Phosphoproteomic Analysis Reveals Differences in Intercellular Spread Among Feline Herpesvirus Type 1 Mutants

Feline herpesvirus-1 (FHV-1) is taxonomically classified within the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus, and species Felid alphaherpesvirus 1. The genome of FHV-1 is 135,797 bp in length and encodes 74 proteins. Among these proteins, serine/threonine protein kinase (pK) and thymidine kinase (TK) have been identified as potential virulence factors in alphaherpesviruses, although these kinases are dispensable for viral replication. As kinases, regulating phosphorylation modification is one of their functions, while the mechanism by which phosphorylation modification affects cell physiological functions and thereby influences viral replication remains unclear. In this study, we generated pK- and TK-deficient FHV-1 mutants by CRISPR/Cas9-mediated homologous recombination. The pK-deficient virus produced significantly smaller plaques than the TK-deficient virus. The replication kinetics of the pK-deficient virus were attenuated in multistep growth compared to the TK-deficient virus. These results indicate that deletion of the pK gene markedly reduces the replicative capacity of FHV-1. We applied data-independent acquisition (DIA) quantitative proteomics to profile changes in global protein expression and phosphorylation in F81 cells upon infection with TK^-, pK^-, and wild-type FHV-1 strain. The pK-deficient virus exhibited 3632 differentially phosphorylated proteins containing 11,936 modification sites; the TK-deficient virus showed 4529 differentially phosphorylated proteins with 19,225 phosphorylation sites. Functional characterization through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses identified significant involvement of phosphoproteins in spliceosome pathways in pK-deficient virus and ATP-dependent chromatin remodeling pathway in TK-deficient virus. Notably, several splicing regulators-including Ess-2 and CDK13, which modulate host spliceosomal function-displayed significantly reduced phosphorylation levels in pK-deficient viruses. A significant enrichment of ATP-dependent factors, such as SMARCA5 and RSF1, was observed in the TK-deficient virus. To our knowledge, this is the first investigation into the effects of FHV-1 infection on the host cell phosphoproteome. These data offer new insights into the phosphoregulatory circuits and signaling networks triggered by FHV-1 and may enhance our understanding of the FHV-1 replication mechanism.