Cihlar T, Fuller M D, Cherrington J M
Gilead Sciences, Foster City, California 94404, USA.
J Virol. 1998 Jul;72(7):5927-36. doi: 10.1128/JVI.72.7.5927-5936.1998.
A number of specific point mutations in the human cytomegalovirus (HCMV) DNA polymerase (UL54) gene have been tentatively associated with decreased susceptibility to antiviral agents and consequently with clinical failure. To precisely determine the roles of UL54 mutations in HCMV drug resistance, recombinant UL54 mutant viruses were generated by using cotransfection of nine overlapping HCMV DNA fragments into permissive fibroblasts, and their drug susceptibility profiles were determined. Amino acid substitutions located in UL54 conserved region IV (N408D, F412C, and F412V), region V (A987G), and delta-region C (L501I, K513E, P522S, and L545S) conferred various levels of resistance to cidofovir and ganciclovir. Mutations in region II (T700A and V715M) and region VI (V781I) were associated with resistance to foscarnet and adefovir. The region II mutations also conferred moderate resistance to lobucavir. In contrast to mutations in other UL54 conserved regions, those residing specifically in region III (L802M, K805Q, and T821I) were associated with various drug susceptibility profiles. Mutations located outside the known UL54 conserved regions (S676G and V759M) did not confer any significant changes in HCMV drug susceptibility. Predominantly an additive effect of multiple UL54 mutations with respect to the final drug resistance phenotype was demonstrated. Finally, the influence of selected UL54 mutations on the susceptibility of viral DNA replication to antiviral drugs was characterized by using a transient-transfection-plus-infection assay. Results of this work exemplify specific roles of the UL54 conserved regions in the development of HCMV drug resistance and may help guide optimization of HCMV therapy.
人类巨细胞病毒（HCMV）DNA聚合酶（UL54）基因中的一些特定点突变，初步认为与抗病毒药物敏感性降低相关，进而与临床治疗失败有关。为了精确确定UL54突变在HCMV耐药性中的作用，通过将九个重叠的HCMV DNA片段共转染到允许性成纤维细胞中，构建了重组UL54突变病毒，并测定了它们的药物敏感性谱。位于UL54保守区域IV（N408D、F412C和F412V）、区域V（A987G）和δ区域C（L501I、K513E、P522S和L545S）的氨基酸替换赋予了对西多福韦和更昔洛韦不同程度的耐药性。区域II（T700A和V715M）和区域VI（V781I）的突变与对膦甲酸钠和阿德福韦的耐药性有关。区域II的突变也赋予了对洛布卡韦的中度耐药性。与其他UL54保守区域的突变不同，特定位于区域III（L802M、K805Q和T821I）的突变与不同的药物敏感性谱有关。位于已知UL54保守区域之外的突变（S676G和V759M）未导致HCMV药物敏感性发生任何显著变化。多个UL54突变对最终耐药表型主要表现为累加效应。最后，通过瞬时转染加感染试验，对所选UL54突变对病毒DNA复制对抗病毒药物敏感性的影响进行了表征。这项工作的结果例证了UL54保守区域在HCMV耐药性发展中的特定作用，并可能有助于指导HCMV治疗的优化。
