A point mutation in the human cytomegalovirus DNA polymerase gene confers resistance to ganciclovir and phosphonylmethoxyalkyl derivatives

Abstract

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.

Characterization of Wild-Type and Cidofovir-Resistant Strains of Camelpox, Cowpox, Monkeypox, and Vaccinia Viruses

“…Experiments showed that cidofovir-PP was 8.5-fold less inhibitory to the cowpox-R (SF) DNA polymerase than it was to cowpox-WT polymerase. Other investigators have determined that human cytomegalovirus resistance to cidofovir was due to mutations in the viral DNA polymerase (29). Thus, it seems likely that orthopoxvirus resistance to cidofovir is due primarily to mutations in the viral DNA polymerase, resulting in a decreased sensitivity of the enzyme to inhibition by cidofovir-PP.…”

“…Mutations were found in the viral DNA polymerase gene that conferred drug resistance (29,32). Resistance to cidofovir results in cross-resistance to other antiviral drugs (29,31).…”

“…Mutations were found in the viral DNA polymerase gene that conferred drug resistance (29,32). Resistance to cidofovir results in cross-resistance to other antiviral drugs (29,31). The most serious clinical consequence of infection with drug-resistant viruses is the inability to effectively treat the disease with the specific medication and sometimes with other similarly acting drugs.…”

Cidofovir Resistance in Vaccinia Virus Is Linked to Diminished Virulencein Mice

“…It is widely assumed that in virus-infected cells, the primary enzymatic targets of these drugs are viral DNA polymerases. Indeed, HPMPC-resistant (HPMPC R ) viruses have been isolated in vitro bearing mutations in the DNA polymerase genes of human cytomegalovirus (HCMV), herpes simplex virus (HSV), and human adenovirus (4,17,35). We have also shown that diphosphoryl HPMPC inhibits primer extension and proofreading reactions catalyzed by vaccinia virus (VAC) DNA polymerase in vitro (18).…”

Cidofovir for treating adenoviral hemorrhagic cystitis in hematopoietic stem cell transplant recipients

“…To date, the majority of ganciclovir-resistant isolates are UL97 gene product (phosphokinase) mutants and remain susceptible to cidofovir 5 . Reduced susceptibility to cidofovir, however, has been reported for DNA polymerase mutants of CMV which are resistant to ganciclovir [6][7][8][9] . To date, all clinical isolates which exhibit high level resistance to ganciclovir, due to mutations in both the DNA polymerase and UL97 genes, have been shown to be cross resistant to cidofovir.…”