Proteomic Analyses Of Swine Testicular Cells Infected With Porcine Transmissible Gastroenteritis Virus

Transmissible gastroenteritis is an acute intestinal infectious disease of swine caused by transmissible gastroenteritis virus(TGEV). Diseased pigs often present with severe vomiting, dehydration, and diarrhea. The mortality rate for piglets under 2 weeks of age is almost 100%. Outbreaks of the disease can cause enormous losses in the pig industry throughout the world. But the molecule mechanisms of TGEV pathogenicity are not fully understood. Also analyses of host cells infected with the virus are involved little. Notably, ST cells are more susceptible to TGEV, and are usually used to multiply and isolate the virus. A proteomic approach using isobaric tags for relative and absolute quantitation(i TRAQ)-coupled two-dimensional liquid chromatography-tandem mass spectrometry(2D LC-MS/MS)identification was conducted on the TGEV-infected and uninfected ST cells. The obtained differentially expressed proteome data were further analyzed by bioinformatics, that will help us to find new targets for the disease diagnosis, protection, cure and provide foundations in the understanding of the molecular pathogenesis of TGEV. The main contents are as following:(1)We determine which time points to investigate for the 4-plex i TRAQ proteomic analysis by observing the cytopathic effect(CPE) and analyzing viral gene expression dynamics in the TGEV-infected ST cells. The results indicated that TGEV induced significant CPEs from 40 to 64 h post infection(pi) in infected cells compared to the mock infected cells. While at 48 hpi more than 80% infected cells showed typical CPE and the m RNA level of the viral gene reached a peak. At 64 hpi, the morphological effects observed were much more pronounced, characterized by even more cellular rounding and detachment. However, the m RNA levels of the viral gene decreased rapidly, a phenomenon we believe may be attributed to the host’s immune response or a decrease in infected cell viability as the TGEV infection progressed. Accordingly, we choose TGEV-infected ST cells at 48 hpi and 64 hpi for later proteomic analysis.(2)Protein separation and identification in the TGEV-infected or uninfected cells were performed using i TRAQ-coupled 2D LC-MS/MS. Further protein quantification were performed with the Protein pilot software 4.0. After searching database, a total of 29,214 peptides and 4,112 confident proteins were detected. In order to identify the proteins that were significantly different post infection, we analyzed the distribution of ratios for the identified proteins and confirmed the distribution range of the differentially expressed proteins(ratios were greater than 4.0 or less than 0.25 compared to the control group). Using the criterion, We identified 316 unique proteins being significantly altered during TGEV infection, 146 of which showed significant changes in expression 48 h after infection. At 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection.(3)GO enrichment analysis of the differentially altered proteins was evaluated against all quantified proteins as background. The results showed that 18 biological processes were significantly enriched(p ≤ 0.05) in this set of proteins, mainly relating to cell adhesion, cellular stress response, generation of precursor metabolites and energy, protein mature, protein complex assembly, immune system processes, cell death, transmembrane transport and cell motility, etc. GO enrichment analysis of the differentially expressed proteins identified at 48 and 64 hpi revealed that six common biological processes were significantly affected(p ≤0.05), while a significant number of the differentially expressed proteins were involved in cellular stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, and immune system processes only at 64 h post infection. The differentially expressed proteins involved in these processes are bound to the virus replication, immune response and cell lesions.(4)To determine the main biochemical metabolism and signal transduction pathways that the differentially expressed proteins are related to, KEGG pathway enrichment analyses of these proteins were conducted. The result indicates 61 significantly enriched pathways(p ≤ 0.05) primarily mapped to the following aspects: 16 pathways distributed in the categories of carbohydrate metabolism, energy metabolism, amino acid metabolism, metabolism of cofactors and vitamins, and lipid metabolism, 10 pathways in the infectious diseases category, 4 pathways in the immune system category, 3 pathways in the digestive and excretory system categories, 2 pathways in the signaling molecules and interaction category, 2 pathways in the signal transduction category. Some pathways were significantly affected only in the differentially expressed proteins for 64 hpi, such as NOD-like receptor and RIG-I-like receptor signaling pathways associating with immunity, pathways associating with infectious disease, TNF signaling pathway, and PI3K-Akt signaling pathway. The differentially expressed proteins that are involved in the above pathways may play an important modulator in the TGEV infection and transmissible gastroenteritis pathogenesis.(5)According to our proteomics data and the GO analysis result, changes in the expression levels of four representative proteins, transforming growth factor beta 1(TGF-β1/TGFB1), Caspase-8, heat shock protein 90 alpha(HSP90α) and probable ATP-dependent RNA helicase DDX58(DDX58/RIG-1) were verified by Western blot analysis. Thus, these results confirm the altered expression observed in the proteomic data for these four representative proteins during TGEV infection, proving the reliability of mass spectrome. These proteins are involved in cellular stress, cell death, innate immune response and cellular protein complex assembly that are induced by TGEV infection.This study is the first time the response profile of ST host cells following TGEV infection has been analyzed using i TRAQ technology. Our description of the comprehensively bioinformatics analysis, the late proteomic changes that are occurring after the time of vigorous viral production and many of the significant immune response related changes in protein expression we discovered are novel, and, to our knowledge, have not been detected in previous proteome study. These results characterize proteomic changes during the host response to TGEV in ST cells and provide a good foundation to uncover the mechanisms of TGEV infection and pathogenesis.