Mechanism Of BinCARD1 Regulating Influenza Virus Replication

Influenza viruses belong to the Orthomyxoviridae family,which is characterized by a segmented,negative sense,and single-stranded RNA(ss RNA)genome.They are categorized into four genera(type A,B,C,and D),among which the natural host of IAV is a wild waterfowl,in addition,it can infect a wide range of poultry as well as mammals,where it is also the etiological agent responsible for human influenza pandemics and seasonal transmission.Currently,there are two main strategies used to control influenza infection:vaccination and antiviral drug therapy.Current influenza vaccines require seasonal updates and offer only partial protection in some at-risk populations.Furthermore,in the event that a new strain causes a pandemic,It will take a while to develop an effective vaccine(it takes at least 6 months using the traditional chicken embryo based approach)and may be too slow to effectively prevent the spread of the virus.In addition,vaccines may confer insufficient protection in some immunocompromised patients.Therefore,the development of novel anti influenza agents against host factors becomes a promising research effort to combat the infection of influenza viruses.The host factor Bin CARD is the 19 th member of the caspase recruitment domain family and is expressed predominantly as two spliceosomes in human derived cells.Bin CARD1 inhibits BCL10 activation and then the activity NFκB is blocked,In turn,Bin CARD2 positively regulates the type I interferon pathway.In this study,si RNA silencing and CRISPR/ cas9 technology were used to demonstrate that down-regulation of expression or knockout of Bin CARD could inhibit the replication of different subtypes of influenza viruses.The overexpression cell lines with the 2 spliceosomes further confirmed that Bin CARD1 can positively regulate influenza virus replication while Bin CARD2 has no effect on influenza virus replication.Immunofluorescence assay confirmed that knockout of Bin CARD1 was able to significantly inhibit the early stage of influenza virus replication.The results of nucleocytoplasmic fractionation assay further confirmed that knockout of Bin CARD1 significantly inhibited the process of NP protein incorporation.On specific mechanistic exploration,the Co-IP assay results showed that Bin CARD1 was able to interact with NP proteins and importins α7.A follow-up trial further confirmed that Bin CARD1 promotes NP association with importins α7 to facilitate the nucleation of v RNPs.Since the function of Bin CARD1 in type I interferon pathway has not been reported,this study continues to explore its function in interferon pathway according to the phenomenon that Bin CARD1 can promote influenza virus replication.In this study,we confirmed that Bin CARD1 was able to activate the type I interferon pathway by using a dual luciferase reporter system with type I interferon stimulation as well as ISRE promoter reporter plasmid.Subsequent Co-IP assay showed that Bin CARD1 was able to interact with TRAF3 and enabled K63 linked polyubiquitination of TRAF3,and the NP protein was able to compete with TRAF3 for binding to Bin CARD1.In the study,it was found that TBK1 was able degrade Bin CARD1.And this degradative effect was mediated through the autophagy pathway.Follow up assay further explored the degradation of Bin CARD1 by TBK1 because the K63 linked polyubiquitination of Bin CARD1 occurred at the 103 lysine site,TBK1 could activate the phosphorylation of p62 at S403 site,and the activated p62 could recognize the Bin CARD1 that occurred ubiquitination,which captured and transported Bin CARD1 to lysosome degradation.Throughout the course of influenza virus infection,TBK1,Bin CARD1 as well as p62 are able to reach a state of dynamic equilibrium,whereby Bin CARD1 is required to assist v RNPs nucleation upon infection.TRAF3,on the other hand,activates the type I interferon pathway,preventing viral replication,when it displaces Bin CARD1.Upon viral infection,TBK1 is activated,which in turn degrades Bin CARD1 by mediating phosphorylation of the autophagy receptor p62.