SINGLE AMINO-ACID CHANGES IN THE DNA-POLYMERASE CONFER FOSCARNET RESISTANCE AND SLOW-GROWTH PHENOTYPE, WHILE MUTATIONS IN THE UL97-ENCODED PHOSPHOTRANSFERASE CONFER GANCICLOVIR RESISTANCE IN 3 DOUBLE-RESISTANTHUMAN CYTOMEGALOVIRUS STRAINS RECOVERED FROM PATIENTS WITH AIDS

Three human cytomegalovirus (HCMV) strains (VR4760, VR4955, and VR5120 ) showing double resistance to ganciclovir (GCV) and foscarnet (PFA) w ere isolated from three patients with AIDS who underwent multiple sequ ential courses of therapy with GCV and PFA (A. Sarasini, F. Baldanti, M. Furione, E. Percivalle, R. Brerra, M. Barbi, and G. Gerna, J. Med. Virol,, 47:237-244, 1995). We previously demonstrated that the three s trains were genetically unrelated and that each of them was present as a single viral population in vivo, Thus, in each of the three cases, a single viral strain was resistant to both GCV and PFA. In the presen t paper, we report the characterization of the molecular bases of the double resistance and demonstrate that the PFA resistance is associate d with a slower replication of HCMV strains in cell cultures. Sequenci ng of the UL97 and UL54 genes, GCV anabolism assays, and marker transf er experiments showed that GCV resistance was due to single amino acid changes in the UL97 gene product (VR4760, Met-460-->Ile; VR4955, Ala- 594-->Val; VR5120, Leu595-->Ser), while single amino acid changes in d omain II of the DNA polymerase (VR4760 and VR5120, Val-715-->Met; VR49 55, Thr-700-->Ala) were responsible for both the PFA resistance and th e slow-growth phenotype, Thus, in these three cases, double resistance to GCV and PFA was not due to a single mutation conferring cross-resi stance or to the presence of a mixture of strains with different drug susceptibilities. The HCMV DNA polymerase recombinant strains carrying the mutations conferring PFA resistance were sensitive to GCV and S)- 1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC). In addition, the same UL54 mutations were responsible for the slow growth of the cl inical isolates, since the recombinant strains showed a marked delay i n immediate-early antigen plaque formation and a reduction of infectio us virus yield compared with AD169, from which they were derived, Thes e results may have some important implications for the successful isol ation, propagation, and characterization of PFA-resistant strains from clinical samples containing mixed viral populations.