Molecular Mechanism Of PI3K/Akt/GSK3 Signal Pathway And Innate Immunity Restriction Factor BST-2 Regulate PEDV Replication

Porcine epidemic diarrhea(PED), caused by porcine epidemic diarrhea virus(PEDV), is an acute and highly contagious enteric disease of pigs. PED is characterized by watery diarrhea, vomiting and dehydration in sucking pigs, and the mortality rate as high as 100% for sucking pigs younger than 7 days of age after the virus infection. Since 2010, various mutated PEDV strains have been identified which have caused large-scale PED outbreaks occurred in swine farms in China. This has made the existing PED vaccine lost its efficacy and caused large economic losses to the swine industry. In order to investigate the PEDV immune escape mechanism and cellular immune response, the 2-DE and MS/MS technology was used to detect the differences in proteomics between PEDV-infected and uninfected Vero cells. We found that signal moleculesparticipating in PI3K/Akt signal pathway and the innate immunity restriction factor BST-2 are significantly altered in these cells suggesting these molecules play important role in PEDV infection. PI3K/Akt signal pathway is one of the most important pathway in regulateing multiple functions of cell process. Multiple viruses infected cells would active PI3K/Akt pathway.The innate Immunity Restriction Factor BST-2 is a transmembrane protein that can be stimulated by type I and II interferon(IFN). This protein can use its structural topology to tie the virus on the cell membrane, and inhibits the virus to infect other cells. The N-terminal domain of BST-2 could also inhibit PEDV proliferation by activating NF-κB.1. The expression of different proteins in PEDV-infected Vero cellsProteomics is one of the most useful technologies to detect virus and host cell interaction. First, we determined the correlation between the cytopathic effect(CPE) and the virus amount in the infected cells to select the most amount of virus infection without causing the CPE for proteomic analysis.We found that the most amount of virus that did not cause CEP was at 5 hours post infection(hpi) and thus chose 5 hpi as the time point for the proteomic analysis.We found 67 of differentially expressed protein spots using 2-DEseparation of infected and uninfected cells, of which 38 were significantly up-regulated and 29 down-regulated. We also found 58 differentially expressed proteins from2-DE separated proteins, of which 33 were significantly up-regulated and 25 down-regulated usingMS/MS analysis. Four representative proteins, LGALS1, Peroxiredoxin-1, SNRPC and PTEN were verified by Real-time PCR and western blotting. The results show the same patterns of the altered protein expression levels as that using MS/MS. These proteins are involved in energy metabolism, protein translation, stress response, cell adhesion, apoptosis, immune system processes and cell killing usingGO enrichment analysis suggesting they participate in virus immune escape and cellular immune response.2. Molecules in PI3K/Akt/GSK3 signaling pathway inhibit PEDV proliferation in Vero cellsPhosphatidylinositol-3-kinase(PI3K)/Akt signal pathway regulates multiple cellular functions, such as cell growth, cell proliferation, inflammation and cell survival/apoptosis. PI3K/Akt signaling pathway not only can be used by cells to inhibit virus proliferation, but also can be used by virus to manipulate the intracellular environment for optimal viral replication. In this study, our data indicated that PI3K-dependent Akt phosphorylation was observed at both the early(5 min – 15 min) and late(4 h) stage of PEDV infected in Vero cells. The PI3K-specific inhibitor LY294002 could obviously inhibit Akt phosphorylation, and promotePEDV proliferation in Vero cells. PI3K/Akt pathway inhibited PEDV proliferation in Vero cells via phosphorylated GSK3. When inhibited GSK3 activity by the specific GSK-3α/β inhibitor CHIR-99021, the PEDV proliferation increased significantly. The downstream targets of Akt, mTOR, Bad and p53 were not regulated by PEDV infection. The results suggested that the PI3K/Akt signal pathway not only can be activated by virus structural proteins in the early stage of PEDV-infected Vero cells, but also can be activated by viral replication in the late stage of virus infection. PI3K/Akt signaling pathway inhibited PEDV proliferation in Vero cells via phosphorylated GSK3.3. BST-2 inhibit PEDV proliferation in Vero cellsBST-2 is a type II transmembrane protein that could induce expression by IFN. T Budding virions using one of the two membrane anchors of the BST-2 protein, whereas the other one membrane anchor remains attached to the plasma membrane of the host cell. To explore the antiviral effect of BST-2 on PEDV infection, the porcine BST-2 complete gene(851 bp, containing 23 bp of 5’-UTR, 534 bp of CDS and 294 bp of 3’-UTR) and promoter(1950 bp) sequence were cloned. The specific MAbs against porcine BST-2 were generated. BST-2 mRNA was detected in most of the porcine tissues, and higher levels of BST-2 mRNA were detected in immunity tissues and organs(lymphonods, thymus, tonsil, and spleen), large intestine, small intestine, and lungs. This suggests that BST-2 play an important role in the early innate immuneresponse. Overexpressing of the porcine BST-2 in Vero cells could inhibit PEDV proliferation. When BST-2 protein expression was inhibited in Vero cells by siRNA targeting BST-2 gene, the virus proliferation was significantly increased. The results from the truncated expressed BST-2in Vero cells and the Luciferase activity test suggest that BST-2 uses its two membrane anchors to tie the virus to the plasma membrane of the host cell, and uses its N-terminal domain to activate NF-κB to inhibit PEDV proliferation in Vero cells.