Identification of a short sequence in the HCMV terminase pUL56 essential for interaction with pUL89 subunit

International audienceThe human cytomegalovirus (HCMV) terminase complex consists of several components acting together to cleave viral DNA into unit length genomes and translocate them into capsids, a critical process in the production of infectious virions subsequent to DNA replication. Previous studies suggest that the carboxyl-terminal portion of the pUL56 subunit interacts with the pUL89 subunit. However, the specific interacting residues of pUL56 remain unknown. We identified a conserved sequence in the C-terminal moiety of pUL56 (671 WMVVKYMGFF 680). Overrepresentation of conserved aromatic amino acids through 20 herpesviruses homologues of pUL56 suggests an involvement of this short peptide into the interaction between the larger pUL56 terminase subunit and the smaller pUL89 subunit. Use of Alpha technology highlighted an interaction between pUL56 and pUL89 driven through the peptide 671 WMVVKYMGFF 680. A deletion of these residues blocks viral replication. We hypothesize that it is the consequence of the disruption of the pUL56-pUL89 interaction. These results show that this motif is essential for HCMV replication and could be a target for development of new small antiviral drugs or peptidomimetics. Human cytomegalovirus (HCMV), a beta herpesvirus, can cause serious diseases in immunocompromised patients. Current antiviral inhibitors (ganciclovir, cidofovir and foscarnet) all target the viral DNA polymerase. They have adverse effects and prolonged treatment can select for drug resistance mutations either in the viral pol-ymerase pUL54, the kinase pUL97 or both of them 1, 2. Thus, we need new drugs targeting others stages of repli-cation. The terminase complex is highly specific for HCMV, has no counterpart in the human organism, and thus represents a target of choice for new antivirals development. This has been confirmed by the recent development of letermovir in the transplant setting 3, 4. DNA packaging process requires several proteins such as pUL56 and pUL89, the large and small termi-nase subunits, respectively. Recently, four additional proteins were shown to be also implicated in this process, namely, pUL51, pUL52, pUL77, pUL93 5-10. This process is driven by specific interactions of protein-DNA and protein-protein to cleave and package unit length genomic DNA into an empty capsid. Evidence suggests that the large subunit pUL56 has a crucial role in DNA cleavage/packaging, containing many of the functional sites required for this process like interaction with the portal protein pUL104, endonu-clease activity, and more interestingly an ATP-binding site (amino acids 709 to 723) 11. Although the association between pUL56 and pUL89 has already been reported, the residues of pUL56 involved in the terminase complex integrity are still unknown 12, 13. Nevertheless, co-immunoprecipitation experiments showing an interaction between the C-terminal half of pUL56 (pUL56-Cter) and pUL89 were confirmed by other results 12, 13. Because knowledge of terminase functional and interaction domains is important both for the development of drugs targeting the DNA packaging stage and for the improvement of existing ones such as letermovir, the aim of the current study is to identify a minimum peptide of pUL56 with a putative key role in its interaction with pUL89. Sequence alignments encouraged us to focus on the putative involvement of one part of the pUL5