Human cytomegalovirus (HCMV) is a major pathogen in immunosuppressed individuals, including patients with acquired immune deficiency syndrome. The nucleoside analogue ganciclovir (9-(1,3-dihydroxy-2-propoxymethyl)-guanine) is one of the few drugs available to treat HCMV infections, but resistant virus is a growing problem in the clinic and there is a critical need for new drugs. The study of ganciclovir-resistant mutants has indicated that the selective action of ganciclovir depends largely on virus-controlled phosphorylation in HCMV-infected cells. The enzyme(s) responsible have not been identified. Here we report that the HCMV gene UL97, whose predicted product shares regions of homology with protein kinases, guanylyl cyclase and bacterial phosphotransferases, controls phosphorylation of ganciclovir in HCMV-infected cells. A four-amino-acid deletion of UL97 in a conserved region, which in cyclic AMP-dependent protein kinase participates in substrate recognition, causes impaired ganciclovir phosphorylation. The implications of these results for antiviral drug development and drug resistance are discussed.

The prophylactic administration of ganciclovir after heart transplantation is safe, and in CMV-seropositive patients it reduces the incidence of CMV-induced illness.

Nature (London) 358:162-164, 1992]. In the present study, we mapped the second mutation to a 4.1-kb DNA fragment containing the DNA polymerase gene and showed that it confers ganciclovir resistance without impairing phosphorylation. Sequence analysis of the 4.1-kb region revealed a single nucleotide change that resulted in a glycine-to-alanine substitution at position 987 within conserved region V of the DNA polymerase. Recombinant viruses constructed to contain the DNA polymerase mutation but not the phosphorylation defect displayed intermediate resistance (4-to 6-fold) to ganciclovir relative to the original mutant 759rD100 (22-fold); the recombinant viruses also displayed resistance to ganciclovir cyclic phosphate (7-fold), 1-(dihydroxy-2-propoxymethyl)-cytosine (12-fold), and the phosphonylmethoxyalkyl derivatives (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (8-to 10-fold). However, the recombinant viruses remained susceptible to certain related compounds. These results imply that the human cytomegalovirus DNA polymerase is a selective target for the antiviral activities of ganciclovir, certain of its derivatives and phosphonomethoxyalkyl derivatives; support a role for region V in substrate recognition; and suggest the possibility of clinical resistance of human cytomegalovirus to these compounds because of polymerase mutations.Human cytomegalovirus (HCMV) is a serious and often life-threatening pathogen of newborn and immunocompromised individuals including transplant recipients and patients with AIDS (47, 56). Only a few drugs are available to treat or suppress HCMV infections; these include the nucleoside analogs ganciclovir {[9-(1,3-dihydroxy-2-propoxymethyl)-guanine]; DHPG} and, at high doses, acyclovir, and the PPi analog foscarnet. These drugs have limited efficacies, they cause toxic side effects, there are difficulties in drug delivery and distribution associated with these drugs, and there is the potential (3, 49) or actual emergence of resistant virus (16,25). These limitations highlight the need both to develop alternative therapies for HCMV infections and to understand the mechanisms of anti-HCMV drug resistance.We have previously described a DHPG-resistant HCMV mutant, 759'D100, derived from strain AD169, which was susceptible to foscamet (3) and recently showed that mutant 759rD100 contains two DHPG resistance mutations (51). One of these lies in the UL97 gene and controls DHPG phosphorylation (51). In the study described here we mapped the second DHPG resistance mutation to a conserved region of the HCMV DNA polymerase (pot) gene and showed that it confers resistance not only to DHPG but also to several other promising antiherpesvirus drugs. The results have implications for drug mechanisms and polymerase function and indicate the potential for drug resistance in the clinical setting.

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