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Impact of Amino Acid Substitutions in Region II and Helix K of Herpes Simplex Virus 1 and Human Cytomegalovirus DNA Polymerases on Resistance to Foscarnet.

ABSTRACT: Amino acid substitutions conferring resistance of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) to foscarnet (PFA) are located in the genes UL30 and UL54, respectively, encoding the DNA polymerase (pol). In this study, we analyzed the impact of substitutions located in helix K and region II that are involved in the conformational changes of the DNA pol. Theoretical substitutions were identified by sequences alignment of the helix K and region II of human herpesviruses (susceptible to PFA) and bacteriophages (resistant to PFA) and introduced in viral genomes by recombinant phenotyping. We characterized the susceptibility of HSV-1 and HCMV mutants to PFA. In UL30, the substitutions I619K (helix K), V715S, and A719T (both in region II) increased mean PFA 50% effective concentrations (EC₅₀s) by 2.5-, 5.6-, and 2.0-fold, respectively, compared to the wild type (WT). In UL54, the substitution Q579I (helix K) conferred hypersusceptibility to PFA (0.17-fold change), whereas the substitutions Q697P, V715S, and A719T (all in region II) increased mean PFA EC₅₀s by 3.8-, 2.8- and 2.5-fold, respectively, compared to the WT. These results were confirmed by enzymatic assays using recombinant DNA pol harboring these substitutions. Three-dimensional modeling suggests that substitutions conferring resistance/hypersusceptibility to PFA located in helix K and region II of UL30 and UL54 DNA pol favor an open/closed conformation of these enzymes, resulting in a lower/higher drug affinity for the proteins. Thus, this study shows that both regions of UL30 and UL54 DNA pol are involved in the conformational changes of these proteins and can influence the susceptibility of both viruses to PFA.

SUBMITTER: Zarrouk K

PROVIDER: S-EPMC8218610 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:UnlabelledDNA 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.ImportanceDNA 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.

Project description:BACKGROUND:Herpesviridae reactivation among non-immunocompromised critically ill patients is associated with impaired prognosis, especially during acute respiratory distress syndrome (ARDS). However, little is known about herpes simplex virus (HSV) and Cytomegalovirus (CMV) reactivation occurring in patients with severe ARDS under veno-venous extracorporeal membrane oxygenation (ECMO). We tried to determine the frequency of Herpesviridae reactivation and its impact on patients' prognosis during ECMO for severe ARDS. RESULTS:During a 5-year period, 123 non-immunocompromised patients with a severe ARDS requiring a veno-venous ECMO were included. Sixty-seven patients (54%) experienced HSV and/or CMV reactivation during ECMO course (20 viral co-infection, 40 HSV alone, and 7 CMV alone). HSV reactivation occurred earlier than CMV after the beginning of MV [(6-15) vs. 19 (13-29) days; p?<?0.01] and after ECMO implementation [(2-8) vs. 14 (10-20) days; p?<?0.01]. In univariate analysis, HSV/CMV reactivation was associated with a longer duration of mechanical ventilation [(22-52.5) vs. 17.5 (9-28) days; p?<?0.01], a longer duration of ECMO [15 (10-22.5) vs. 9 (5-14) days; p?<?0.01], and a prolonged ICU [29 (19.5-47.5) vs. 16 (9-30) days; p?<?0.01] and hospital stay [44 (29-63.5) vs. 24 (11-43) days; p?<?0.01] as compared to non-reactivated patients. However, in multivariate analysis, viral reactivation remained associated with prolonged MV only. When considered separately, both HSV and CMV reactivation were associated with a longer duration of MV as compared to non-reactivation patients [29 (19.5-41) and 28 (20.5-37), respectively, vs. 17.5 (9-28) days; p?<?0.05]. Co-reactivation patients had a longer duration of MV [58.5 (38-72.3); p?<?0.05] and ICU stay [51.5 (32.5-69) vs. 27.5 (17.75-35.5) and 29 (20-30.5), respectively] as compared to patients with HSV or CMV reactivation alone. In multivariate analysis, HSV reactivation remained independently associated with a longer duration of MV and hospital length of stay. CONCLUSIONS:Herpesviridae reactivation is frequent among patients with severe ARDS under veno-venous ECMO and is associated with a longer duration of mechanical ventilation. The direct causative link between HSV and CMV reactivation and respiratory function worsening under ECMO remains to be confirmed.

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Impact of Amino Acid Substitutions in Region II and Helix K of Herpes Simplex Virus 1 and Human Cytomegalovirus DNA Polymerases on Resistance to Foscarnet.

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