Phosphoproteomic landscape of pseudorabies virus infection reveals multiple potential antiviral targets

Phosphorylation, a significant posttranslational modification, is implicated in nearly two-thirds of all proteins. The involvement of phosphorylation in various cellular responses is known to play a crucial role in the replication of pseudorabies virus (PRV). However, the phosphoproteomic landscape during PRV infection remains unknown. To address this, a tandem mass tag-based liquid chromatography-tandem mass spectrometry was performed. It was found that a total of 7,342 phosphosites were detected; among these, 1,197 phosphosites were significantly upregulated, and 1,120 phosphosites were significantly downregulated when the threshold was set at 1.5. Gene ontology analysis of these significantly changed proteins showed that they mainly participated in mRNA processing, RNA splicing, stress granule assembly, and other pathways. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that these proteins were associated with cell cycle, spliceosome, tight junction, mRNA surveillance, and other pathways. Additionally, an immunoblotting assay confirmed the phosphoproteomic results by detecting the protein level of several upregulated phosphorylated proteins, which included pBRAF, pRPS6KA1, and pSTMN1. Further experiments using siRNA showed that knockdown of BRAF, RPS6KA1, and STMN1 significantly inhibited the replication of PRV. In summary, this study provides insight into the phosphoproteomic landscape during PRV infection and contributes to a better understanding of the mechanisms underlying PRV replication.IMPORTANCEPseudorabies virus (PRV) is a kind of alpha herpesvirus that infects a wide range of animals and even human beings. Therefore, it is important to explore the mechanisms behind PRV replication and pathogenesis. By conducting a tandem mass tag-based phosphoproteome, this study revealed the phosphorylated proteins and cellular response pathways involved in PRV infection. Findings from this study shed light on the relationship between the phosphorylated cellular proteins and PRV infection, as well as guiding the discovery of targets for the development of antiviral compounds against PRV. Pseudorabies virus (PRV) is a kind of alpha herpesvirus that infects a wide range of animals and even human beings. Therefore, it is important to explore the mechanisms behind PRV replication and pathogenesis. By conducting a tandem mass tag-based phosphoproteome, this study revealed the phosphorylated proteins and cellular response pathways involved in PRV infection. Findings from this study shed light on the relationship between the phosphorylated cellular proteins and PRV infection, as well as guiding the discovery of targets for the development of antiviral compounds against PRV.