at the top. The columns to the right indicate which drug treatment was associated with the emergence of a variant. (B) Sequence of polymerase mutants of WHV produced for phenotypic analyses. These particular variants have not been found, so far, to occur in drug-treated woodchucks. The YIDD, YVDD, and L565M+YVDD mutants were analogous to HBV mutations that arise in patients subjected to lamivudine therapy. (C) Comparison of amino acid sequences in conserved regions of the HBV and WHV genomes. Amino acids known to be mutated in drug-resistant variants are shown in boldface. (D) Predicted mutations in the overlapping envelope protein due to drug resistance mutations of the WHV polymerase. Amino acid changes in the envelope were predicted for all but the type VI mutant. Among these, a tryptophan-to-stop codon change was predicted for type I and X mutants. In panels A and C, amino acid positions in the WHV polymerase ORF are numbered from the first AUG of polymerase and from the beginning of the reverse transcriptase region (23); only the latter convention is used for HBV.

FIG. 2.

Replication of wild-type (wt) and mutant WHV in HepG2 cells. (A) Transfected cells maintained in drug-free medium. (B) Cells cultured with growth medium containing 100 μM lamivudine. (C) Cells cultured with growth medium containing 100 μM l-FMAU. (D) Cells cultured with growth medium containing 100 μM lamivudine and 100 μM l-FMAU. Molecular weight markers are shown as bars at the left. UT, DNA extracted from negative control; PC, 25 pg of linear, full-length WHV DNA. Amino acid sequences of the mutants are shown in Fig. 1. Asterisks indicate mutants that have not been found in woodchucks.

FIG. 3.

Quantitative summary of transfection results. Signal intensities for the Southern blots shown in Fig. 2 were quantified with a Fujix BAS 1000 Bio-imaging analyzer. A standard amount of cloned WHV DNA (25 pg), lane PC in Fig. 2, served as a hybridization control. (A) Inhibition of accumulation of virus DNA replication intermediates in the presence and absence of drug(s). (B) Accumulation of virus DNA replication intermediates in the presence and absence of drug(s), normalized to wild type (no drug). Asterisks indicate mutants that have not been found in woodchucks.

FIG. 4.

Additional mutations in the polymerase A, B, and E regions. Three additional amino acid changes were predicted for some of the mutants summarized in Table 2, based on the sequencing of the polymerase active site. Their frequency and coding capacity are summarized here in comparison to the sequence of a laboratory strain of wild-type (wt) WHV. The mutation V471I in the A region of polymerase would be accompanied by an M281I change in the overlapping S-ORF. The F551L change in the B region does not change the amino acid sequence of the overlapping S gene product. The E region does not overlap S.

FIG. 5.

Effect of the V471I, F551L, and M635L mutations on WHV DNA synthesis in HepG2 cells. Southern blots of core DNA from transfected cells maintained in drug-free medium (A), medium containing 100 μM lamivudine (B), 100 μM l-FMAU (C), or 100 μM lamivudine plus 100 μM l-FMAU (D). Hybridization signals were quantified, with the results summarized in Fig. 6. wt, wild type. UT, DNA extracted from negative control; PC, 25 pg of linear full-length WHV DNA. Asterisks indicate mutants that have not been found in woodchucks.

FIG. 6.

Quantitative summary of analyses of V471I, F551L, and L565M mutations. (A) Inhibition of accumulation of virus DNA replication intermediates in the presence and absence of drug(s). (B) Accumulation of virus DNA replication intermediates in the presence and absence of drug(s), normalized to wild type (no drug). wt, wild type. Asterisks indicate mutants that have not been found in woodchucks.

FIG. 7.

Replication of WHV genomes containing the complete polymerase gene of serum-derived virus from drug-treated woodchucks. WHV genomes were amplified by PCR with primers located in WHV nt 1941 to 1960 and 1945 to 1925. PCR products were cloned, and their nucleotide sequence was determined (accession no. AF410859 [wt-335a], AF410860 [wt-335b], AF410861 [wt-342a], AF410858 [342b], AF410857 [type I], AF410855 [type II], AF410856 [type IV]). Clones with the indicated mutations in the polymerase active site were cleaved upstream at AflII at nt 2303 and downstream at the NsiI site at nt 1914, releasing the complete WHV polymerase gene. This fragment was then substituted for the homologous region of an expression plasmid containing the laboratory strain of WHV. Accumulation of virus DNA was measured (A) in drug-free medium, (B) in medium containing 100 μM lamivudine, (C) in medium containing 100 μM l-FMAU, and (D) in medium containing 100 μM lamivudine and 100 μM l-FMAU. One of the wild-type clones, 342b, did not replicate; sequencing revealed a 6-nt (2 amino acid) deletion in the spacer region of the polymerase ORF. UT, DNA extracted from a negative control; PC, 25 pg of linear full-length WHV DNA. wt-335a, wt-335b, and type I polymerase genes were cloned from woodchuck 335 (Table 2) serum collected 2 months after termination of lamivudine therapy; wt-342a and wt-342b polymerase genes were cloned from woodchuck 342 serum collected 2 months after termination of lamivudine therapy. The type II and IV polymerase genes were cloned from woodchuck 331 serum collected upon termination of lamivudine therapy.

FIG. 8.

Quantitative summary of transfection assays in Fig. 7. (A) Inhibition of accumulation of virus DNA replication intermediates in the presence and absence of drug(s). (B) Accumulation of virus DNA replication intermediates in the presence and absence of drug(s), normalized to wild type (wt [no drug]).

FIG. 9.

Summary of data on persistence of lamivudine-resistant WHV in the serum following cessation of drug treatment. Results are summarized from reference . The order in which the variants are listed reflects their relative abundance in the serum.

FIG. 10

Models for the emergence of wild-type (wt) WHV after the termination of drug treatment. (A) Replication space model for the emergence of wild-type virus after termination of drug treatment. The curves were calculated from the formula of Zhang and Summers (26): t = (total liver regeneration)/(rate of hepatocyte death) = ln {F_wt · E^[1/(1 − k_rel)] + F_mut · E^[k_rel/(1 − k_rel)]}/(rate of cell death)F_wt and F_mut refer to the fraction of wild-type and mutant cccDNA at time 0, and E refers to the relative enrichment of wild type to mutant (that is, the ratio at time t divided by the ratio at time 0). k_rel is the ratio of the rate of replication of mutant to wild type, and is less than 1. k_d is the rate constant (fraction of hepatocytes/day) for death of infected hepatocytes. Calculations were made assuming that wild-type virus was initially 10% of the total in the serum and that the daily rate of cell destruction was 1%. (B) Cellular model 1 for the emergence of wild-type virus following the termination of drug treatment. The model (sim4) is described in Materials and Methods. Calculations were made assuming that wild-type virus is initially 10% of the total in the serum and that cccDNA amplification following cell division occurs via reverse transcription of pregenomic RNA synthesized from inherited cccDNAs. (C) Cellular model 2 for the emergence of wild-type virus following the termination of drug treatment. In contrast to model 1, this model assumes that both cccDNA and replicative DNAs are inherited by daughter cells and that amplification of cccDNA in the daughter cells utilizes the inherited cytoplasmic DNAs.