RapidIn VitroEvolution of Human Cytomegalovirus UL56 Mutations That Confer Letermovir Resistance

Abstract

Letermovir (LMV) is an experimental cytomegalovirus terminase inhibitor undergoing phase 3 clinical trials. Viral mutations have been described at UL56 codons 231 to 369 that confer widely variable levels of LMV resistance. In this study, 15 independent experiments propagating an exonuclease mutant viral strain in escalating LMV concentrations replicated 6 of the 7 published UL56 mutations and commonly elicited additional resistance-conferring mutations at UL56 codons 231, 236, 237, 244, 257, 261, 325, and 329. Mutations were first detected earlier in LMV (median, 3 passages) than in 8 parallel experiments with foscarnet (median, 15 passages). As LMV concentrations increased, the typical initial UL56 change F261L, which confers low-grade resistance, combined or was replaced with mutations conferring higher-grade resistance, eventually enabling normal viral growth in 30 M LMV (>5,000-fold the 50% effective concentration [EC 50 ] for the wild type). At high LMV concentrations, the UL56 changes C325F/R were commonly detected, as well as a combination of changes at codons 236, 257, 329, and/or 369. Recombinant viruses containing individual UL56 mutations and combinations were constructed to confirm their resistance phenotypes and normal growth in cell culture. Several double and triple mutants showed much higher LMV resistance than the respective single mutants, particularly those including changes at both codons 236 and 257. The multiplicity of pathways to high-grade LMV resistance with minimal viral growth impact suggests a low viral genetic barrier and the need for close monitoring during treatment of active infection.T he prevention and treatment of human cytomegalovirus (CMV) infection and disease are an important aspect of the medical care of immunosuppressed individuals. The viral DNA polymerase inhibitors ganciclovir, its oral prodrug valganciclovir, foscarnet, and cidofovir have long been used for this purpose, with generally satisfactory outcomes but also well-known limitations of toxicity, intravenous treatment complexity, and risk of drug resistance after prolonged therapy (1). Cross-resistance among current drugs may develop because they have the same DNA polymerase target (2). Therefore, priority has been given to the development of alternative CMV drug targets. The viral terminase complex, including components encoded by the CMV genes UL56, UL89, and UL51, acting in concert with UL104 and others, is responsible for the cleavage of concatemeric DNA formed during viral replication into unit-length genomes and packaging them into preformed viral capsids (3). This essential process is an attractive target for specific viral inhibition. Drug discovery programs have identified diverse chemical structures that turned out to be CMV terminase inhibitors. Earlier candidates, a benzimidazole pyranoside GW275175X (4) and a chemically unrelated compound, Bay38-4766 (tomeglovir) (5), were tested in phase I trials but were not advanced to later-stage clinical development. Resistance mutations were mapped to the gen...

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

“…Two independent A591V recombinants were constructed, one derived from baseline clone BA1, as for all other strains in Table 1, and a second one based on clone BD1, which differs from BA1 in the restoration of missing US7 to US16 sequences and compensatory deletion of the extra copy of genes RL1 through RL13 (14). The BD1-derived baseline strain T4198 has the same GCV EC 50 as the BA1-derived strain T3261 (Table 1), whether assayed in HFFs (1.1 M) (10) or in ARPEp cells (1.2 M Ϯ 0.31; 11 replicates).…”

“…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). A second A591V mutant was derived using the same methods from a separate BD1 clone of strain AD169 (10,14). Some baseline and mutant strains also contain the UL97 amino acid variation H587Y that has been shown not to affect the GCV susceptibility phenotype (12).…”

Treatment of CMV infection after allogeneic hematopoietic stem cell transplantation

“…The study is expected to complete the accrual in January 2017 for primary outcome (NCT02137772). Overall, in vitro LMV resistant CMV strains have been reported [79,80,81].…”

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

“…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 for the parental wild-type strain T3265 (8,9), raising the possibility that additional mutations on this genetic background may not represent evolution in the wild-type virus.…”