Evolution-guided prioritization identifies a tissue-specific phosphorylation switch on herpes simplex virus 1 UL7 regulating viral replication and pathogenicity.

Although numerous phosphorylation sites on herpes simplex virus 1 (HSV-1) proteins have been identified through phosphoproteomic analyses, their virological significance remains largely unclear. Here, we developed an evolution-guided prioritization strategy that integrates phosphoproteomic data with residue-level conservation across the genus IMPORTANCE: Intricate phosphorylation-dependent regulatory mechanisms enable viruses to diversify the functions of their proteins, profoundly shaping viral replication and pathogenicity. Although phosphoproteomic analyses have produced an expanding catalog of phosphorylation sites on viral proteins, a considerable proportion of these modifications are likely non-functional. This creates a pressing need for a prioritization strategy to predict functionally relevant phosphorylation sites. To address this, we developed an evolution-guided prioritization strategy that integrates phosphoproteomic data with genus-level conservation. Using this strategy, we prioritized two tyrosine phosphorylation sites for functional analysis. Notably, phosphorylation at one of these sites, UL7 Tyr-89, functions as a tissue-specific regulatory mechanism that fine-tunes UL7 activity and modulates herpes simplex virus 1 replication and pathogenicity in the central nervous system. This strategy provides a practical framework for prioritizing candidate regulatory sites for functional investigation in viral replication and pathogenicity.