Functional and structural insights into HCMV terminase accessory proteins pUL77 and pUL93

ABSTRACT Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in immunocompromised and congenitally infected patients. Current antiviral therapies, primarily targeting the viral DNA polymerase, are limited by toxicity and resistance, underscoring the need for novel therapeutic strategies. Letermovir (LTV), which targets the viral terminase complex (pUL56-pUL89-pUL51), has improved prophylaxis in transplant recipients, but resistance mutations have already emerged. This complex is responsible for cleaving and packaging the viral genome into neo-formed capsids in close interaction with several other proteins. This study focuses on two essential terminase-associated proteins, pUL77 and pUL93, investigating their natural polymorphism, mutations arising under LTV prophylaxis, and the functional relevance of their putative nuclear localization signals (NLS). Sequence alignments across 18 herpesviruses revealed conserved and variable regions in both proteins. New mutations emerging in patients treated with LTV did not confer resistance, but the pUL77 R43C mutation, when present with pUL56 C325F, significantly reduces LTV resistance compared to C325F alone. In addition, nuclear localization motifs were assessed in the viral replication context and in autonomous cell production after plasmid transfection and revealed motifs essential for viral replication: 43 RVRKRYLRQ 55 and 225 PRWKRV 231 in pUL77 and 505 RDRRGRLRR 513 in pUL93. Finally, AI-based modeling provided some insights into the functions of these motifs for both proteins, notably in interaction with other viral proteins. Collectively, these data provide a wealth of information about putative functional sites in pUL77 and pUL93 that could be new targets for anti-HCMV strategies such as inhibitory peptides. IMPORTANCE Human cytomegalovirus (HCMV) remains a significant health concern, particularly for immunocompromised individuals and in cases of congenital infection. The emergence of resistance to current antivirals targeting viral polymerase has necessitated the development of novel therapeutic approaches. Letermovir, which targets the HCMV terminase complex, represents a promising alternative. However, a deeper understanding of the virus’s structural components and their interactions is crucial for identifying new potential drug targets. Here, we focused on two proteins, pUL77 and pUL93, which are associated with the viral capsid and interact with the terminase complex. By analyzing their polymorphism, structure, and functional motifs, we identified essential nuclear localization motifs and conserved regions in these proteins, providing valuable insights for the development of innovative anti-HCMV strategies, such as inhibitory peptides, which could complement existing treatments and address the ongoing challenge of antiviral resistance.