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

Wenfeng He,Guoqing Yang,Liangliang Dong,Chen Li,Huimin Liu

doi:10.3390/genes12101557

Abstract

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.

Highlights

Pseudorabies (PR), an acute, febrile infectious disease caused by pseudorabies virus (PRV), which is a member of the subfamily Alphaherpesvirinae in the family Herpesviridae [1]
Our findings provide valuable information for understanding the Interferon-stimulated gene 15 (ISG15) knockout-induced proteome changes following PRV infection, and these findings are expected to shed some light on potential antiviral targets and signaling pathways involved with PRV pathogenesis
Gene sequencing showed that the ISG15 knockout PK15 cell line was successfully constructed, and the knockout efficiency was evaluated by Western blot analysis

Summary

Introduction

Pseudorabies (PR), an acute, febrile infectious disease caused by pseudorabies virus (PRV), which is a member of the subfamily Alphaherpesvirinae in the family Herpesviridae [1]. PRV can cause a high mortality rate in newborn pigs, neurological and respiratory system disorders in growing pigs, and reproductive failure in pregnant sows [3]. Pigs are the only natural hosts for PRV and are the main source of its transmission, PRV can infect a broad range of hosts, including livestock and wildlife [2,4,5]. It has been reported that PRV can infect humans with weakness, fever, sweating, dysphagia, and neurological dysfunction as typical symptoms [6,7,8,9]. Since 2011, highly pathogenic PRV variants that emerged in several pig farms in China have caused substantial economic losses to the pig industry worldwide [10]. The threat of PRV should not be ignored, and new targets are urgently needed to control PRV spread are urgently needed

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Results

Conclusion