Rapid In Vitro Evolution 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 μM LMV (>5,000-fold the 50% effective concentration [EC50] 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.

FIG 1

SEAP growth curves of recombinant CMV strains. Cell-free stocks of wild type (wt) and UL56 mutant virus stocks were inoculated onto human foreskin fibroblast monolayers at an MOI of ∼0.01. Culture media were assayed daily at 4 to 8 days postinoculation for SEAP activity using a chemiluminescent substrate. Each point on the growth curves is the mean and standard deviation from 4 replicates. SEAP, secreted alkaline phosphatase; RLU, relative light units; wt, control wild type with an Frt motif upstream of UL56; K355del, in-frame deletion of catalytic lysine residue resulting in loss of UL97 kinase activity and severe growth impairment, for comparison with previously published growth curves (14).