Proteomic alteration of Marc-145 cells and PAMs after infection by porcine reproductive and respiratory syndrome virus

Abstract

Viral infections usually result in alterations in the host cell proteome, which determine the fate of infected cells and the progress of pathogenesis. To uncover cellular protein responses in porcine reproductive and respiratory syndrome virus (PRRSV), infected pulmonary alveolar macrophages (PAMs) and Marc-145 cells were subjected to proteomic analysis involving two-dimensional electrophoresis (2-DE) followed by MALDI-TOF-MS/MS identification. Altered expression of 44 protein spots in infected cells was identified in 2D gels, of which the 29 characterised by MALDI-TOF-MS/MS included 17 up-regulated and 12 down-regulated proteins. Some of these proteins were further confirmed at the mRNA level using real-time RT-PCR. Moreover, Western blot analysis confirmed the up-regulation of HSP27, vimentin and the down-regulation of galectin-1. Our study is the first attempt to analyze the cellular protein profile of PRRSV-infected Marc-145 cells using proteomics to provide valuable information about the effects of PRRSV-induced alterations on Marc-145 cell function. Further study of the affected proteins may facilitate our understanding of the mechanisms of PRRSV infection and pathogenesis.

Fig. 1

2-DE analysis of PRRSV-infected Marc-145 cells (A) and PAMs (C), mock-infected Marc-145 cells (B) and PAMs (D). Arrows indicate the isolated and identified protein spots with at least 1.5-fold up-regulation (A and C) or down-regulation (B and D). Spots are numbered according to Table 2. Equal amounts of total protein from infected and uninfected whole cell lysates were resolved by 2-D PAGE. The protein spots were visualized by silver staining.

Fig. 2

Transcript alteration of seven selected genes in PAMs and Marc-145 cells from the PRRSV-infected group compared with the mock-infected group. Total RNA extracted from PAMs or Marc-145 cells was measured by real-time RT-PCR analysis; relative expression levels were calculated according to the 2^−ΔΔCT method, using GAPDH as an internal reference gene and the mock-infected group as a calibrator (relative expression = 1). Error bars represent the standard deviation. For identification of gene symbols representing different genes, refer to Table 2.

Fig. 3

Western blot confirmation of representative proteins in PRRSV-infected Marc-145 cells (A) and PAMs (B). Aliquots of 50 μg of protein extracts were loaded per lane and resolved by 12% SDS-PAGE gel. Western blot analysis was then performed using antibodies to the proteins of HSP27, vimentin, galectin-1 and β-actin. The latter was used as an internal control to normalize the quantitative data.