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

Abstract

Diagnostic mutations in the cytomegalovirus UL97 kinase gene are used to assess the level of associated ganciclovir resistance and therapeutic options. The best-known mutations at codons 460, 520, or 591 to 607 individually confer 5-to 10-fold-decreased ganciclovir susceptibility, except that a 3-fold decrease occurs in the case of the amino acid substitution C592G. Less common point and in-frame deletion mutations at codons 591 to 603 remain incompletely characterized. The ganciclovir susceptibilities of 17 mutants in this codon range were evaluated by use of the same recombinant phenotyping system and extensive assay replicates in two types of cell cultures. Amino acid substitutions K599E and T601M conferred no ganciclovir resistance, while A591V conferred 3.8-fold-decreased susceptibility. In-frame deletions of three or more codons conferred at least 8-fold-increased ganciclovir resistance, while the level of resistance conferred by one-or two-codon deletions varied from 4-to 10-fold, depending on their location. Measured levels of ganciclovir resistance were closely comparable when assays were performed in either fibroblasts or modified retinal epithelial cells. The significant revision of a few previously published resistance phenotypes and the new data strengthen the interpretation of genotypic testing for cytomegalovirus drug resistance.KEYWORDS cytomegalovirus, drug resistance mechanisms, ganciclovir P rolonged treatment of human cytomegalovirus (CMV) infection with ganciclovir (GCV) or its oral prodrug, valganciclovir, may result in antiviral drug resistance when viral replication is incompletely suppressed. Without the routine availability of susceptibility testing on CMV culture isolates, the laboratory diagnosis of drug resistance is dependent on the direct detection of characteristic viral mutations in clinical specimens (1, 2). GCV resistance mutations usually develop first in the UL97 kinase gene involved in the initial phosphorylation of GCV that is necessary for its antiviral action. One of seven UL97 mutations (M460V/I, H520Q, C592G, A594V, L595S, and C603W) is initially detected in Ͼ80% of cases of GCV resistance in clinical practice, with most of the remainder consisting of less common amino acid substitutions or in-frame deletions at codons 590 to 607 or of mutations in the UL54 DNA polymerase gene (2). The canonical UL97 mutations confer 5-to 10-fold increases in the GCV concentration required for 50% viral growth inhibition (50% effective concentration [EC 50 ]), except that C592G confers a 3-fold increase. Given the limited alternative treatment options, mutations conferring lower-grade resistance may be amenable to GCV dose escalation, according to consensus treatment guidelines (3). Therefore, accurate calibration of the levels of resistance conferred by specific mutations is desirable for treatment planning purposes

Patient-Derived Cytomegaloviruses with Different Ganciclovir Sensitivities from UL97 Mutation Retain Their Replication Efficiency and Some Kinase Activity In Vitro

“…The results showed UL97-A594V led to the highest EC 50 increase for GCV compared to that for the wild-type virus (5.7-fold), followed by UL97-A594S (4.2-fold), whereas UL97-G598D did not show an increase (Table 1). A recent study systematically assessed 17 mutants over the codon range 591 to 603 with high replicate numbers to demonstrate mutations in UL97 resulted in different levels of GCV resistance (19). The reported fold change between wild-type and pUL97 A594V-carrying virus was 6.4-fold, therefore slightly higher than our findings (19).…”

A third component of the human cytomegalovirus terminase complex is involved in letermovir resistance

“…Mutant recombinant viruses were constructed as described below for phenotypic assays of specific mutations and combinations of interest. Drug susceptibility and growth assays were performed in retinal epithelial cells transduced to over-express platelet-derived growth factor receptor α (ARPEp), which facilitate assay standardization as described (Chou, 2017; Chou et al, 2017). …”

The Third International Consensus Guidelines on the Management of Cytomegalovirus in Solid-organ Transplantation

“…Deletion of less than 3 codons may confer varying degrees of GCV resistance (fourfold to 10-fold). 302 d D605E is a baseline sequence polymorphism common in east Asia, unrelated to drug resistance. e Maribavir cross-resistance documented; all except F342S are markedly growth-inhibited.…”