<p>Human cytomegalovirus (HCMV) causes life-threatening infections, particularly in immunocompromised patients. Letermovir targets the viral terminase complex (pUL56, pUL89, pUL51), essential for DNA packaging, but resistance mutations in pUL56 underline the need for new antiviral strategies. Targeting critical interaction domains within the complex, such as the pUL56 peptide ₆₇₁WMVVKYMGFF₆₈₀, has emerged as a promising approach. This study identifies two additional pUL56 motifs, ₅₁₄EVNVRKRAY₅₂₂ and ₇₅₇YLLLYRHL₇₆₄, as key mediators of terminase assembly. These motifs were deleted or mutated in an HCMV-BAC clone, and protein-protein interactions were assessed using NanoBit^® PPI/PCI-neo assays in HEK293T cells. Interaction domains were mapped on a 3D model of HCMV terminase and compared with the cryo-EM structure of HSV-1 homologs (PDB 6M5R, 6M5S). Mutations in these motifs strongly impaired viral replication. Functional assays showed that ₅₁₄EVNVRKRAY₅₂₂ directly mediates interaction with pUL89, while modeling indicated that ₆₇₁WMVVKYMGFF₆₈₀ and ₇₅₇YLLLYRHL₇₆₄ stabilize the multimeric ring-shaped complex through contacts with pUL89 and pUL51. Our results suggest that two novel pUL56 regions are essential for terminase assembly and conformation and may represent promising targets for future antiviral development.</p>