Mechanism Of Inhibiting Interferon-β Induction By Infectious Bursal Disease Virus Infection

Infectious bursal disease virus (IBDV) is an Avibirnavirus belonging to the Birnavirus family. IBDV infects 3-6 weeks young chicken mainly, and causes the immunosuppression. The exact mechanisms of immunosuppression caused by IBDV infection has not been elucidated clearly. In this study, we investigated the interinfluence between IBDV infection and the type Ⅰ interferon (IFN) system in vitro and the mechanism of VP3 inhibiting the antiviral innate immune response, in order to supply the foundation for exploring the immunosuppression mechanism of IBDV and finding the potential targets to develop the efficient therapeutic drugs for the IBD.Type Ⅰ IFN is the first line for the host antiviral immune response, pattern recognition receptors (PRRs), including RLRs and TLRs, mediate the type Ⅰ IFN induction during the viral infection. Chicken Interferon-α/β (chIFN-α/β) was firtly discovered and named, but the the pathways involved the chIFN-α/β induction had not been clearly described. In this study, we firstly cloned the chicken MAVS (chMAVS) whose mammalian counterpart supplies important platform for the IFN-β induction during the viral infection, and the following bioinformaitcs analysis showed that the chMAVS possesses the similar constitution domains and the same subcellular localization with the human MAVS. Furthermore, the chMAVS mediated the chIFN-β induction upon the intracellular Poly(I:C) stimulation. Coexpression of the chMAVS and the transcription factor chicken IRF3 (chIRF3) which nuclearly translocates in the mammalian during IFN-β induction showed that the chIRF3 did not change its subcelluar localization. So, the chMAVS mediates the chIFN-β production, however, chIRF3 nuclear tranlocation is not involved in the process.Previous reports showed that the chicken IFN-α/β (chIFN-α/β) showed the anti-IBDV effect in vivo. In this study, we showed that chIFN-α/β inhibted the IBDV replication significantly but not the viral entry in vitro. Despite the naked IBDV genomic dsRNA could be sensed by cytosolic PRR, chicken MDA5 (chMDA5) and upregulates the chIFN-β expression, it did not induce the chIFN-α/β upregulation during IBDV infection of DF-1 cells, neither with different hours (range from 1 h.p.i.[hours post infection] to 12 h.p.i.) nor with different MOI (multiplicity of infection). Collectively, IBDV genomic dsRNA is PAMP and could be sensed by the host PRR chMDA5, some viral proteins of IBDV may block the recognition of chMDA5 to the IBDV genomic dsRNA or the downstream signal transduction. VP3, VP4 and VP5 were found to be the candidates of the inhibitors to block the intracellular dsRNA induced chIFN-β expression by the dual-luciferase assay. Combined with the results from the endogenous chIFN-β expression induced by the intracellular IBDV genomic dsRNA stimulation based on the viral proteins inducible espression, we found out that VP3 and VP4 are the inhibitors that block the intracellular IBDV genomic dsRNA induced chIFN-β expression. dsRNA pulldown assay showed that the VP3 possesses the dsRNA binding activity and this phenomenon existed commonly in the VP3 from the other members of birnavirus. Construction of site-direct mutants of VP3 and performing dsRNA pulldown assay demonstrate that the basic amino acids of K99R102K105K106 were involved in the dsRNA binding activity together. Overexpression the mutants of VP3 with the decreased dsRNA binding capability reduced the blockage of the chIFN-β induction by the intracellular IBDV genomic dsRNA stimulation. Adding the IBDV genomic dsRNA as competitor to perform the dsRNA pulldown assay, VP3 showed the higher affinity than the chMDA5 binding to the IBDV genomic dsRNA, and the coimmunoprecipitation assay showed that VP3 and chMDA5 did not form the complex eventhough in the presence of the IBDV genomic dsRNA, that means the chMDA5 and VP3 did not bind to the same dsRNA. All of the results indicated that the VP3 with the higher affinity binding to dsRNA blocks the chMDA5 to sense the IBDV genomic dsRNA and induce the chIFN-β production.