Recombinant Phenotyping of Cytomegalovirus UL97 Kinase Sequence Variants for Ganciclovir Resistance

Chou, Sunwen

doi:10.1128/aac.00186-10

Cited by 71 publications

(125 citation statements)

References 35 publications

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“…The baseline GCV EC 50 of 1.1 M in HFFs matches previous data obtained from the same reporter-based phenotyping system since its inception (10)(11)(12). Unlike nonclonal ARPE-19␣ cells, where the baseline GCV EC 50 was 2.4 M (10), the new ARPEp cells gave a GCV EC 50 of 1.2 M, almost identical to that in standard HFF cultures.…”

Section: Resultssupporting

confidence: 74%

“…These findings are the same as those previously observed for substitutions E596D, N597D, K599R, L600I, and D605E (11,12,17,18) and unlike those for E596G/Y and K599T, which were reported to confer GCV resistance (5,17,19). Except for the common UL97 sequence polymorphism D605E, all of these substitutions are rarely encountered (2).…”

Section: Discussionsupporting

confidence: 76%

Section: Methodsmentioning

confidence: 99%

“…All mutations selected for phenotyping have been detected in one or more clinical specimens. UL97 mutations selected as controls were those with amino acid substitutions C592G and A594V (11). Previously phenotyped mutations included A591V (5, 13), 591del4 (in-frame deletion of four consecutive codons beginning with 591 [7,33]), 595del (8), 595del9 (4), 597del3 (13), 600del (5), and 601del3 (6).…”

Section: Methodsmentioning

confidence: 99%

“…Baseline CMV strain AD169 was cloned as bacterial artificial chromosome (BAC) BA1 containing a secreted alkaline phosphatase (SEAP) reporter gene for viral quantitation (11). Control BAC clones and derived strains representing wild-type baseline virus and UL97 mutants C592G and A594V were the same as reported previously (11). For the remaining 15 mutations, including those previously phenotyped in this laboratory using nonclonal strains, new mutant BAC clones and derived strains were constructed as previously described for the control strains (11).…”

Section: Methodsmentioning

confidence: 99%

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

2017

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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

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Section: Resultssupporting

confidence: 74%

Section: Discussionsupporting

confidence: 76%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

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

2017

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Cytomegalovirus Infection

Zaia¹

2015

Thomas’ Hematopoietic Cell Transplantation

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Human cytomegalovirus antiviral drug resistance in hematopoietic stem cell transplantation: current state of the art

Campos

Ribeiro

Boutolleau

et al. 2016

Reviews in Medical Virology

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Human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant recipients. The significant clinical impact of HCMV infection and progression to HCMV disease among allogeneic hematopoietic stem cell transplant recipients has been reduced by prophylactic, preemptive, and curative treatments using ganciclovir, valganciclovir, foscarnet, and cidofovir. Resistance to (val)ganciclovir results from mutations localized in HCMV UL97 gene (encoding the pUL97 phosphotransferase), UL54 gene (encoding the pUL54 DNA polymerase), or both genes, whereas foscarnet and cidofovir resistance results from mutations localized within UL54 gene only. This review is focused on HCMV antiviral drug resistance, including the functions of target genes of antivirals, the mechanisms of antiviral resistance, the different mutations in pUL97 and pUL54 that have been identified in either clinical isolates or laboratory strains, and their impact on HCMV susceptibility to antiviral drugs. It emphasizes the importance of proving that observed genetic changes confer resistance so they can be distinguished from polymorphisms. Because of the emergence of HCMV resistance to currently available drugs, novel drugs are urgently needed for the therapeutic management of HCMV-resistant infections in hematopoietic stem cell transplant patients.

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Recombinant Phenotyping of Cytomegalovirus UL97 Kinase Sequence Variants for Ganciclovir Resistance

Cited by 71 publications

References 35 publications

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

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

Cytomegalovirus Infection

Human cytomegalovirus antiviral drug resistance in hematopoietic stem cell transplantation: current state of the art

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