Competitive Fitness of Cytomegalovirus Mutants Bearing Changes in the UL56 Terminase Subunit, Associated with Letermovir-Resistance, in Presence and Absence of Antivirals

Letermovir (LMV), a cytomegalovirus (CMV) terminase inhibitor, has a potentially low genetic barrier to the emergence of resistance, with single mutations in the terminase subunits (UL51, UL56, UL89) being associated with LMV resistance (LMV-R). We determined the fitness of LMV-R viruses (UL56 C325F/W/Y or M236V mutants) with different DNA polymerase (pol) (UL54) mutants in the presence and absence of anti-CMV drugs in dual-infection competition assays. After 7 days of growth, viral variants were quantified by targeted sequencing of the UL54 or UL56 genes by next-generation sequencing. Without antivirals, the UL56 C325F mutant was equally fitted as the wild-type virus, in contrast to C325W/Y mutants that had a reduced replication capacity. The UL56 C325F mutant showed the highest replication capacity among all terminase mutants tested when grown in competition with wild-type virus under LMV. The UL56 terminase mutants gained replication capacity when grown in competition with DNA pol mutant viruses under LMV pressure, with the terminase mutant UL56 C325F being able to overgrow various DNA pol mutants. Furthermore, the UL56 C325F mutant showed the highest replication capacity when grown in competition with another UL56 terminase mutant. Our results are in line with the C325F UL56 mutant being the most frequent LMV-R mutation identified in the clinic.