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

species, respectively. Phosphomimetic mutations at either site significantly reduced progeny virus yields in cultured cells, suggesting that phosphorylation at these sites can function as an inhibitory switch regulating HSV-1 replication. In agreement with this, in-depth analyses of phosphorylation at UL7 Tyr-89 during HSV-1 infection revealed that the phosphomimetic mutation led to phenotypes similar to those of the UL7 null mutation in virion morphogenesis, HSV-1 replication in the central nervous system (CNS) and eyes of mice, as well as CNS pathogenicity and ocular pathogenic manifestations. Notably, the non-phosphorylatable mutation at this site had little effect on HSV-1 infection in cultured cells, whereas it significantly reduced HSV-1 replication and pathogenicity in the CNS, but not in the eyes of mice. These results suggest that our strategy effectively prioritizes phosphorylation sites for functional investigation and that phosphorylation at UL7 Tyr-89 serves as a context-dependent inhibitory switch, fine-tuning UL7 activity in a tissue-specific manner, particularly in the CNS. 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.