Serine 13 of the human cytomegalovirus viral cyclin-dependent kinase UL97 is required for regulatory protein 14-3-3 binding and UL97 stability

The human cytomegalovirus (HCMV) UL97 protein is a conserved herpesvirus protein kinase (CHPK) and a viral cyclin-dependent kinase (v-CDK). However, mechanisms regulating its activity in the context of Infection are unknown. Here we identified several cellular regulatory 14-3-3 proteins as UL97-interacting partners that promote UL97 stability. Humans are known to encode 7 isoforms of 14-3-3 proteins (β, ε, η, γ, σ, θ, and ζ) that bind phosphoserines or phosphothreonines to impact protein structure, stability, activity, and localization. Our proteomic analysis of UL97 identified 49 interacting partners, including 14-3-3 isoforms β, η, and γ. Furthermore, co-immunoprecipitation with western blotting assays demonstrated that UL97 interaction with 14-3-3 isoforms β, ε, η, γ, and θ occurs in a kinase activity-dependent manner. Using mutational analysis, we determined the serine residue at amino acid 13 of UL97 is crucial for 14-3-3 interaction. We demonstrate UL97 S13A (serine to alanine substitution at residue 13) retains kinase activity, but the mutant protein accumulated at lower levels than wild type UL97. Finally, we show both laboratory (AD169) and clinical (TB40/E) strains of HCMV encoding UL97 S13A replicated with wild type kinetics in fibroblasts but showed decreased UL97 accumulation. Taken together, we conclude that 14-3-3 proteins interact with and stabilize UL97 during HCMV Infection.