Recombinant Phenotyping of Cytomegalovirus UL54 Mutations That Emerged during Cell Passages in the Presence of either Ganciclovir or Foscarnet

Gilbert, Clare; Azzi, Arezki; Goyette, Nathalie; Lin, Sheng‐Xiang; Boivin, Guy

doi:10.1128/aac.00334-11

Cited by 28 publications

(29 citation statements)

References 53 publications

(105 reference statements)

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“…The regions of UL54 and UL97 that were targeted by primers in the library preparation step were selected based on the locations of phenotypically validated drug resistance mutations (6,32,37,38). The optimization of these primer sets was performed using the AD169 strain to ensure that all amplicons produced bands of similar intensities when examined by agarose gel electrophoresis.…”

Section: Resultsmentioning

confidence: 99%

Detection of Cytomegalovirus Drug Resistance Mutations by Next-Generation Sequencing

et al. 2013

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Antiviral therapy for cytomegalovirus (CMV) plays an important role in the clinical management of solid organ and hematopoietic stem cell transplant recipients. However, CMV antiviral therapy can be complicated by drug resistance associated with mutations in the phosphotransferase UL97 and the DNA polymerase UL54. We have developed an amplicon-based high-throughput sequencing strategy for detecting CMV drug resistance mutations in clinical plasma specimens using a microfluidics PCR platform for multiplexed library preparation and a benchtop next-generation sequencing instrument. Plasmid clones of the UL97 and UL54 genes were used to demonstrate the low overall empirical error rate of the assay (0.189%) and to develop a statistical algorithm for identifying authentic low-abundance variants. The ability of the assay to detect resistance mutations was tested with mixes of wild-type and mutant plasmids, as well as clinical CMV isolates and plasma samples that were known to contain mutations that confer resistance. Finally, 48 clinical plasma specimens with a range of viral loads (394 to 2,191,011 copies/ml plasma) were sequenced using multiplexing of up to 24 specimens per run. This led to the identification of seven resistance mutations, three of which were present in <20% of the sequenced population. Thus, this assay offers more sensitive detection of minor variants and a higher multiplexing capacity than current methods for the genotypic detection of CMV drug resistance mutations. H uman cytomegalovirus (CMV)is an important cause of severe systemic and tissue-invasive disease in immunocompromised patients (reviewed in reference 1), such as solid organ transplant (SOT) and hematopoietic stem cell transplant (HSCT) recipients. In these patients, prophylactic or preemptive treatment with antiviral agents for CMV significantly reduces the rates of CMV disease, the risk of other viral and bacterial infections, and all-cause mortality (2-5), but these treatments place patients at risk for the development of CMV drug resistance (6). In addition to anti-CMV therapy lasting for Ն3 months, other risk factors for drug resistance are intensive immunosuppression, multiple episodes of CMV reactivation, high viral counts, and suboptimal antiviral concentrations due to poor compliance or low drug absorption (7).The number of antiviral drugs approved for CMV treatment is limited and includes ganciclovir (GCV) and its prodrug valganciclovir (VCV), foscarnet (FOS), and cidofovir (CDV). Intravenous GCV and oral VCV are used as first-line agents for both prophylaxis and treatment, largely because of their lower toxicities than those of FOS and CDV (8-13). All of the available CMV drugs ultimately target the viral DNA polymerase UL54; however, GCV and VCV also require phosphorylation by the phosphotransferase UL97 for their antiviral activity (6). Thus, mutations in the UL97 and UL54 genes can confer resistance to GCV and VCV, while CDV and FOS resistance is only associated with mutations in UL54. Large numbers of sequence variants ha...

