PHOSPHORYLATION OF ACYCLOVIR, GANCICLOVIR, PENCICLOVIR AND S2242 BY THE CYTOMEGALOVIRUS UL97 PROTEIN - A QUANTITATIVE-ANALYSIS USING RECOMBINANT VACCINIA VIRUSES

We used recombinant vaccinia viruses (rVV) containing the UL97 open re ading frame (ORF) of the human cytomegalovirus (HCMV) to investigate t he UL97-dependent phosphorylation of different nucleoside analogs. The rVV T1 expressed the wild-type UL97 protein whereas rVV A5 contained a 12 bp deletion in the UL97 which had been known to be responsible fo r resistance of HCMV to ganciclovir (GCV). The rVV Tlopal was generate d which contained a stop codon at position 1089 of the UL97 ORF and wh ich expressed a truncated UL97 protein. We quantitatively analyzed the capability of these rVVs to phosphorylate GCV, penciclovir (PCV), aci clovir (ACV) and 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl] purine (S 2242) as well as the natural nucleosides deoxycytidine and deoxythymid ine. Moreover, we compared their phosphorylating capability with that of herpes simplex virus type 1 strains. In thymidine kinase (TK)-defic ient 143B cells infected with rVV T1, the three compounds GCV, ACV and PCV were phosphorylated with different efficiency whereas in cells in fected with the rVV A5 a markedly reduced but not completely abolished phosphorylation of these compounds was observed. In rVV Tlopal-infect ed cells no specific phosphorylation of the compounds was detectable a t all. Neither S2242 nor the natural substrates of TKs were phosphoryl ated by any of the vaccinia recombinants. The rVVs proved to be a suit able tool for analysis of UL97-dependent phosphorylation of nucleoside analogs and also allowed to quantitatively study the influence of UL9 7 mutations on drug phosphorylation. (C) 1997 Elsevier Science B.V.