Contrasting effects of W781V and W780V mutations in helix N of herpes simplex virus 1 and human cytomegalovirus DNA polymerases on antiviral drug susceptibility.

UNLABELLED: DNA polymerases of the Herpesviridae and bacteriophage RB69 belong to the α-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 [EC50s], respectively). The recombinant HCMV mutant harboring the W780V mutation was slightly resistant to foscarnet (a 1.9-fold increase in the EC50) 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. IMPORTANCE: DNA polymerases of the Herpesviridae and bacteriophage RB69 belong to the α-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.

Characterization of multiple cytomegalovirus drug resistance mutations detected in a hematopoietic stem cell transplant recipient by recombinant phenotyping.

Cytomegalovirus resistance to antivirals is a major problem in transplant recipients. We evaluated the impact of five mutations (A594V, L595F, and E655K in the UL97 gene and V526L and E756K in the UL54 gene), detected in a blood sample from a stem cell transplant recipient, on drug susceptibilities and replicative capacities of recombinant viruses.

Novel method based on "en passant" mutagenesis coupled with a gaussia luciferase reporter assay for studying the combined effects of human cytomegalovirus mutations.

Human cytomegalovirus (HCMV) resistance to antivirals is a major problem in immunocompromised patients. Drug resistance is characterized by phenotypic testing or genotypic analysis of the phosphotransferase (UL97) and DNA polymerase (UL54) genes. However, genotypic assays require further characterization of unknown mutations in the drug resistance phenotype. Here, we describe a novel method for generating single or multiple mutations anywhere in the HCMV genome and for studying their effects on drug susceptibility. This method is based on cloning of the reference AD169 strain in a bacterial artificial chromosome and the use of "en passant" mutagenesis in bacteria to introduce mutations in recombinant HCMV without scar sequences. We also used this methodology to introduce the Gaussia luciferase reporter gene into the genome of the recombinant virus. To validate our system, the well-characterized single mutants with UL97 A594V and UL54 E756K mutations as well as the undescribed A594V/E756K double mutant were generated and their drug susceptibility profiles were determined by measuring the luciferase activity in cell culture supernatants. Drug susceptibility phenotypes for the A594V (8.2-fold increase in ganciclovir 50% effective concentration [EC50]) and E756K (1.9-, 3.9-, and 3.0-fold increases in ganciclovir, foscarnet, and cidofovir EC50s, respectively) mutants were similar to those previously reported, while the double mutant exhibited 10.8-, 4.1-, and 2.0-fold increases in ganciclovir, foscarnet, and cidofovir EC50s, respectively. The combination of the Gaussia luciferase reporter-based assay with the markerless "en passant" mutagenesis methodology constitutes an efficient system for studying phenotypes with single or multiple HCMV mutations.

Artesunate demonstrates in vitro synergism with several antiviral agents against human cytomegalovirus.

BACKGROUND: Human cytomegalovirus (HCMV) infections remain a major problem in immunocompromised patients. Three antiviral agents, ganciclovir (GCV), foscarnet (FOS) and cidofovir (CDV), are currently approved for the treatment of HCMV infections. They all target the viral DNA polymerase and are associated with significant side effects. Combinations of novel antiviral compounds acting on different targets such as artesunate (ART) with currently approved drugs or eventually letermovir or maribavir (MBV) may result in synergistic effects. Here, we evaluated the in vitro activity of a series of two-drug combinations against a wild-type recombinant HCMV strain by the Gaussia luciferase (GLuc) reporter assay. METHODS: The in vitro activity of each drug was first tested individually against HCMV by using the GLuc reporter assay. The activity of two-drug combinations consisting of ART and currently approved drugs, as well as letermovir or MBV, was then analysed by the Chou-Talalay method. RESULTS: The concentrations of GCV, FOS, CDV and ART that reduced the GLuc activity by 50% (EC50 values) were 3.92 ±1.64 µM, 62.45 ±8.39 µM, 0.68 ±0.19 µM and 3.86 ±1.25 µM, respectively, whereas those of MBV and letermovir were 64 ±22 nM and 2.50 ±0.83 nM, respectively. The combination of ART with GCV, CDV or MBV was associated with synergism, whereas combination of ART with FOS or letermovir resulted in moderate synergism. As expected, the combination of MBV with GCV was antagonistic. CONCLUSIONS: These results suggest that the combination of ART with the antiviral agents tested in this study could be an interesting strategy for the treatment of HCMV infections to reduce toxicity and drug-resistance development.