Contrasting Effects of W781V and W780V Mutations in Helix N of Herpes Simplex Virus 1 and Human Cytomegalovirus DNA Polymerases on Antiviral Drug Susceptibility

Abstract

DNA polymerases of the Herpesviridae and bacteriophage RB69 belong to the ␣-like DNA polymerase family. In spite of similarities in structure and function, the RB69 enzyme is relatively resistant to foscarnet, requiring the mutation V478W in helix N to promote the closed conformation of the enzyme to make it susceptible to the antiviral. Here, we generated recombinant herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) mutants harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility and viral replicative capacity. The mutation W781V in HSV-1 induced resistance to foscarnet, acyclovir, and ganciclovir (3-, 14-, and 3-fold increases in the 50% effective concentrations [EC 50 s], respectively). The recombinant HCMV mutant harboring the W780V mutation was slightly resistant to foscarnet (a 1.9-fold increase in the EC 50 ) and susceptible to ganciclovir. Recombinant HSV-1 and HCMV mutants had altered viral replication kinetics. The apparent inhibition constant values of foscarnet against mutant UL30 and UL54 DNA polymerases were 45-and 4.9-fold higher, respectively, than those against their wild-type counterparts. Structural evaluation of the tryptophan position in the UL54 DNA polymerase suggests that the bulkier phenylalanine (fingers domain) and isoleucine (N-terminal domain) could induce a tendency toward the closed conformation greater than that for UL30 and explains the modest effect of the W780V mutation on foscarnet susceptibility. Our results further suggest a role of the tryptophan in helix N in conferring HCMV and especially HSV-1 susceptibility to foscarnet and the possible contribution of other residues localized at the interface between the fingers and N-terminal domains. IMPORTANCEDNA polymerases of the Herpesviridae and bacteriophage RB69 belong to the ␣-like DNA polymerase family. However, the RB69 DNA polymerase is relatively resistant to the broad-spectrum antiviral agent foscarnet. The mutation V478W in helix N of the fingers domain caused the enzyme to adopt a closed conformation and to become susceptible to the antiviral. We generated recombinant herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) mutants harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility. The W781V mutation in HSV-1 induced resistance to foscarnet, whereas the W780V mutation in HCMV slightly decreased drug susceptibility. This study suggests that the different profiles of susceptibility to foscarnet of the HSV-1 and HCMV mutants could be related to subtle conformational changes resulting from the interaction between residues specific to each enzyme that are located at the interface between the fingers and the N-terminal domains.A ll antiviral agents currently approved for the treatment of herpes simplex virus (HSV) and human cytomegalovirus (HCMV) infections ul...

Distribution and effects of amino acid changes in drug-resistant α and β herpesviruses DNA polymerase

“…, the IC 50 of FOS for VZV DNApol was measured bellow 1 μM (49) while in another study, an IC 50 of 180 nM was observed (51). In contrast, K I values ranging from 300 nM to 3.5 μM were obtained with HCMV UL54 DNApol (62,74,76,78). This difference in inhibitory effects might be due to the assay used, its sensitivity or to the quality of the protein purification.…”

“…investigated the effects of W780V in HCMV UL54 and W781V in HSV-1 UL30, which confer resistance to FOS, GCV and/or ACV (76). In HSV-1 UL30, W781V increased the K I from 0.04 to 1.8 μM (45-fold), while in HCMV UL54, W780V increased the K I from 0.55 to 2.7 μM (4.9-fold).…”

Novel Method Based on Real-Time Cell Analysis for Drug Susceptibility Testing of Herpes Simplex Virus and Human Cytomegalovirus

“…The replicative capacities of HSV-1 and HCMV strains that harbored mutations in the DNA polymerase were reduced compared to those of their respective WT strains when determined by RTCA. Furthermore, we have already demonstrated that there was good concordance between viral replicative capacity determined by RTCA and genome copy numbers in cell lysates measured by real-time PCR for recombinant WT and mutant HSV-1 strains in Vero cells (38). The RTCA technology has the advantages of requiring less work and a shorter manipulation time to evaluate the viral replicative capacities of HSV-1 and HCMV strains than the measurement of virus yields by classical plaque assays or intracellular genome copy numbers by real-time PCR.…”

Human cytomegalovirus antiviral drug resistance in hematopoietic stem cell transplantation: current state of the art

“…In addition, some mutations with a resistance index of less than 2 are considered either to confer a reduction of susceptibility to HCMV antiviral drugs or to have no effect on drug susceptibility. For example, A505V, T552N, Q578L, I726V/T, L802V, and L862F mutations are considered to be associated with a lower drug susceptibility, because the majority of them are located within conserved sites of DNA polymerase; some are located at the same position than confirmed resistance‐associated amino acid changes, and all of them have been observed in patients experiencing antiviral treatment failure .…”

“…Moreover, resistance mutations in UL97 and/or UL54 have been associated with treatment failure and progression of HCMV disease and also with poor outcome and in some cases with an attributable mortality of almost 2% . In both pediatric and adult allo‐HSCT recipients, several resistance mutations have been reported, and while some are shared with other immunocompromised individuals , others are only found in HSCT recipients (Tables and ).…”