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Section: Resultsmentioning

confidence: 99%

Detection of Cytomegalovirus Drug Resistance Mutations by Next-Generation Sequencing

et al. 2013

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“…The initial recombinant phenotyping of V812L showed a low-grade (2.5-to 3-fold) increased EC 50 for GCV, CDV, and FOS (10). Although a more recent study did not confirm the FOS-resistant phenotype (19), the current data (Table 1) are consistent with the initial report and the general tendency of mutations in this functional domain to confer variable degrees of FOS resistance and low-grade cross-resistance to GCV and CDV (5). This phenotype probably results from altered substrate recognition during polymerization of the incoming base into replicating DNA, based on the structural locus of residue 812 (16).…”

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confidence: 94%

“…V812L was previously described after in vitro selection under GCV (10,19) and in a clinical isolate after GCV and FOS therapy (20). The initial recombinant phenotyping of V812L showed a low-grade (2.5-to 3-fold) increased EC 50 for GCV, CDV, and FOS (10).…”

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confidence: 99%

Novel Cytomegalovirus UL54 DNA Polymerase Gene Mutations Selected In Vitro That Confer Brincidofovir Resistance

Chou

Ercolani

Lanier

2016

Antimicrob Agents Chemother

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Eight in vitro selection experiments under brincidofovir pressure elicited the known cytomegalovirus DNA polymerase amino acid substitutions N408K and V812L and the novel exonuclease domain substitutions D413Y, E303D, and E303G, which conferred ganciclovir and cidofovir resistance with 6-to 11-fold resistance to brincidofovir or 17-fold when E303G was combined with V812L. The new exonuclease domain I resistance mutations selected under brincidofovir pressure add to the single instance previously reported and show the expected patterns of cross-resistance. Brincidofovir (BCV or CMX001, Chimerix) is an experimental orally bioavailable hexadecyloxypropyl conjugate of the nucleotide analog cidofovir (CDV) (1). A phase 3 clinical trial of BCV for the prevention of human cytomegalovirus (CMV) infection in bone marrow transplant recipients (ClinicalTrials registration no. NCT01769170) was recently completed, and BCV unexpectedly failed to meet its primary efficacy endpoint despite showing a statistically significant antiviral effect during the ontreatment phase (2). These results and those of a prior successful phase 2 trial (3) showed that short-term prophylactic use of BCV did not result in the detection of CMV UL54 DNA polymerase gene mutations conferring drug resistance (4). The genetic pathways and biochemical mechanisms of resistance to BCV are expected to be the same as those of the parent compound CDV (5), into which BCV is metabolized prior to formation of the active antiviral CDP diphosphate (CDV-PP). Intracellular levels of CDV-PP differ markedly after exposure to CDV or BCV (6), as reflected in the much lower BCV concentrations (0.2 to 0.5 nM for BCV versus 200 nM for CDV) required to reduce CMV replication by 50% in cell culture (50% effective concentration [EC 50 ]). In vitro exposure to BCV may select for mutations different from those previously reported for CDV or ganciclovir (GCV), as higher intracellular active drug concentrations may select for mutants with higher associated levels of drug resistance at the cost of reduced growth fitness. In one report, the UL54 exonuclease domain III amino acid substitution D542E (7) was detected after many cell culture passages under BCV and shown to confer cross-resistance to CDV but not to GCV or foscarnet (FOS). Eight additional in vitro selection experiments under BCV are reported here with characterization of the resistance and relative growth phenotypes of the emergent mutations.The reference BCV compound was provided by Chimerix and added to the human fibroblast cultures starting with 0.2 nM BCV for all experiments after CMV inoculation at a low multiplicity of infection (MOI), followed by weekly propagation under increasing drug concentrations as permitted by interim viral growth, as previously described (8). We commonly use an error-prone UL54 exonuclease mutant (e.g., D413del, strain T4138) to accelerate the evolution of resistance mutations, but this mutant is already resistant to BCV, CDV, and GCV (Table 1), with EC 50 s ϳ5-fold increased over those f...

