Molecular Mechanism Of Interaction Between Avian Influenza Virus PB2 Protein And Duck MAVS Protein

Avian influenza virus(AIVs)is an acute pathogen causing infection of birds and mammals,which seriously endangers poultry industry and threatens human health.Avian influenza viruses were divided into low pathogenic avian influenza viruses(LPAIVs)and highly pathogenic avian influenza viruses(HPAIVs)according to their pathogenicity in chickens.From January 2015 to November 2018,the H5N1 subtype avian influenza virus caused 11 poultry outbreaks,including at least 51,000 poultry infections,32,800 deaths,and 123,900 kills in China.Thus,H5N1 subtype avian influenza is still a severe avian infectious disease in China.At present,the study on the innate immune of poultry is still at an early stage,and the mechanism of the interaction between avian influenza virus and innate immune system of poultry is still unclear.This study aimed at investigating the molecular mechanism of duck Mitochondrial antiviral signaling protein(MAVS)protein affecting the replication of H5N1 subtype avian influenza virus.To identify the duck MAVS gene and predict its domains,duck MAVS gene was amplificated from DEF cells by RT-PCR.The results showed that the CDS of duck MAVS gene was 1860 bp and encoded 619 amino acids.The amino acid homology of duck MAVS gene was only 10%-18% with that of other mammalian MAVS genes.Structural domains prediction showed that duck MAVS gene was similar to human MAVS gene,including a caspase activation and recruitment domain(CARD),a proline-rich region domain(PRR)and a transmembrane domain(TM).To verify the function of duck MAVS(DKMAVS)protein,duck MAVS gene and its gene fragments were overexpressed in DEF cells.The results showed that duck MAVS protein could significantly upregulated the m RNA levels of TRAF3,IRF-7,IFN-β and IL-6,and could also activated avian IFN-β promoter and IRF-7 promoter.The 1-92 and 1-175 amino acid regions of DKMAVS were the key regions for inducing the expression of immune gene and activating avian IFN-β promoter and IRF-7 promoter.Therefore,DKMAVS can activate the duck TRAF3-IRF-7-IFN-β signaling pathway.To study the molecular mechanism of interaction between avian influenza virus PB2 protein and duck MAVS protein,H5N1 subtype avian influenza virus PB2(212 PB2)gene and duck MAVS gene were co-transfected in DEF cells.The results showed that DK212PB2 could significantly inhibited the expression of MAVS,TRAF3,IRF-7,IFN-β and IL-6,and could also significantly inhibited the activity of IFN-β promoter and IRF-7 promoter.Co-IP analysis showed that 212 PB2 could directly interacted with DKMAVS by the 1-175 amino acid region.Further analyzed showed that the 1-177 and 535-759 amino acid regions of 212 PB2 protein could inhibited the m RNA expression of MAVS,TRAF3,IRF-7,IFN-β,IL-6 and inhibited the activity of avian IFN-β promoter and avian IRF-7 promoter.Co-IP analysis also found that the 1-177 and 535-759 amino acid regions of DK212PB2 protein could directly interacted with DKMAVS,but the 1-177,318-483 and 535-759 amino acid regions of DK212PB2 could directly interacted with mouse MAVS protein.Therefore,the interaction regions of 212 PB2 protein with duck MAVS and mouse MAVS are different.In summary,duck MAVS protein is a key adaptor protein in duck RLRs signaling pathway,and the 1-175 amino acid region of duck MAVS is the key region for its function.The PB2 protein of H5N1 subtype HPAIVs can directly interact with duck MAVS protein,and significantly inhibit duck MAVS-mediated innate immunity signaling pathway.The 1-177 and 535-759 amino acid regions of DK212PB2 protein could directly interacted with DKMAVS,but the 1-177,318-483 and 535-759 amino acid regions of DK212PB2 could directly interacted with mouse MAVS protein,which indicates that the interaction mechanism between duck and mouse MAVS protein and DK212PB2 are different.This will help us to understand the molecular mechanism of avian influenza virus regulating duck MAVS signaling pathway.