Mutations in human cytomegalovirus UL97 gene confer clinical resistance to ganciclovir and can be detected directly in patient plasma.

Abstract

Specific mutations in the UL97 region of human cytomegalovirus (HCMV) have been found to confer resistance to laboratory-adapted strains subjected to ganciclovir selection. In this study, mutations in the UL97 region of HCMV isolates obtained from patients receiving ganciclovir therapy were examined to determine whether they would confer ganciclovir resistance, and if these mutations could be detected directly in the plasma of AIDS patients with progressive HCMV disease despite ganciclovir treatment. A single nucleotide change within a conserved region of UL97 was found in five resistant isolates, resulting in an amino acid substitution in residue 595: from leucine to phenylalanine in one, and from leucine to serine in four resistant isolates. A sixth resistant isolate demonstrated a single nucleotide change, leading to a threonine to isoleucine substitution in residue 659. The role of the 595 amino acid substitution in conferring ganciclovir resistance was confirmed by marker transfer experiments. In further studies, direct sequencing of HCMV DNA present in plasma obtained from persons with resistant viruses revealed the identical amino acid substitutions in plasma as those present in the cultured viruses. These findings indicate that clinical resistance to ganciclovir can result from specific point mutations in the UL97 gene, and that the emergence of the resistant genotype can be detected directly in patient plasma. (J. Clin. Invest. 1995. 95:257-263.)

Differentiated Levels of Ganciclovir Resistance Conferred by Mutations at Codons 591 to 603 of the Cytomegalovirus UL97 Kinase Gene

“…A591V has not been documented as a polymorphism in baseline UL97 sequences from untreated subjects but has occasionally been detected since the early use of GCV (5,20,21), including the emergence of A591V as a new mutation after GCV therapy (22). The first published phenotype was from plaque reduction assays showing a GCV EC 50 of 7.9 M, which was 1.3-fold increased over control wild-type EC 50 s (5) but above the cutoff of 6 M originally proposed for classifying GCV resistance (23).…”

Functional regions of the human cytomegalovirus protein pUL97 involved in nuclear localization and phosphorylation of ganciclovir and pUL97 itself.

“…This could indicate that this region might be involved in substrate binding. Point mutations and small deletions in this domain have been detected in phenotypically GCV-resistant HCMV (Lurain et al, 1994 ;Wolf et al, 1995 ;Baldanti et al, 1995 ;Chou et al, 1995 ;Hanson et al, 1995). The 4 aa deletion and the point mutations investigated here in the vaccinia virus system had no detectable influence on phosphorylation of pUL97.…”

“…which induce the resistance of HCMV strains to GCV in biological assays (Sullivan et al, 1992 ;Lurain et al, 1994 ;Wolf et al, 1995 ;Baldanti et al, 1995 ;Chou et al, 1995 ;Hanson et al, 1995). Until now, only point mutations between amino acids 460 and 607 have been detected in GCV-resistant clinical isolates, and neither isolation of clinical HCMV strains lacking the UL97 ORF or carrying extended deletions in the UL97 ORF has been reported, nor has anyone succeeded in generating such mutants in the laboratory (He et al, 1997 ;Michel et al, 1996).…”

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

“…The facts, that specific mutations in the UL97 and UL54 genes are associated with antiviral drug resistance, have led to the development of genetic methods. These are based on PCR amplification of the specific region of the genome followed by restriction enzyme analysis or direct sequencing of the amplification product/products (Alain et al, 1997;Lurain et al, 2002;Mendez et al, 1999b;Wolf et al, 1995). An obvious advantage of these assays is the possibility to use clinical specimens directly, rather than virus isolates, which significantly shortens the time required for performance of the test.…”