Molercular Mechanisms Of Autophagy Regulation By Infectious Bursal Disease Virus Structural Proteins VP2and VP3

Autophagy is a conserved cellular protein and organelles degredation process, whichalso plays important role in clearing intracellular pathogen during infection.However,the relationship between IBDV and autophagy is still unveil. Here, we report thatautophagy was induced at early stage of IBDV infection. Autophagy detection showedthat early infection not only increased the conversion of light chain3(LC3)-I to LC3-IIusing western blot analysis, but also results in increasing formation of GFP-LC3punctas in cytoplasm using corfocal microscopy analysis. Induction of autophagycould be confirmed that level of P62, a substrate of autophagy, decreased greatly inresponse to IBDV infection. We also found that induction of autophagy usingrapamycin, but not blockade of autophagy using wortmanin, inhibited replication ofIBDV. Thus, our data firstly demonstrated that early IBDV infection results in stronglyinduction of autophagy, which plays critical role in controlling pathogen infection.Further study showed us that autophagy was induced through AKT/mTORpathway in response to IBDV infection. We found that heat inactivate virus or IBDVVP2protein expressed in E.Coli could induce autophagy constantly. Moreover,inactivated virus or IBDV VP2protein treatment resulted in inactivation of AKT,followed by dephosphorylation of mTOR, which is a key regulator responsible forinduction of autophagy. We therefore infer that recognition of IBDV by certainmembrane receptor is a key step for pathogen dependent induction of autophagy. Thestudy showed that Hsp90could recognize IBDV or IBDV VP2. What’s more,knockdown of Hsp90, Akt and mTOR led to failure of infection dependent autophagy.We also found that incubation of Hsp90antibody in DF-1cells resulted in stronginduction of autophagy and impairment of AKT/mTOR activation.Therefore, wecoucluded here that pathogen receptor Hsp90, linking the pathogen recognition tomTOR dependent autophagy, a critical step for control autophagy.However, IBDV could inhibit autophagy during robust replication process. IBDV VP3could interact with chicken or human Beclin-1. This interaction directly interferethe binding of VPS34to Beclin-1. Disruption of interaction between VPS34andBeclin-1led to reduction of PI3P, which is required for autophagy. What’s more, wealso found that VP3transfection resulted in phosphorylation of AKT, which led toactivation of mTOR. The results showed that VP3transfection contributed to therecruitment of AKT or PDK1to ER by VPS34. PDK1inhibitor conteract theVP3-inducing phosphorylation of AKT, suggesting PDK1kinase is required for VP3dependent activation of AKT. By contrast, PDK1inhibitor had no effect on VP3dependent disruption of VPS34-Beclin-1complex.However, PDK1kinase inhibitorrestore the autophagy in presence of VP3, suggesting that VP3inhibit autophagy majorthrough VPS34dependent phosphorylation of AKT, but not from disruption ofVPS34-Beclin-1. We concluded here that IBDV VP3contribute to formation of novelcomplex, VPS34-PDK1-AKT complex, which led to phosphorylation of AKT andconsequently inhibit autophagy, which plays an antivirus role during IBDV infection.Thus, autophagy, facilitating host cells to clear virus, was induced at earlyinfection process through inactivation of AKT/mTOR pathway via recognition ofIBDV by Hsp90receptors. However, this defense system was finally inhibited throughactivation of AKT/mTOR by IBDV VP3during replication process. Collectively, AKT/mTOR pathway control the defense program during IBDV infection.