Mass Spectrometry-Based Proteomic Analysis of Potential Host Proteins Interacting with GP5 in PRRSV-Infected PAMs

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus causing a large economic impact on the swine industry. The structural protein GP5 of PRRSV plays a pivotal role in its pathogenicity and immune evasion. Virus-host interactions play a crucial part in viral replication and immune escape. Therefore, understanding the interactions between GP5 and host proteins are significant for porcine reproductive and respiratory syndrome (PRRS) control. However, the interaction network between GP5 and host proteins in primary porcine alveolar macrophages (PAMs) has not been reported. In this study, 709 GP5-interacting host proteins were identified in primary PAMs by immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatics analysis revealed that these proteins were involved in multiple cellular processes, such as translation, protein transport, and protein stabilization. Subsequently, immunoprecipitation and immunofluorescence assay confirmed that GP5 could interact with antigen processing and presentation pathways related proteins. Finally, we found that GP5 may be a key protein that inhibits the antigen processing and presentation pathway during PRRSV infection. The novel host proteins identified in this study will be the candidates for studying the biological functions of GP5, which will provide new insights into PRRS prevention and vaccine development.

Keywords: GP5; LC-MS/MS; PRRSV; antigen processing and presentation; protein–protein interaction.

Figure 1

IP-MS analysis of GP5-interacting proteins. PAMs were infected with PRRSV for 36 h and then harvested for Co-IP. The resultant cell lysates were subjected to immunoblotting using the indicated antibody. Meanwhile, after SDS-PAGE electrophoresis, the gel was revealed by silver staining, and the gel fragment was excised to identified by LC-MS/MS.

Figure 2

GO annotation of GP5-interacting proteins. (A) GO enrichment analysis for biological process. (B) GO enrichment analysis for cellular component. (C) GO enrichment analysis for molecular function. The Top 20 enriched terms were revealed. The abscissa in the figure is the enrichment. The color of the dot represents the p value of the hypergeometric test. The color ranges from green to red. The redder the color is, the smaller the value is. The size of the point represents the number of proteins.

Figure 2

GO annotation of GP5-interacting proteins. (A) GO enrichment analysis for biological process. (B) GO enrichment analysis for cellular component. (C) GO enrichment analysis for molecular function. The Top 20 enriched terms were revealed. The abscissa in the figure is the enrichment. The color of the dot represents the p value of the hypergeometric test. The color ranges from green to red. The redder the color is, the smaller the value is. The size of the point represents the number of proteins.

Figure 3

KEGG pathway annotation of GP5-interacting proteins. The Top 20 enriched pathway terms were revealed.

Figure 4

PPI analysis of GP5-interacting proteins. The GP5 interaction network was built from the STRING database with confidence score > 0.7 (high confidence). These proteins were mainly concentrated in three PPI network clusters, including protein translation, protein transport, and protein processing, which were, respectively, circled with three different shapes.

Figure 5

Identification of the selected GP5-interacting proteins. (A) HEK293T cells were co-transfected with Flag-vector encoding the indicated host proteins and Myc-GP5 for 24 h. The cell lysates were immunoprecipitated with anti-Flag IgG beads and subjected to immunoblotting using the indicated antibody. (B) MARC-145 cells were co-transfected with Flag-vector encoding the indicated host proteins and Myc-GP5 for 36 h. Cells were immunostained and observed using confocal microscopy. Scale bar indicates 10 μm.

Figure 6

The effects of PRRSV and GP5 on expression levels of identified proteins. (A) PAMs were infected with PRRSV at an MOI of 1 for 24 h, and cells were harvested for a TMT-based quantitative proteomic analysis. (B) PAMs were infected with PRRSV at an MOI of 1 for 24 h, and the cell lysates were subjected to immunoblotting using the indicated antibody. (C) HEK293T cells were co-transfected with Flag-SLA-II and Myc-GP5 for 24 h. The resultant cell lysates were subjected to immunoblotting using the indicated antibody. EV: Empty vector. The protein bands were relatively quantified by ImageJ software (version 1.46). Error bars: mean ± SD of 3 independent tests. Student’s t-test; *** p < 0.001 compared to control.