Tandem Mass Tag-Based Quantitative Proteomic Analysis of ISG15 Knockout PK15 Cells in Pseudorabies Virus Infection

Pseudorabies virus (PRV) is recognized as one of the most important pathogens of swine and poses a serious threat to the swine industry worldwide. Available commercial vaccines fail to protect against the emergence of new PRV strains. Therefore, the new protein targets against PRV highlight the urgent need for uncovering the molecular determinants of host cellular proteins following PRV infection. Interferon-stimulated gene 15 (ISG15) demonstrates an outstanding antiviral response. However, the molecular mechanism of ISG15 that affects PRV replication is incompletely known. Here, we performed a tandem mass tag (TMT)-based approach to quantitatively identify protein expression changes in PRV-infected ISG15 knockout PK15 (ISG15^-/--PK15) cells. In total, 4958 proteins were identified by using TMT coupled with LC-MS/MS in this study. In the PRV- and mock-infected groups, 241 differentially expressed proteins (DEPs) were identified, 162 upregulated and 79 downregulated proteins at 24 h post-infection (hpi), among which AFP, Vtn, Hsp40, Herc5, and Mccc1 may play important roles in PRV propagation. To ensure the validity and reliability of the proteomics data, the randomly selected DEPs were verified by RT-qPCR and Western blot analysis, and the results were consistent with the TMT results. Bioinformatics analyses further demonstrated that the DEPs are mainly involved in various biological processes and signaling pathways, such as signal transduction, the digestive system, and the PI3K-AKT pathway. These findings may provide new insight into molecular mechanisms for PRV infection, which is helpful for identifying potential protein targets for antiviral agents.