Research Center in Infectious Diseases, CHU de Québec-Laval University, Quebec City, Quebec, Canada.
PROTEO and Institute of Integrative and Systems Biology, Laval University, Quebec City, Quebec, Canada.
Antimicrob Agents Chemother. 2021 Jun 17;65(7):e0039021. doi: 10.1128/AAC.00390-21.
Amino acid substitutions conferring resistance of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) to foscarnet (PFA) are located in the genes and , 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 (ECs) 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 ECs 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.
氨基酸取代使单纯疱疹病毒 1（HSV-1）和人类巨细胞病毒（HCMV）对膦甲酸（PFA）产生耐药性，这些取代分别位于编码 DNA 聚合酶（pol）的 和 基因中。在这项研究中，我们分析了位于螺旋 K 和区域 II 中参与 DNA pol 构象变化的取代对病毒的影响。通过对（对 PFA 敏感的）人类疱疹病毒和（对 PFA 耐药的）噬菌体的螺旋 K 和区域 II 的序列比对，确定了理论取代，并通过重组表型引入病毒基因组。我们对 HSV-1 和 HCMV 突变体对 PFA 的敏感性进行了特征分析。在 UL30 中，取代 I619K（螺旋 K）、V715S 和 A719T（均位于区域 II）使 PFA 的 50%有效浓度（EC）分别比野生型（WT）增加了 2.5、5.6 和 2.0 倍。在 UL54 中，取代 Q579I（螺旋 K）使病毒对 PFA 高度敏感（变化 0.17 倍），而取代 Q697P、V715S 和 A719T（均位于区域 II）使 PFA 的 EC 分别比 WT 增加了 3.8、2.8 和 2.5 倍。这些结果通过使用携带这些取代的重组 DNA pol 进行的酶分析得到了证实。三维建模表明，位于 UL30 和 UL54 DNA pol 的螺旋 K 和区域 II 中的取代，赋予了对 PFA 的耐药性/超敏性，使这些酶更倾向于开放/闭合构象，从而降低/提高了蛋白质对药物的亲和力。因此，这项研究表明，UL30 和 UL54 DNA pol 的这两个区域都参与了这些蛋白质的构象变化，并可能影响这两种病毒对 PFA 的敏感性。
