Proteomic Characterization of PAMs with PRRSV-ADE Infection

Abstract

The antibody-dependent enhancement (ADE) effect of a PRRSV infection is that the preexisting sub- or non-neutralizing antibodies specific against PRRSV can facilitate the virus entry and replication, and it is likely to be a great obstacle for the selection of immune strategies and the development of high-efficiency PRRSV vaccines. However, the proteomic characterization of primary alveolar macrophages (PAMs) with a PRRSV-ADE infection has not yet been investigated so far. Therefore, we performed a tandem mass tag (TMT)-based quantitative proteomic analysis of PAMs with a PRRSV-ADE infection in this study. The results showed that a total of 3935 differentially expressed proteins (DEPs) were identified in the PAMs infected with PRRSV-ADE, including 2004 up-regulated proteins and 1931 down-regulated proteins. Further, the bioinformatics analysis for these DEPs revealed that a PRRSV-ADE infection might disturb the functions of ribosome, proteasome and mitochondria. Interestingly, we also found that the expression of the key molecules in the innate immune pathways and antiviral proteins were significantly down-regulated during a PRRSV-ADE infection. This study was the first attempt to analyze the proteomic characterization of PAMs with a PRRSV-ADE infection in vitro. Additionally, the findings will provide valuable information for a better understanding of the mechanism of virus-antibody-host interactions during a PRRSV-ADE infection.

Keywords: PAMs; PRRSV; PRRSV-ADE infection; proteomics; virus–antibody–host interactions.

Figure 1

The establishment of the PAMs model with PRRSV-ADE infection. (A) viral loads of the cell culture supernatants in different infection groups were monitored by RT-qPCR. (B) Confirmation of the PRRSV proliferation in PAMs of the mock group, the PRRSV-NI group and the PRRSV-ICs group by IFA. The N protein (red) and the nucleus (blue) of these PAMs were counterstained. Scale bar = 50 μm. ** p < 0.01.

Figure 2

The volcano plot of DEPs in PAMs of the PRRSV-NI group and the PRRSV-ICs group. Upregulated proteins are shown in red, downregulated proteins are shown in blue and unchanged proteins are shown in gray.

Figure 3

Subcellular annotation of the DEPs in PAMs infected with PRRSV-ADE infection. Upregulated proteins are shown in red, and downregulated proteins are shown in blue.

Figure 4

GO enrichment analysis of the DEPs in PAMs with PRRSV-ADE infection for biological process (A), molecular function (B) and cellular component (C). The abscissa in the figure is the rich factor which is the ratio of the number of different proteins in the corresponding pathway to the total number of proteins identified by the pathway. 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 differential proteins in the corresponding pathway.

Figure 5

KEGG enrichment analysis of the DEPs in PAMs with PRRSV-ADE infection. Rich factor refers to the ratio of DEPs annotated in this pathway to all identified protein numbers annotated in this pathway. A larger rich factor with a lesser p value indicates a greater intensiveness. The top 26 enriched pathway terms were revealed.

Figure 6

The PPI network of 143 DEPs. The connections between proteins indicate that they interact with each other and the confidence interval is greater than 0.9. DEPs were mainly concentrated in three PPI network clusters including ribosome, proteasome and mitochondria, which were circled with red, yellow and blue, respectively.

Figure 7

The validation of the DEPs in PAMs with PRRSV-ADE infection. (A) RT-qPCR analysis of six selected DEPs (COX-5B, MMP-9, ISG56, OAS1, Mx1 and RSAD2) in PAMs with PRRSV-ADE infection. (B) Western blot analysis of four DEPs (TBK-1, STAT-1, STAT-2 and Mx1) in PAMs with PRRSV-ADE infection. *** p < 0.001.