|Abstract:||Porcine epidemic diarrhea (PED) is a highly contagious and acute enteric disease of swine featured by vomiting, watery diarrhea, and severe dehydration causing high mortality in piglets. In the U.S., PED emerged for the first time in 2013 and severely affected most pig-producing states. The causative agent is PED virus (PEDV), which is an enveloped, positive-sense, single-strand RNA virus in the genus Alphacoronavirus of the family Coronaviridae in the order Nidovirales. The innate immune system is the first line of host defense in response to viral infections. Type I interferons (IFN-α/β) are the major components of the innate immune system, and in turn many viruses have evolved to modulate the host interferon responses.
In the present study, the PEDV IFN antagonism was investigated. We found that IFN-β production was significantly suppressed during PEDV infection, and of 16 PEDV nonstructural proteins (nsps), nsp1, nsp3, nsp7, nsp14, nsp15, and nsp16 were found to inhibit the IFN-β and IRF3 promoter activities. The sole accessory protein ORF3 and the envelope (E), membrane (M), and nucleocapsid (N) proteins were also determined to inhibit such activities. Since nsp1 is the first viral protein synthesized during PEDV infection and appears to be the most potent IFN antagonist, nsp1 was further investigated for its action. PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation upon stimulation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP) by degrading CBP. The CBP degradation by nsp1 was proteasome-dependent.
We further showed that PEDV inhibited both NF-κB and proinflammatory cytokine production in porcine epithelial cells. PEDV blocked the p65 activation in infected cells and suppressed the PRD II-mediated NF-κB activity. Nine proteins were identified as NF-κB antagonists, and nsp1 was the most potent suppressor of proinflammatory cytokines. Nsp1 interfered the phosphorylation and degradation of IκBα, and thus blocked the p65 activation. Mutational studies demonstrated the essential requirements of the conserved residues of nsp1 for NF-κB suppression.
Recent reports showed that type III interferons (IFN-λs) play a vital role to maintain the antiviral state of the mucosal epithelial surface in the gut. The intestinal epithelial cells selectively produce and respond to type III IFNs. To study the type III IFN response to PEDV, a line of porcine intestinal epithelial cells was developed as a cell model for PEDV replication. Recombinant proteins IFN-λ1 and IFN-λ3 inhibited PEDV replication, indicating the anti-PEDV activity of type III IFNs. Of the total of 21 PEDV proteins, nsp1, nsp3, nsp5, nsp8, nsp14, nsp15, nsp16, ORF3, E, M, and N were found to suppress the type III IFN response. PEDV specifically inhibited IRF1 nuclear translocation. Peroxisomes are the innate antiviral signaling platforms for activation of IRF1-mediated IFN-λ production, and they were found to decrease in number in PEDV-infected cells. PEDV nsp1 blocked the nuclear translocation of IRF1 and reduces the number of the peroxisomes to suppress the IRF1-mediated type III IFN production. The conserved residues of nsp1 were crucial for IRF1-mediated IFN-λ suppression. Taken together, these studies provide the evidence that PEDV and its nsp1 protein, a potent multifunctional IFN antagonist, evade both type I and type III IFN responses and the molecular basis for this antagonism.