Elsevier

Journal of Clinical Virology

Volume 43, Issue 1, September 2008, Pages 107-109
Journal of Clinical Virology

Short communication
Contrasting drug resistance phenotypes resulting from cytomegalovirus DNA polymerase mutations at the same exonuclease locus

https://doi.org/10.1016/j.jcv.2008.04.005Get rights and content

Abstract

Background

Diverse mutations in the cytomegalovirus (CMV) DNA polymerase (pol) gene confer resistance to one or more of the antiviral drugs ganciclovir, foscarnet or cidofovir. The levels of resistance conferred by specific mutations are variable, ranging from insignificant resistance to triple-drug resistance.

Objectives

Three pol mutations, I521T, P522A and P522L, detected in patients who received antiviral therapy for CMV infection, were studied by recombinant phenotyping to characterize their associated drug resistance.

Study design

The individual mutations were transferred by homologous recombination into a reference CMV strain modified with a reporter gene and the drug concentrations required to reduce the reporter signal by 50% (IC50) were determined.

Results

The mutations I521T and P522A each conferred 3- to 4-fold increases in IC50 to both ganciclovir and cidofovir, while mutation P522L conferred no significant resistance to either drug. None of these mutations conferred foscarnet resistance.

Conclusions

The resistance phenotypes of mutations I521T and P522A are as predicted from the known mutation P522S, but divergent results with P522L indicate that different amino acid substitutions at the same position may not have the same effect on drug resistance. New mutations must be individually validated for proper interpretation of genotypic resistance testing.

Introduction

The human cytomegalovirus (CMV) DNA polymerase, encoded by the UL54 (pol) gene, is the target for all current systemic CMV drugs, including ganciclovir (GCV), foscarnet (FOS) and cidofovir (CDV). A considerable diversity of mutations in pol can confer resistance to any or all of these drugs, although GCV resistance usually begins with mutations in the viral UL97 kinase gene.1 Because they confer different levels of resistance and cross-resistance, over 30 individual pol mutations have been characterized by recombinant phenotyping (marker transfer), a process in which they are transferred in isolation to reference drug sensitive CMV strains to determine the resulting level of resistance to each drug.2, 3, 4 The results are used for genotypic CMV resistance testing, where the UL97 and pol DNA sequences amplified from clinical specimens are checked for mutations known to confer drug resistance. Yet new mutations continue to be identified, and some published ones remain uncharacterized, resulting in less reliable interpretations of drug resistance based on similarities with known mutations. The traditional phenotypic approach of testing clinical isolates directly for drug sensitivity in cell culture is limited by current clinical laboratory practices that do not provide a live viral isolate, and the technical complexity of such assays. Continuing our practice of providing recombinant phenotyping data where they are lacking, we report here on three mutations at an exonuclease domain of pol that illustrate the divergent phenotypes that can result from different mutations at the same locus.

Section snippets

Methods

Strains and procedures for performing recombinant phenotyping have been published.5 Briefly, reference strain T2211 was derived from standard strain AD169 by introducing unique restriction sites in pol and UL97, along with a secreted alkaline phosphatase (SEAP) expression cassette at US6, which enabled viral quantitation by assay of supernatant SEAP activity using a chemiluminescent substrate. PCR products containing the desired mutations were created by using specific mutagenic primers and

Results

CMV pol mutations at codons I521T, P522A and P522L in the exonuclease III domain were studied. Mutation I521T was published as part of a genotype of multiple UL97 and pol mutations in a pediatric stem cell recipient who developed multi-drug resistant CMV and fatal CMV encephalitis.6 More recently, another pediatric stem cell recipient in Denver received 7 months of GCV treatment and had recurrent low-level CMV DNA in the peripheral blood, which contained pol mutation I521T and UL97 mutation

Discussion

The interpretation of CMV DNA polymerase drug resistance mutations is complicated by their diversity and a track record of suspected resistance phenotypes that were not subsequently confirmed by recombinant phenotyping, including mutations at codons 515, 737, 845 and 971.3, 4, 9 The three mutations studied here are located in the exonuclease III domain, as recognized by sequence homology to the herpes simplex DNA polymerase structure.10 Based on several established resistance mutations in this

Acknowledgements

This work was supported by NIH grant AI39938 and Department of Veterans Affairs research funds. Daniel Mitchell and Laura Van Wechel provided technical assistance.

References (10)

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    The results of this study will help to understand the molecular mechanisms of HCMV drug-resistance and increases the number of mutations that can now be detected by genotypic assays. Patient 1 is an 11-year old stem-cell transplant recipient (D−/R+) from our institution who developed a disseminated HCMV-infection during conditioning (Eckle et al., 2000; Chou et al., 2008; Cihlar et al., 1998; Fischer et al., 2013; Chou et al., 1995c; Fischer et al., 2015). Since the patient did not respond to GCV, therapy was switched to FOS 100 days post transplantation.

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