Label-Free Quantitative Phosphoproteomic Analysis Reveals Differentially Regulated Proteins and Pathway in PRRSV-Infected Pulmonary Alveolar Macrophages

Porcine reproductive
and respiratory syndrome virus (PRRSV) is
an important pathogen of swine worldwide and causes significant economic
losses. Through regulating the host proteins phosphorylation, PRRSV
was found to manipulate the activities of several signaling molecules
to regulate innate immune responses. However, the role of protein
phosphorylation during PRRSV infection and the signal pathways responsible
for it are relatively unknown. Here liquid chromatography–tandem
mass spectrometry for label-free quantitative phosphoproteomics was
applied to systematically investigate the global phosphorylation events
in PRRSV-infected pulmonary alveolar macrophages. In total, we identified
2125 unique phosphosites, of which the phosphorylation level of 292
phosphosites on 242 proteins and 373 phosphosites on 249 proteins
was significantly altered at 12 and 36 h pi, respectively. The phosphoproteomics
data were analyzed using ingenuity pathways analysis to identify defined
canonical pathways and functional networks. Pathway analysis revealed
that PRRSV-induced inflammatory cytokines production was probably
due to the activation of mitogen-activated protein kinase and NF-κB
signal pathway, which were regulated by several protein kinases during
virus infection. Interacting network analysis indicated that altered
phosphoproteins were involved in cellular assembly and organization,
protein synthesis, molecular transport, and signal transduction in
PRRSV infected cells. These pathways and functional networks analysis
could provide direct insights into the biological significance of
phosphorylation events modulated by PRRSV and may help us elucidate
the pathogenic mechanisms of PRRSV infection.