CHARACTERIZATION OF DRUG RESISTANCE-ASSOCIATED MUTATIONS IN THE HUMANCYTOMEGALOVIRUS DNA-POLYMERASE GENE BY USING RECOMBINANT MUTANT VIRUSES GENERATED FROM OVERLAPPING DNA FRAGMENTS

A number of specific point mutations in the human cytomegalovirus (HCM V) DNA polymerase (UL54) gene have been tentatively associated with de creased susceptibility to antiviral agents and consequently with clini cal failure. To precisely determine the roles of UL54 mutations in HCM V drug resistance, recombinant UL54 mutant viruses were generated by u sing cotransfection of nine overlapping HCMV DNA fragments into permis sive fibroblasts, and their drug susceptibility profiles were determin ed. Amino acid substitutions located in UL54 conserved region IV (N408 D, F412C, and F412V), region V (A987G), and delta-region C (L501I, K51 3E, P522S, and L545S) conferred various levels of resistance to cidofo vir and ganciclovir. Mutations in region II (T700A and V715M) and regi on VI (V781I) were associated with resistance to foscarnet and adefovi r. The region II mutations also conferred moderate resistance to lobuc avir. In contrast to mutations in other UL54 conserved regions, those residing specifically in region LII (L802M, K805Q, and T821I) were ass ociated,vith various drug susceptibility profiles, Mutations located o utside the known UL54 conserved regions (S676G and V759M) did not conf er any significant changes in HCMV drug susceptibility. Predominantly an additive effect of multiple UL54 mutations,vith respect to the fina l drug resistance phenotype was demonstrated. Finally, the influence o f selected UL54 mutations on the susceptibility of viral DNA replicati on to antiviral drugs was characterized by using a transient-transfect ion-plus-infection assay. Results of this work exemplify specific role s of the UL54 conserved regions in the development of HCMV drug resist ance and may help guide optimization of HCMV therapy.