Analysis Of Porcine Reproductive And Respiratory Syndrome Virus Infection Influencing TypeⅠInterferon Expression

Porcine reproductive and respiratory syndrome(PRRS), an economically swine disease caused by porcine reproductive and respiratory syndrome virus(PRRSV), was characterized by high fever, respiratory disorder in nursery swine and severe reproductive disorder in sows. Inhibitory effect on innate immunity of PRRSV was owning to non-structural protein interacting with pathways of antiviral innate immunity. However the molecular mechanism of PRRSV infection inhibited immunosuppression has not been completely resolved. HuN4 strain of highly pathogenic porcine reproductive and respiratory syndrome virus with HuN4-F112 strain of attenuated strain, which had a clear genetic background, were used to infect Marc-145 cells. It is reported that the IFN-β expression on cells infected with HuN4-F112 was more than that incfcted with HuN4 by relative real-time PCR detection. To make clear the difference IFN-β expression between two PRRSV strains, detected activity of IFN-β promoter after transfection eukaryotic expression plasmid of NSPs by dual luciferase detection system, we showed that two PRRSV strains had the same NSPs to inhibite innate immunity, including NSP1α, NSP1β, NSP2, NSP4 and NSP12, but these same NSPs make no difference on inhibition capacity. Howver we showed NSP12 could inbite IFN-β promoter activity, which had never been reported,the fuction of NSP12 was poorly understood. We confired that inhibitor capacity of NSP12 depended on transfection dose. Cotransfection with espression vecter, including RIG-I, VISA, IKKβ, IKKε and cis-acting elements of IRF3 or NF-κB, luciferase assay reported that NSP12 was failed to inhibite IFN-β promoter activity which was stimulated by RIG-I pathway proteins. The reseason of NSP12 inbitor capacty has yet to be in-depth study.As previous conclusion, PRRSV could up-regulate IL-10 expresison, prosess of which needed CREB interaction with CBP, however phosphorylated p65 also could combine CBP to transcrapt I-IFN genes, it was confused us which one could combine CBP when infected with PRRSV.CREB activity analysis showed that level of CREB phosphorylation was up-regulated in PAMs infected with PRRSV and phosphorylated CREB remainted all the time, while CREB phosphorylation was found only in 15min-30 min after infected in Marc-145. To confirm that p38 MAPK and PKA singnal pathway were activated in PRRSV infected, p38 and ERK phosporylation were analyzed by western blot and CREB phosporylation were ananlyzed by incubation PKA inhibitor. The result showed that p38 phosporylation was significantly increased and PKA inhibior suppressed CREB phosporylation after Marc-145 infcrted with HuN4 strain. In PAMs, p38 phosporylation was remarkable increased and PKA inhibitor failed to inhibite CREB phosporylation. Co-Immunoprecipitation expriment showed that PRRSV infection stimulated the combition of CREB phosporylation and CBP, while inhibited the combition of CBP with p65 phosporylation, which stimulated by human tumor necrosis factor α(hTNF-α). Our results showed that HuN4 could activite p38 MAPK pathway to phosporylat CREB competitive binding CBP to inhibite I-IFN transcription. This study would boost our further understanding of innate immune suppression of PRRSV.