title: 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 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.
  
  
  OBJECTIVE
  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.
authors: Research Center in Infectious Diseases, CHU de Québec and Laval University, Quebec City, QC, Canada.
authors: Jocelyne
authors: J
authors: Piret
authors: Research Center in Infectious Diseases, CHU de Québec and Laval University, Quebec City, QC, Canada.
authors: Nathalie
authors: N
authors: Goyette
authors: Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, Vermont, USA.
authors: Brian E
authors: BE
authors: Eckenroth
authors: Research Center in Infectious Diseases, CHU de Québec and Laval University, Quebec City, QC, Canada.
authors: Emilien
authors: E
authors: Drouot
authors: Department of Microbiology & Immunology, McGill University, Montreal, QC, Canada Department of Biochemistry, McGill University, Montreal, QC, Canada Department of Medicine, McGill University, Montreal, QC, Canada.
authors: Matthias
authors: M
authors: Götte
authors: Research Center in Infectious Diseases, CHU de Québec and Laval University, Quebec City, QC, Canada Guy.Boivin@crchudequebec.ulaval.ca.
authors: Guy
authors: G
authors: Boivin
mesh_terms: D000212:Acyclovir; D000704:Analysis of Variance; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002522:Chlorocebus aethiops; D003587:Cytomegalovirus / Q000201:enzymology*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D024882:Drug Resistance, Viral / Q000235:genetics*; D017245:Foscarnet; D015774:Ganciclovir; D018014:Gene Transfer Techniques; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D020125:Mutation, Missense / Q000235:genetics*; D017433:Protein Structure, Secondary / Q000235:genetics*; D014709:Vero Cells
chemical_list: D000998:Antiviral Agents; D017245:Foscarnet; D004259:DNA-Directed DNA Polymerase; D015774:Ganciclovir; D000212:Acyclovir