Divide et Impera: Identification of Small-Molecule Inhibitors of HCMV Replication Interfering with Dimerization of DNA Polymerase Processivity Factor UL44

Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS and transplanted patients, and in congenitally infected newborns. Despite the availability of several antiviral drugs, their utility is limited by poor bioavailability, toxicity, and resistant strains emergence. Therefore, it is crucial to identify new targets of therapeutic intervention. The dimerization of HCMV DNA polymerase processivity factor UL44 plays an essential role in the viral life cycle being required for oriLyt-dependent DNA replication. We validated the existence of UL44 homodimers both in vitroand in living cells by a variety ofapproaches, including GST pulldown, thermal shift, FRET and BRET assays. Dimerization occurred with an affinity comparable to that of the UL54/UL44 interaction, and was impaired by amino acid substitutions at the dimerization interface. Subsequently, we performed an in-silicoscreeningto select 18 small molecules (SMs) potentially interfering with UL44 homodimerization. Antiviral assays using recombinant HCMV TB4-UL83-YFP in the presence of the 18 selected SMs led to the identification of four active SMs. The most active one also inhibited AD169 in plaque reduction assays, and impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistantcounterpart to a similar extent. As assessed by Western blotting experiments, treatment of infected cells specifically reduced viral gene expression starting from 48 h post infection, consistent with activity on viral DNA synthesis. Therefore, SMs inhibitors of UL44 dimerization could represent a new class of HCMV inhibitors, alternative to those targeting the DNA polymerase catalytic subunit or the viral terminase complex.