Identification of differentially expressed proteins in porcine alveolar macrophages infected with virulent/attenuated strains of porcine reproductive and respiratory syndrome virus

Abstract

The highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) is still a serious threat to the swine industry. However, the pathogenic mechanism of HP-PRRSV remains unclear. We infected host porcine alveolar macrophages (PAMs) with the virulent HuN4 strain and the attenuated HuN4-F112 strain and then utilized fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) to screen for intracellular proteins that were differentially expressed in host cells infected with the two strains. There were 153 proteins with significant different expression (P<0.01) observed, 42 of which were subjected to mass spectrometry, and 24 proteins were identified. PAM cells infected with the virulent strain showed upregulated expression of pyruvate kinase M2 (PKM2), heat shock protein beta-1 (HSPB1), and proteasome subunit alpha type 6 (PSMA6), which were downregulated in cells infected with the attenuated strain. The upregulation of PKM2 provides sufficient energy for viral replication, and the upregulation of HSPB1 inhibits host cell apoptosis and therefore facilitates mass replication of the virulent strain, while the upregulation of PSMA6 facilitates the evasion of immune surveillance by the virus. Studying on those molecules mentioned above may be able to help us to understand some unrevealed details of HP-PRRSV infection, and then help us to decrease its threat to the swine industry in the future.

Figure 1. 2D-DIGE analysis of PRRSV-infected PAMs and mock-infected PAMs.

Arrows indicate isolated and identified protein spots that were up- or downregulated by at least 1.2-fold (P<0.01). Spots are numbered according to Table 3. Equal amounts of total protein from infected and uninfected whole cell lysates were resolved by 2D-DIGE.

Figure 2. Functional classification.

Column diagrams showing the gene ontology (GO) distribution of differentially expressed proteins according to major biological process categories (A), molecular function categories (B), and cellular component categories (C). The Y axis represents the percentage (%) of GO terms.

Figure 3. The protein-protein interaction network as analyzed by String software.

An edge was drawn with up to seven differently colored lines that represent the existence of the seven types of evidence used in predicting the associations. A red line indicates the presence of fusion evidence; a green line indicates neighborhood evidence; a blue line indicates co-occurrence evidence; a purple line indicates experimental evidence; a yellow line indicates text-mining evidence; a light blue line indicates database evidence; and a black line indicates coexpression evidence.

Figure 4. Confirmation of the transcriptional regulation of DEPs by real-time RT-PCR.

Transcript alteration of three selected genes in PAM cells from the PRRSV-infected group compared with the mock-infected group. Total RNA extracted from PAM cells was measured by real-time RT-PCR analysis; relative expression levels were calculated according to the 2^ΔΔCT method, using β-actin as an internal reference gene and the mock-infected group as a calibrator (relative expression = 1). Error bars represent the standard deviation. Please refer to Table 2 for the identification of gene symbols that represent different genes.