SILAC-based quantitative proteomic analysis of secretome of Marc-145 cells infected with porcine reproductive and respiratory syndrome virus

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which causes severe reproductive failure in sows, respiratory disease in young and growing pigs, and enormous economic losses to the global swine industry. In this study, SILAC combined with MS/MS was used to quantitatively identify the secretory proteins differentially expressed in PRRSV-infected Marc-145 cells compared with mock-infected controls. In total, we identified 204 secretory proteins showing significant differences in infected cells (163 upregulated, 41 downregulated). Intensive bioinformatic analysis of secretome data revealed that PRRSV infection strongly activated nonclassical protein secretion, especially vesicle-mediated release of exosomal proteins, including different danger-associated molecular pattern molecules and the majority of secreted proteins involved in protein binding and transport, regulation of response to stimulus, metabolic processes, and immune responses. According to the functional proteins analysis, we speculate that proteins functioning in binding, transport, and the immune response are exploited by PRRSV to facilitate virus replication and immune evasion. Our study for the first time analyzes the secretory protein profile of PRRSV-infected Marc-145 cells and provides valuable insight into the host response to PRRSV infection.

Proteomic Analysis of the Secretome of Porcine Alveolar Macrophages Infected with Porcine Reproductive and Respiratory Syndrome Virus

“…In response to PRRSV infection, cells secrete a broad range of proteins . To avoid contamination by other proteins, PRRSV‐ and mock‐infected PAMs were cultured in serum‐free medium and the supernatants were collected at an optimal time point of viral replication before the appearance of cell damage.…”

“…It is known that PRRSV infection causes vasculitis in multiple organs of pigs, even though blood vessels are not directly targeted by PRRSV infection. It has been speculated that the secreted proteins, including inflammatory cytokines, are responsible for the inflammatory lesions during PRRSV infection . PRRSV‐infected intravascular macrophages are presented between the noninfected endothelial cells and close to blood vessels .…”

“…The proteome of Marc‐145 cells infected with PRRSV was recently described by Zhao et al . The differentially expressed proteins identified by Zhao et al.…”

“…The secretomic approach is a useful means to draw a profile of the proteins secreted by virus‐infected cells . A profile of the secreted proteins of PRRSV‐infected Marc‐145 cells was recently described, in which a total of 204 secreted proteins involved in different biological progresses was identified, thereby providing an interesting clue into the mechanisms underlying the host immune response against PRRSV infection . However, Marc‐145 cells, which originate from monkey kidney cells, are not the primary target cells of PRRSV infection and the immune response elicited by PRRSV infection is different from that of PAMs, which are the primary target cells of PRRSV infection .…”

iTRAQ-based Proteomic Analysis of Porcine Kidney Epithelial PK15 cells Infected with Pseudorabies virus

“…Proteomic approaches provide effective tools for facilitating a comprehensive characterization of virus-host interactions, for example, two-dimensional gel electrophoresis (2DE) and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) approaches have been used to provide the proteomic expression profiles of host cells in response to viral infections, including classical swine fever virus 10 , severe acute respiratory syndrome 11 , H1N1 influenza virus 12 and spleen and kidney necrosis virus 13 . Stable isotope labeling of amino acids in cell culture (SILAC) has also been used in identifying the differentially expressed proteins of viral infections, including dengue virus type 2 14 , porcine reproductive and respiratory syndrome virus 15 , Enterovirus 71 16 and herpes simplex virus type 1 17 . These studies have provided extensive insight into understanding the host response to viral infection and have also highlighted the potential antiviral agents capable of targeting the various kinds of viral infection.…”