Study On The Interaction Between IBV S1 And Host Membrane Proteins And Primary Analysis Of The Function Of HSPA8 On Virus Infection

Infectious bronchitis(IB)is an acute infectious disease in chickens caused by Infectious bronchitis virus(IBV).In this study,we successfully identified the host proteins HSPA8,Fibronectin,HSP90AB1,HSPD1,and VPS 18 that can interact with the IBV-S protein using IP experiments combined with mass spectrometry.The results of Co-IP assay showed that HSPA8,HSP90AB1 and HSPD1 can interact with M41-RBD(Receptor binding domain).In view of the results of silver stain,we selected HSPA8 protein for further identification.The results of detection of HSPA8 expression in chicken tissues showed kidney with highest expression level.The results of flow cytometry showed that HSPA8 protein could be expressed on the surface of CEK,Vero and 293T cells.The results of confocal assay showed that HSPA8 and M41-RBD were co-localized.The Gst-pull-down assay further proved that the interaction between HSPA8 protein and M41-RBD is directly.Further research found that HSPA8 protein can interact with S1 protein of different IBV strains(QX,H120,Beaudette strains),HSPA8 originate from pig and human could interact with M41-RBD.These results suggest that the host HSPA8 protein may plays an important role during the infection of different IBV strains to host cells.The results of infection inhibition assay showed that the HSPA8 recombinant protein and HSPA8 antibody could inhibit IBV infection.The results of continuously virus passage experiment indicated that HSPA8 is a binding molecule of IBV rather than a functional receptor for IBV infection.HSPA8 overexpression and RNA interference were used to verify the effect of HSPA8 on IBV replication.The results showed that overexpression of HSPA8 protein can inhibit the replication of IBV-Beaudette strain in Vero cells.Whereas HSPA8 knock down by using a specific small interfering RNA increased IBV infection in Vero cells.The above results also indicate HSPA8 is not a functional receptor for IBV infection.And HSPA8 may perform additional functions after IBV entry into cells.Then we explored the effect of HSP90AB1 on the replication of IBV-Beaudette.The results showed that over expression of HSP90AB1 inhibited IBV replication,whereas knockdown of HSP90AB1 increased IBV replication in host cells.These results indicated that HSP90AB1 is a potential anti-viral host factor.Taken together,our study successfully identified HSPA8,HSP90AB1 and HSPD1 are interacted protein of IBV S1 protein.Further research found that HSPA8 protein is an attachment factor for IBV and HSP90AB1 is a potential anti-viral host factor.This study laid the foundation for further identification of IBV receptor and finding suitable cell culture system for IBV.Moreover,identification of the founction of HSPA8 during infection of IBV may increase our knowledge to the pathogenic mechanism of IBV.