Transient Selection of A Hepatitis B Virus Polymerase Gene Mutant Associated With A Decreased Replication Capacity and Famciclovir Resistance

Abstract

Prolonged therapy for chronic hepatitis B (HBV) with nucleoside analogs may result in the emergence of HBV mutants resistant to antivirals. Here, we describe the transient selection of an HBV polymerase gene mutant that was associated with viral persistence in an immune competent patient treated with famciclovir. Viral polymerase gene sequence was analyzed directly on polymerase chain reaction (PCR) products and also after cloning. The results showed the transient selection of a V542I mutant in the C domain of the viral polymerase. This mutation was associated with a stop codon at amino acid position 199 in the overlapping S gene. The mutated sequence was subcloned in a vector expressing the entire HBV pregenome to study its replication capacity after transient transfection in cultured hepatoma cells. The results showed that the V542I mutant has a decreased replication capacity compared with wild type virus and does not produce HBsAg. The sensitivity of the V542I mutant to penciclovir, the active metabolite of famciclovir, was further studied in tissue culture. This mutant was shown to be resistant to penciclovir, but remained sensitive to lamivudine, as was subsequently observed in vivo. Recently, the antiviral efficacy of new nucleoside analogs, famciclovir and lamivudine, has been assessed in chronically hepatitis B virus (HBV)-infected patients. 1,2 Famciclovir is the oral prodrug of penciclovir (9-[4-hydroxy-3 hydroxymethylbut-1-yl] guanine; BRL 39123)-triphosphate, which was shown to inhibit hepadnavirus replication in vitro in tissue culture and in vivo in animal models of HBV infection. 3,4 It was further shown that penciclovir triphosphate not only inhibits the DNA dependent DNA polymerase activity of the HBV polymerase, 5 but also the RNA-dependent activity of this enzyme, i.e., reverse transcription, by inhibiting the synthesis of the short DNA primer for reverse transcription. 6 Preliminary evidence suggested that famciclovir may have clinical utility in the treatment of chronic hepatitis B patients 7 and in orthotopic liver transplant patients. 8 A first placebocontrolled pilot study showed a fall of HBV DNA levels by more than 90% in 6 out of 11 evaluable patients. 7 Kruger et al. have reported, in 11 patients with HBV recurrence after orthotopic liver transplantation, that famciclovir treatment could induce a significant decrease of serum viral DNA levels in 90% of the patients and a clearance of HBV DNA detected by PCR in 36%, without significant side effect. 8 A large multicenter placebo controlled trial including 333 patients showed the clinical efficacy of a 16-week course of famciclovir with a dose-dependent antiviral effect accompanied by a decrease of serum ALT levels and a significant increase of anti-HBe antibody seroconversion compared with the placebo group. 9 By using the results of clinical trials for the evaluation of another promising nucleoside analog, lamivudine (3TC or [(Ϫ)-␤-L-2Ј, 3Ј-Dideoxy-3Јthiacytidine]), Nowak et al. have determined the kinetics of viral clearance during an...

In vitro evaluation of hepatitis B virus polymerase mutations associated with famciclovir resistance

“…18,20 However, several recent reports have indicated that penciclovir has high IC 50 values in the range of 82 µmol/L to 100 µmol/L. 38,47,48 The lower potency of penciclovir, compared with adefovir and lamivudine, for the inhibition of HBV polymerase and HBV replication in vitro is more consistent with the apparent lower efficacy of famciclovir compared with adefovir and lamivudine in recently published clinical studies. Patients treated with 30 mg adefovir daily for 12 weeks displayed Ն99.99% decreases in median serum HBV-DNA levels from baseline.…”

“…Our in vitro enzymatic assays indicated that the V555I mutation in HBV polymerase was clearly associated with a phenotypic resistance to famciclovir. This mutation has also recently been shown to be resistant to penciclovir in a cell culture model of HBV replication by Pichoud et al 38 The valine to isoleucine mutation at position 555 may directly interfere with the binding of PCVTP to HBV polymerase or indirectly interfere by inducing a conformational change in the PCVTP binding site. The exact mode of PCVTP binding to HBV polymerase is unknown because of the lack of a crystal structure for the complex HBV polymerase.…”

“…[33][34][35][36][37][38][39][40] However, because these were aggregate sequences of polymerase chain reaction products it is unclear whether multiple mutations were present in individual viral clones. A third possible explanation is that these mutations are natural HBV variants that have nothing to do with famciclovir resistance; however, all 5 of these mutations have been reported in famciclovir-treated patients on at least 2 separate occasions and none of them were present in a survey of 49 isolates of wild-type HBV taken from Genbank.…”

“…Mutations in the C-domain of HBV polymerase have been found to be associated with strong phenotypic resistance to another nucleoside analog, whereas the lamivudine-resistant M552I and M552V mutations are also located in the conserved C-domain of HBV polymerase. The V555I mutation has been reported in both famciclovir-and lamivudine-treated HBV patients 26,38 and interestingly, 2 different sets of codons were used for the isoleucine mutation at 555. In the famciclovirtreated patient, adenosine-uridine-adenosine was used to encode isoleucine, which leads to a stop codon in the overlapping reading frame for HBsAg at amino acid 199, and this was shown to be defective in HBsAg production in cell culture.…”

“…1). [33][34][35][36][37][38] These mutations were associated with an increase in serum HBV-DNA levels, suggesting that they may confer reduced sensitivity to famciclovir. In early reports, famciclovir-associated mutations were found most frequently in the conserved domain B of HBV polymerase.…”