Novel mutation in the UL97 gene of a clinical cytomegalovirus strain conferring resistance to ganciclovir

Abstract

Characterization of a ganciclovir-resistant cytomegalovirus strain from a patient with AIDS showed a histidine-to-glutamine change at residue 520 of UL97 (Q520 mutation). In anabolism studies, Q520 was associated with impaired phosphorylation of ganciclovir. Transfer of Q520 to a recombinant virus resulted in a ganciclovir-resistant phenotype.Resistance of clinical and laboratory cytomegalovirus (CMV) strains to ganciclovir has been associated with impaired phosphorylation of this compound in virus-infected cells (11). Characterization of ganciclovir-resistant CMV laboratory strains has demonstrated the presence of amino acid deletions (residues 590 to 593) or substitutions (residue 460) in conserved regions of the UL97 protein and/or point mutations in the DNA polymerase of the virus (2, 9, 10, 12, 13). In clinical CMV strains, resistance to ganciclovir has been associated with substitutions (residues 460, 594, and 595) or deletions (residue 595) in UL97 (1,4,14). We have identified a novel mutation in the UL97 protein of a ganciclovir-resistant CMV strain and demonstrated that this mutation is responsible for resistance to ganciclovir.(This work was presented in part at the 34th Interscience Conference on Antimicrobial Agents and Chemotherapy, Orlando, Fla., 6 October 1994.) Viruses studied were from an AIDS patient with progressive CMV disease (retinitis and gastrointestinal disease) and included an early isolate obtained prior to ganciclovir therapy and a late isolate (C-9330) from a sample of rectal tissue obtained after 421 days of ganciclovir therapy. Ganciclovir susceptibilities were determined by a DNA hybridization method (3). A fragment of the catalytic domain of UL97 was amplified by PCR using viral DNA purified from fibroblasts infected with the patient's CMV isolates and primers designed on the basis of published data on strain AD169. Primer sequences were 5Ј-CATCGACGTTTCCACACAGAC-3Ј (Z2671, forward) and 5Ј-TTGCGCCGCCAGAATGAGCAG-3Ј (Z2672, reverse). PCRs were performed with Taq polymerase and consisted of 35 cycles at 95ЊC for 1 min, 55ЊC for 2 min, and 72ЊC for 1 min. Amplified products were filter purified and sequenced with a commercial kit (Prism Ready Reaction Dyedeoxy Terminator Cycle sequencing kit; Applied Biosystems) and sequencing primers Z2671 (forward), Z2828 (5Ј-ATCCGGATTACAACGAGCGCT-3Ј, forward), Z2672 (reverse), and Z2829 (5Ј-TAACATTCGCGCAGACGGTGC-3Ј, reverse). UL97 sequences were aligned with that of strain AD169 to determine whether mutations were present in the region analyzed. A recombinant virus (C-9330-5) was obtained in marker transfer experiments after cotransfection of MRC5 cells with full-length AD169 DNA and an 858-bp UL97 DNA fragment (encompassing codon 520) amplified by PCR from strain C-9330 using primers VS9714 (5Ј-ATGTTCTTGCGC CTTACGCA-3Ј, forward) and CT9729 (5Ј-CCATGCGCAC CTCGTCC-3Ј, reverse) (12). For ganciclovir anabolism studies, MRC5 cells infected with isolate C-9330 or the recombinant strain C-9330-5 were pulse-labeled with purified 8-14 C-ganciclovir, and ...

Sequencing of Cytomegalovirus UL97 Gene for Genotypic Antiviral Resistance Testing

“…A consequence of this antiviral drug therapy is the development of GCVresistant CMV strains, which have been well documented in human immunodeficiency virus-infected patients (3,20,21,24,36). More recently resistance has been increasingly detected in isolates from transplant recipients following extended prophylactic or preemptive therapy (1,2,9,16,17,19,23,25).The UL97 phosphotransferase (kinase), a virus-encoded product, activates GCV by monophosphorylation (32). Subsequent di-and triphosphorylation are carried out by cellular enzymes.…”

Point Mutations and Antiviral Drug Resistance

“…However, it cannot identify new mutations associated with resistance. This method is more applicable to routine detection of the most common mutations, particularly those in codons 520, 460, 594, 595, 591, and 592 that confer GCV resistance (Hanson et al, 1995;Prix, 1999).…”

Drug Resistance due to Mutation in UL 97 and Certain Other Specific Genes in Cytomegalovirus Strains: An Overview

“…The mutations in both the viral phosphotransferase gene (UL97) and the viral polymerase gene (UL54) may confer antiviral drug resistance in HCMV. The mutations of the UL97 coding sequence, which may confer resistance only to ganciclovir, occur mainly in the region including codons 460-607 Hanson et al, 1995;Lurain et al, 1994). The more rare mutations in the UL54 coding sequence may confer resistance to any or all of the three most commonly used drugs (ganciclovir, foscarnet or cidofovir) (Cihlar et al, 1998b;Mousavi-Jazi et al, 2003).…”