Differential Phosphoproteome Of Porcine Alveolar Macrophage Associated With Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus Infection

Porcine reproductive and respiratory syndrome (PRRS) is the most important infectious disease of swine in present worldwide, and its etiological agent is porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV is characterized by antigen variability, macrophage tropism, antibody dependent enhancement(ADE) and persistent infection. In comparison with the classical PRRSV isolated in China, the pathogenicity of HP-PRRSV was significantly enhanced,but the reasons are still unclear.Protein is the basic molecule in cellular activity, and phosphorylation is an important chemical modification of protein which plays vital roles in completion and change of protein function. Infection of host cells by virus, as the extracellular signal, affects the normal cell activities by a series of cell signal transduction, of which protein phosphorylation is the most extensive and most important posttranslational modifications. Porcine alveolar macrophages (PAMs) are the primary target cells for PRRSV infection in vivo, and investigation on the differential phosphoproteins between normal PAMs and HP-PRRSV-infected PAMs may provide some valuable reference data for explaining the molecular mechanism of HP-PRRSV enhanced pathogenicity.In this study, we harvested PAMs from healthy piglets of three 30 days-old without both PRRSV and its antibody. The PAMs were divided into two groups, uninfected group (C group) and infected group (T group). The phosphoproteins for liquid chromatograph equipped with tandem mass spectrometer (LC-MS/MS) were purified by PhosphoProtein Purification Kit(QIAGEN). Finally, we identified 306 proteins in C group and 420 proteins in T group through searching in non-redundant suina database of international protein indicators (IPI) of MS/MS spectral data. Simultaneously, seting 79.9799 STY dynamic modification, we found 58 phosphorylation peptides of 56 proteins in C group and 48 phosphorylation peptides of 46 proteins in T group. Searching in the DAVID Bioinformatics Resources 6.7 database (NIAID/NIH), we found that in C group, 113 proteins have functional categories, and 27 of them are unique; and in T group,145 proteins have functional categories, and 59 of them are unique. From the functional point of view, those functional categories proteins were mainly classified into 9 types: immune response, stress response, signal transduction, protein metabolism, nucleic acid metabolism, metabolism, cell activity, cytoskeleton and organelles component. We compared the list of functional categories proteins to the biological pathways annotated by the Kyoto Encyclopedia of Genes and Genomes (KEGG), and identified proteins in C group involved in 6 biological pathways and proteins in T group involved in 5 biological pathways.In uninfected control group, we found six specific membrane proteins or receptor proteins: CD163, TLR5, CD45, CD9, Pglyrp2/pPGRP-LB and LOX-1/OLR1. In infected group, we found a few differential phosphoproteins in regulation of actin cytoskeleton pathway, antigen processing and presentation pathway, and proteasome pathway. The exact role of these differential phosphoproteins in uninfected PAMs and HP-PRRSV-infected PAMs still needs to be determined further.