Herpes simplex virus alkaline nuclease interacts directly with ubiquitin-specific protease 15 through a conserved C-terminal domain.

Herpes simplex virus replicates in the nucleus of the host cell and utilizes many cellular proteins to facilitate DNA replication. The deubiquitinating enzyme ubiquitin-specific protease 15 (USP15) is one of the most enriched proteins on replicating viral DNA and is recruited to the nucleus by a direct interaction with the viral alkaline nuclease, UL12. To better define this protein-protein interaction, we applied genetic and structural approaches and have mapped the USP15 binding domain to three conserved amino acids on the UL12 C-terminal alpha helix. A small internal deletion in this helix disrupts the UL12 interaction with USP15 but not other UL12-interacting proteins. Bioinformatic analysis reveals that this alpha helix is strongly conserved in all alphaherpesviruses but not in betaherpesviruses or gammaherpesviruses. The Kaposi's sarcoma-associated herpesvirus homolog, SOX, has an alpha helix in the same position, but the amino acid composition is highly divergent compared with UL12. Furthermore, overlaying the HerpesFolds predictions of UL12 with SOX shows high structural similarity in the nuclease domains but marked divergence in the C-terminal helix. We demonstrate that the UL12 C-terminal alpha helix can be swapped with another alphaherpesvirus UL12 helix and maintain the ability to recruit USP15 to the nucleus. Conversely, substitution with the divergent SOX helix maintains a folded protein with nuclease activity but fails to recruit USP15 to the nucleus. Analysis of the sequence between the alphaherpesvirus UL12 helices identified six identical residues. We functionally validated three of these amino acids as required for USP15 interaction. These observations identify the USP15 binding domain on UL12.