The Hepatitis B Virus Polymerase Mutation rtV173L Is Selected during Lamivudine Therapy and Enhances Viral Replication In Vitro

Abstract

Therapy of chronic hepatitis B virus (HBV) infection with the polymerase inhibitor lamivudine frequently is associated with the emergence of viral resistance. Genotypic changes in the YMDD motif (reverse transcriptase[rt] mutations rtM204V/I) conferred resistance to lamivudine as well as reducing the in vitro replication efficiency of HBV. A second mutation, rtL180M, was previously reported to partially restore replication fitness as well as to augment drug resistance in vitro. Here we report the functional characterization of a third polymerase mutation (rtV173L) associated with resistance to lamivudine and famciclovir. rtV173L was observed at baseline in 9 to 22% of patients who entered clinical trials of adefovir dipivoxil for the treatment of lamivudine-resistant HBV. In these patients, rtV173L was invariably found as a third mutation in conjunction with rtL180M and rtM204V. In vitro analyses indicated that rtV173L did not alter the sensitivity of wild-type or lamivudine-resistant HBV to lamivudine, penciclovir, or adefovir but instead enhanced viral replication efficiency. A molecular model of HBV polymerase indicated that residue rtV173 is located beneath the template strand of HBV nucleic acid near the active site of the reverse transcriptase. Substitution of leucine for valine at this residue may enhance polymerization either by repositioning the template strand of nucleic acid or by affecting other residues involved in the polymerization reaction. Together, these results suggest that rtV173L is a compensatory mutation that is selected in lamivudine-resistant patients due to an enhanced replication phenotype.Until the recent approval of adefovir dipivoxil, lamivudine (a dideoxycytidine analog in the unnatural L configuration) was the only approved oral therapy for the treatment of chronic hepatitis B. Antiviral therapy for chronic hepatitis B with famciclovir and lamivudine has been limited by the emergence of viral resistance in significant proportions of patients. Although lamivudine therapy results in potent reductions in viremia, relapse is common, as resistant viruses emerge in approximately 24% of patients after 1 year of therapy and 70% after 4 years of therapy (20). Sequencing of hepatitis B virus (HBV) isolates from patients for whom lamivudine treatment failed revealed a mutation of methionine to valine or isoleucine at position rt204 (rtM204V/I) in the YMDD motif of the C subdomain of HBV polymerase (3, 21); amino acid residues in HBV polymerase are numbered according to the consensus nomenclature developed by Stuyver et al. (34). A second mutation, of leucine 180 to methionine (rtL180M), in the upstream B subdomain of HBV polymerase frequently accompanies rtM204 mutations. The rtM204V mutation almost invariably occurs in tandem with rtL180M, while rtM204I can occur as a single mutation or in conjunction with rtL180M.In vitro analyses have confirmed and characterized the role of the major HBV polymerase mutations in lamivudine resistance. Cell culture and enzyme assays have revealed that rtM20...

Resistance mutations of hepatitis B virus in entecavir‐refractory patients

“…On the other hand, the replication efficiency was strongly reduced by mutations of rtL180M/M204V but rescued by rtV173L. These observations of compensation by rtV173L were analogous with the case of LAM resistance,19 and we could clarify that these mutations are also associated with ETV resistance. The ETV‐resistant level of clone A1+MV seemed to be high, although the essential role of LAM‐resistant mutations to ETV is well known.…”

“…Among these, rtL180M/M204V have been reported previously to confer LAM resistance associated with a decline in replication efficiency 20. The rtV173L mutation did not affect susceptibility to LAM but instead enhanced viral replication 19. Similarly, the introduction of the rtL180M/M204V mutations in clone A1 could confer ETV resistance.…”

“…In case A, emerged HBV at VBT (clone A2) had three mutations compared with the prototype clone A1 that was isolated at the beginning of treatment. The identified mutations, rtV173L/L180M/M204V, are known to be LAM resistant 19. Among these, rtL180M/M204V have been reported previously to confer LAM resistance associated with a decline in replication efficiency 20.…”

Antiviral drug-resistant HBV

“…15,16 For example, HBV replication competent clones encoding the rtV173L (valine to leucine substitution) ϩ rtL180M ϩ rtM204V were demonstrated to have an increased replication yield phenotype but no change in sensitivity to lamivudine relative to HBV clones encoding rtL180M ϩ rtM204V. 17 Therefore, the rtV173L change should be considered a compensatory mutation in the HBV DNA genome.…”

“…12 Other compensatory mutations in-clude the rtV173L and rtL80I changes. 9,17 The rtA181T change has been reported to occur in the absence of rtM204I/V and is considered a primary resistance mutation. 44,45 The rtA181T change is also selected during ADV treatment.…”