First evidence of efficacy of peptides targeting the pUL56-pUL89 interaction domain of the human cytomegalovirus terminase complex

Human cytomegalovirus (HCMV) is a major cause of morbidity in immunocompromised patients and the leading viral cause of congenital infection. The toxicity and emergence of resistance associated with current antivirals underscore the need for alternative therapeutic strategies. The viral terminase complex (pUL56-pUL89-pUL51), essential for genome cleavage and packaging and without homologs in mammalian cells, represents a promising antiviral target. This study evaluated the antiviral potential of peptides targeting the pUL56-pUL89 interaction domain. Peptides derived from the minimal interaction domain between pUL56 and pUL89 (WF10: ₆₇₁WMVVKYMGFF₆₈₀) or an extended sequence (PD17: ₆₆₈PSEWMVVKYMGFFNFSD₆₈₄) were synthesized to interfere with this interaction. To optimize intracellular delivery, peptides were conjugated to a cell-penetrating peptide (CPP) derived from either the HIV-1 transactivator of transcription (TAT) or the Antennapedia homeodomain of Drosophila melanogaster (penetratin; PT). Peptide candidates- PT-WF10, TAT-WF10, and PT-PD17- were evaluated in cellular models for cytotoxicity, hemolysis, antiviral activity, and intracellular distribution. TAT-WF10 and PT-PD17 significantly reduced the cytopathic foci in HCMV-infected cells, with IC₅₀ values of 58 μM and 39 μM, respectively. PT-WF10 lacked antiviral activity, induced significant cytotoxicity and hemolysis, and was mainly localized in the cytoplasm, with only minimal nuclear signal. TAT-WF10 showed cytoplasmic and nuclear distribution, no hemolysis, but induced long-term cytotoxicity from 40 μM. PT-PD17 exhibited cytoplasmic and nuclear distribution, with no significant cytotoxicity or hemolysis up to 80 μM. This study provides the first proof of concept that a peptide targeting the pUL56-pUL89 interaction domain can inhibit HCMV replication. PT-PD17 demonstrated antiviral activity, intracellular distribution, and a favorable safety profile.