The Mechanism By Which Porcine Epidemic Diarrhea Virus Inhibits Interferon-β Production And Activates NF-κB In Porcine Intestinal Epithelial Cells

Porcine epidemic diarrhea virus(PEDV) is the pathogen of swine that causes severe diarrhea and dehydration resulting in substantial morbidity and mortality in piglets. Although in recent years progress has been made in elucidation of the molecular epidemiology, diagnosis, prevention,and treatment of PED, its outbreaks continue to rise and pose problems to the swine industry and international trade. Thus, there is a need to understand the interaction between PEDV and host innate immune system. Such information would be valuable in understanding the molecular mechanisms regulating virulence, pathogenesis and disease outcomes.PEDV did not induce type I IFNs production, therefore, PEDV acquired the most of this opportunity to evade innate immune response. In addition, viruses have developed various strategies which lead to either activation or inhibition of NF-κB-dependent gene transcription for their benefits. However, the specific mechanisms of PEDV infection to induce type I IFN or weather activate NF-κB remain unclear. It is well known that PEDV mainly infects and damages porcine intestinal epithelial tissue and causing intestinal villus atrophy and congestion, resulting in formation of cytoplasm vacuoles in intestinal epithelial cells and subsequent desquamation. Porcine intestinal epithelial cells(IECs) are the target cells of PEDV.To clarify the mechanism that PEDV evade host innate immune responses, we chose IECs as an infection model to research the signal pathways in innate immune responses during PEDV infection.During viral infection and replication, the host innate immune response is the first line of defense; therefore, the ability of viruses to suppress or avoid this response is crucial for pathogenic potential. IFN is one of the key production of the host immune response. Here, we reported that PEDV infection of its target cell line, IECs did not induce IFN-β production and inhibited poly(I:C)-mediated IFN-β gene expression. IFN regulatory factor 3(IRF-3) and NF-κB are two essential IFN-β transcription factors, we found that NF-κB was activated during PEDV infection, but not IRF-3. In addition, PEDV inhibited ds RNA-induced IRF3 nuclear translocation and IFN-β production. To identify the molecule mechanism of PEDV intervention with IRF-3activation more accurately, the individual molecule of the retinoic acid-inducible gene I(RIG-I)and TIR domain-containing adapter inducing IFN-β(TRIF) of Toll-like receptor 3(TLR3)signaling pathway induced IFN-β production was investigated. Upstream of IRF3, the activity of TANK-binding kinase 1(TBK1) or inhibitor of κB kinase-ε(IKKε) was not blocked by PEDV,however, the adapter molecule mitochondrial antiviral signaling protein(MAVS) of RIG-I and RIG-I induced IFN-β transcription was completely suppressed by PEDV. Furthermore, we also found that PEDV infection only partially affected the adapter molecule TRIF(TIR domaincontaining adapter inducing IFN-β)-induced IFN-β production, which is part of the TLR3 signaling pathway. Taken together, our results indicated that PEDV infection did not induce IFN-βproduction mainly by interfering with the RIG-I signaling pathway.NF-κB regulates expression of numerous components of the immune system, which could modulate the expression of multiple pro-inflammatory cytokines, chemokines, adhesion molecules and inducible enzymes, as well as cell survival/proliferation. However, overactivation of NF-κB can result in inflammatory and autoimmune diseases, therefore, activation of this transcription factor is thought to be a hallmark of most infection. So far, there is no evidence showed that PEDV infection IECs could induce activation of NF-κB. The present study demonstrated for the first time that PEDV activated NF-κB in IECs. In PEDV-infected cells, NF-κB activation was characterized by translocation of NF-κB from the cytoplasm to the nucleus, increased NF-κBregulated gene expression. NF-κB activation was increased as PEDV infection progressed and in a viral dose-dependent manner. UV-inactivation of PEDV significantly reduced the level of NF-κB activation. Degradation of IκBα protein was detected late in PEDV infection, suggesting that PEDV-induced NF-κB activation had a certain relationships with the degradation of IκBα protein.To evaluate the specific mechanisms of this, we investigated NF-κB activation in RLRs and TLRs signal pathway during PEDV infection. By using small interfering RNA(si RNA) screening, we demonstrated that the adaptor TRIF and My D88 in TLRs signal pathway contributed to the activation of NF-κB in response to PEDV infection, but not RIG-I in RLRs signal pathway. In addition, we also investigated that PEDV-induced NF-κB activity was related with TLR2, TLR3 and TLR9, but not TLR4 and TLR8 in TLRs signal pathway by using siRNA technology. It is clear that viral nucleic acids encode proteins that to positively modulate NF-κB activities, thus, we investigated that the roles of PEDV structural proteins in the regulation of NF-κB. Screening PEDV structural proteins for their abilities to induce NF-κB activities by using luciferase activity assay, we investigated that in IECs overexpression of PEDV N protein significantly increased NF-κB activity and this increase was in a dose-dependent manner, and the middle region of the N protein was essential for NF-κB activation. Furthermore, TLR2 was involved in PEDV N-induced NF-κB activation in IECs. Our results presented here provided a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by PEDV.