A novel inhibitor of human cytomegalovirus acts through a specific antiviral mechanism that involves the helicase-primase complex

Human cytomegalovirus (HCMV) remains the leading viral cause of congenital birth defects and a significant pathogen in transplant recipients. The most commonly used drugs inhibit the viral DNA polymerase, but result in severe side effects and the emergence of drug resistance. The terminase inhibitor (letermovir) and viral UL97 kinase inhibitor (maribavir) have improved the CMV armamentarium; however, both select for resistance, and have limited therapeutic indications. We recently identified MLS8091, a highly selective HCMV inhibitor. While its maximal time of HCMV inhibition overlaps with ganciclovir (GCV), it remains active against GCV-resistant strains, suggesting a distinct mechanism involving the CMV replisome. Using resistance selection protocols and next-generation sequencing of mutant viruses that escaped MLS8091 inhibition, we identified point mutations in the HCMV primase (UL70 D486H, D487Y) and the primase-associated factor (UL102 P165S, F275L) of the helicase-primase (H/P) complex. Marker transfer experiments confirmed that UL70 D486H, UL70 D487Y, and UL102 F275L each conferred resistance to MLS8091. UL102 P165S was identified together with UL70 D487Y; however, its independent contribution to resistance was not determined. Sequencing of 17 wild-type and GCV-resistant clinical isolates identified a UL102 polymorphism, A294G, that did not confer reduced susceptibility to MLS8091 upon marker transfer. Among the herpesviruses tested, only guinea pig CMV (GPCMV) was moderately inhibited by MLS8091. Sequence alignment revealed some similarity between GPCMV and HCMV UL70 486-487. In summary, we identified an inhibitor of HCMV replication that acts through a novel mechanism involving the H/P complex, opening new avenues in the development of HCMV-specific drugs.