| Transmissible gastroenteritis is a highly contagious acute infectious diseases characterized by vomiting, diarrhea and severe dehydration. Pigs of different ages are susceptible to the disease; the lethal infection rate of two weeks old piglets is up to 100%. TGEV often co-infected with porcine epidemic diarrhea virus, porcine δ coronavirus, which has caused tremendous economic losses to the global swine industry.Type I interferon(IFN-α / IFN-β) and interferon-stimulating factor are the potent anti-viral effectors. In order to infect the host and proliferation, many viruses have evolved various countermeasures to inhibit the IFN production,but some virus infection can induce interferon production. Previous studies have shown that TGEV infection can induce interferon production, but the molecular mechanism remains unclear. Hence, deepen study on the molecular mechanisms of TGEV inducing IFN-β will help to clarify the immunological mechanisms and pathogenesis of TGEV and provide the scientific basis for further prevention and control of TGE. 1. TGEV stimulate IFN-β expression dose-dependently in PK-15 cellsAs the main antiviral effector molecule, type I interferon is widely expressed in the host cells and involved in the regulation of host antiviral immune response. Through the detection of IFN-β、IRF3 and NF-κB promoter activity by dual luciferase experiment, TGEV infection could significantly activate IFN-β in a dose-dependent manner, depending on IRF3 and NF-κB. 2. TGEV nsp14 significantly induce IFN-β activationMany virus-encoded proteins were involved in regulation of IFN-β. We used the interferon promoter-reporter system to screen TGEV encoded proteins for their activities to regulate the interferon signaling. The results showed that TGEV nsp14 was the most significant inducer of IFN-β production. In addition, TGEV nsp14 induced the activation of NF-κB more obviously than IRF3, which indicated that the activation of IFN-β mainly depended on NF-κB. 3. The molecular mechanisms of TGEV nsp14 inducing NF-κB activationPhosphorylation and nuclear translocation of p65 is the typical marker of NF-κB activation. By western blot and indirect immunofluorescence assay, we found that TGEV nsp14 increased phosphorylation of p65 and promoted nuclear translocation of p65 to activate NF-κB. By constructing a series of nsp14 mutants and analyzing their NF-κB activation ability, we confirmed that nsp14 regulated immune response depending on its exonuclease activity and N7-methyl transferase activity.4. The mechanism of TGEV activation of IFN-β signaling pathwayHelicase protein DDX1 is a novel discovered recognition receptor in IFN-β production and was found interacted with SARS or IBV nsp14 protein. Hence, we analyzed whether TGEV induce IFN-β expression via DDX1.By co-immunoprecipitation assay and indirect immunofluorescence assay, we confirmed DDX1 could interact with TGEV nsp14. By over-expressing or knocking down DDX1 and analysing the effect on nsp14 or TGEV inducing IFN-β, ISGs and inflammatory cytokine, we confirmed that the nsp14 and DDX1 interaction mediated the recognition of TGEV by host immune system, in turn triggering anti-viral response. 5. The effect of DDX1 on the proliferation of TGEVMany helicase proteins can interact with the viral protein to help or inhibit the proliferation of the virus. By over-expressing or knocking down DDX1, we analyze the effect of DDX1 on level of TGEV infection titer, genome replication and protein expression. The results confirmed that DDX1 inhibited the proliferation of TGEV.6. The effect of TGEV infection on expression and localization of DDX1In the long-term fight of the virus against the host, virus have evolved various strategies to conduct their proliferation. Many viruses can degrade or alter the distribution of the host anti-viral protein to evade antivirus protection. In this study, we found TGEV infection inhibited DDX1 expression, but had no impact on the distribution of DDX1. |
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