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“…The mutations we identified, V715M (patient 14) and N408D (patients 3 and 38), are well-characterized resistance mu- tations (2,10,12,13,18). Patient 14 is a lung transplant recipient who developed multiple-drug-resistant CMV.…”

Section: Discussionmentioning

confidence: 99%

“…Clinically, GCV resistance usually arises first from a UL97 mutation resulting in decreased accumulation of the activated drug. Subsequent UL54 mutations can confer high levels of GCV resistance and various degrees of cross-resistance to the second-line drugs cidofovir (CDV) and foscarnet (FOS) (2).…”

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Rapid Detection of Human Cytomegalovirus UL97 and UL54 Mutations Directly from Patient Samples

et al. 2013

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cHuman cytomegalovirus (CMV) is a significant contributor to morbidity and mortality in immunocompromised patients, particularly in the transplant setting. The availability of anti-CMV drugs has improved treatment, but drug resistance is an emerging problem. Here, we describe an improved, rapid, sequencing-based assay for the two genes in CMV where drug resistance occurs, the UL97 and UL54 genes. This assay is performed in 96-well format with a single master mix and provides clinical results within 2 days. It sequences codons 440 to 645 in the UL97 gene and codons 255 to 1028 in the UL54 gene with a limit of detection of 240 IU/ml. With this assay, we tested 43 specimens that had previously been tested for UL97 drug resistance and identified 3 with UL54 mutations. One of these patients had no concurrent UL97 mutation, pointing toward the need for an assay that facilitates dual UL97/UL54 gene testing for complete resistance profiling. Human cytomegalovirus (CMV) causes morbidity and mortality in immunocompromised patients, including transplant and HIV-infected patients (1). The first-line drug therapy for CMV infection is ganciclovir (GCV) or its prodrug valganciclovir (VGCV). GCV or VGCV must be activated by phosphorylation before they act on human CMV. This phosphorylation is carried out by the viral kinase UL97, and activated GCV subsequently inhibits the viral DNA polymerase UL54. Clinically, GCV resistance usually arises first from a UL97 mutation resulting in decreased accumulation of the activated drug. Subsequent UL54 mutations can confer high levels of GCV resistance and various degrees of cross-resistance to the second-line drugs cidofovir (CDV) and foscarnet (FOS) (2).Our group previously published a rapid PCR-and sequencingbased detection method for UL97 mutations conferring ganciclovir resistance (3). This test, currently used to detect mutations directly from clinical specimens, results in the sequence for UL97 gene codons 440 to 645. This region covers the known drug resistance mutation sites in the UL97 gene. However, because UL54 is the target of all currently marketed anti-CMV drugs, a detection method for UL54 mutations is also needed. Polymerase mutations are more likely to emerge after prolonged GCV treatment and typically add to the level of resistance conferred by UL97 mutations or introduce cross-resistance to CDV or FOS (4).Despite its predicted utility in treatment management, clinical tests for UL54 drug resistance mutations have been slower to develop for a number of reasons. First, the coding sequence of UL54 is almost twice as long as that of UL97. In addition, the baseline sequences are more variable and the number and variety of resistance mutations are greater in the UL54 gene than in the UL97 gene. Polymerase genotypic testing therefore requires more-extensive sequencing, typically covering codons 300 to 1,000. To meet the clinical need for complete CMV drug resistance profiles, we adapted our previous UL97 sequencing method (3) to develop a single assay that amplifies six region...

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Recombinant Phenotyping of Cytomegalovirus UL54 Mutations That Emerged during Cell Passages in the Presence of either Ganciclovir or Foscarnet

Cited by 28 publications

References 53 publications

Detection of Cytomegalovirus Drug Resistance Mutations by Next-Generation Sequencing

Detection of Cytomegalovirus Drug Resistance Mutations by Next-Generation Sequencing

Novel Cytomegalovirus UL54 DNA Polymerase Gene Mutations Selected In Vitro That Confer Brincidofovir Resistance

Rapid Detection of Human Cytomegalovirus UL97 and UL54 Mutations Directly from Patient Samples

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