Letermovir is a human cytomegalovirus (CMV) terminase inhibitor recently 
approved as prophylaxis in stem cell transplant recipients. In further studies 
of emerging drug resistance, a baseline laboratory CMV strain was serially 
propagated in cell culture under a combination of letermovir and ganciclovir. In 
eight experiments, UL56 terminase gene mutations were detected beginning at 10 
passages and included novel amino acid substitutions V236A, L328V, and A365S in 
a region previously associated with letermovir resistance. Outside this region, 
the UL56 substitution C25F was detected at moderate drug concentrations in two 
experiments as either the first detected mutation or an addition to a 
preexisting V231L substitution. In all cases, mutation at UL56 codon 325 
conferring absolute letermovir resistance eventually developed at a median of 20 
passages. No UL97 kinase or UL54 DNA polymerase mutations relevant to 
ganciclovir resistance were detected until many passages after the first 
detection of the UL56 mutations. UL56 substitutions V236A, L328V, and A365S were 
shown to confer borderline or low-grade letermovir resistance, while C25F 
conferred a 5.4-fold increase in letermovir resistance (50% effective 
concentration [EC50]) by itself and a 46-fold increase in combination with 
V231L. The evolution of resistance mutations sooner in UL56 than in UL54 or UL97 
is consistent with prior in vitro observations, and UL56 codon 25 is a genetic 
locus for letermovir resistance distinct from loci previously described.