Diverse Cytomegalovirus UL27 Mutations Adapt to Loss of Viral UL97 Kinase Activity under Maribavir

In vitro resistance to maribavir (MBV), a cytomegalovirus UL97 kinase inhibitor currently in clinical trials, is known to result from viral UL97 mutations that confer moderate to high-level resistance and UL27 mutations that confer low-level resistance. To add to the four reported UL27 mutations, cytomegalovirus isolates or strains were propagated under MBV. Four clinical isolates evolved UL27 mutations, which were first detected after 8 to 30 passages under drug selection. In three separate experiments, laboratory strain T2294, which contained an exonuclease mutation, developed UL27 mutations at 10 to 12 passages under MBV. Most of these isolates and strains also developed a UL97 mutation, commonly T409M, before or after the appearance of the UL27 mutation. The passage of two laboratory strains genetically defective in UL97, in the absence of MBV, likewise resulted in UL27 mutations. The nine UL27 mutations observed included multiple instances of point, stop, and frameshift mutations, which were individually transferred to a reference CMV strain and which were shown to confer two- to threefold increases in MBV inhibitory concentrations. In contrast, seven common UL27 amino acid changes found in baseline clinical isolates conferred no MBV resistance. The mutants with UL27 mutations had slightly attenuated growth. The frequent mutation of UL27 suggests that its normal expression is mildly disadvantageous to the virus in the absence of UL97 kinase activity, whether the latter results from MBV inhibition or a genetic defect. Although the function of UL27 is unknown, it does not appear to be a direct antiviral target for MBV.