Selection and characterization of varicella-zoster virus variants resistant to (R)-9-[4-hydroxy-2-(hydroxymethy)butyl]guanine

Abstract

(R)-9-[4-Hydroxy-2-(hydroxymethy)butyl]guanine (H2G) is a potent and selective inhibitor of herpesvirus replication. It is a nucleoside analog, and its triphosphate derivative (H2G-TP) is a competitive inhibitor of herpesvirus DNA polymerases. In this study, the antiviral activities of H2G and acyclovir (ACV) and the development of viral resistance to these agents were compared in varicella-zoster virus (VZV)-infected cells. In plaque reduction assays, the 50% effective concentration of H2G for VZV was 60- to 400-fold lower than that of ACV, depending on the virus strain and the cell line tested. The enhanced efficacy of H2G against VZV can be accounted for in part by the fact that the intaracellular H2G-TP level (>170 pmol/10(6) cells) is higher than the intracellular ACV-TP level (<1 pmol/10(6) cells). In addition, H2G-TP has extended half-lives of 3.9 and 8.6 h in VZV-infected MRC-5 and MeWo cells, respectively. To assess the emergence of H2G-resistant VZV in vitro, VZV was passaged in the presence of increasing concentrations of H2G. Earlier in the passage, when the concentration of H2G was relatively low, the predominant variant had the (A)76 deletion in the viral thymidine kinase (TK) gene. This mutant was identical to an ACV-resistant mutant generated in parallel experiments. However, higher concentrations of H2G appeared to favor a novel mutant, which had deletions of two consecutive nucleotides at positions 805 and 806 of the TK gene. All of these changes introduced frameshift mutations in the TK gene resulting in the expression of truncated polypeptides. H2G-resistant viruses were cross-resistant to ACV, and vice versa.

FIG. 1

Accumulation of H2G-TP and ACV-TP in VZV-infected MeWo cells. MeWo cells were infected with VZV and labeled with medium containing 5 μM [³H]H2G or [³H]ACV at 4 h postinfection. At the indicated time points postlabeling, infected cells were extracted and the samples were assayed by HPLC as described in Materials and Methods.

FIG. 2

Half-lives of H2G-TP in VZV-infected MRC-5 cells (A) and MeWo cells (B). Cells were infected with VZV and labeled with medium containing 5 μM [³H]H2G at 4 h postinfection. At 20 h postlabeling, the infected cells were washed and then incubated with fresh medium. At the indicated time points after the removal of the labeled medium, infected cells were extracted and the samples were assayed by HPLC as described in Materials and Methods. The half-lives of H2G-TP were calculated by a first-order decay formula.