Protein-protein Interactions Of Per Os Infectivity Factors Of Insect Baculovirus And Identification Of P74 Interacting Proteins In The Host Midgut

In the baculovirus infection cycle,they produce two different virus phenotypes,called budded virus(Budded virus,BV)and occlusion-derived virus(Occlusion-derived virus,ODV).Though BV and ODV have the same genome,they differ in phenotypes.When the occlusion bodies containing ODVs are ingested by insect larvae,the primary infection occurs and it is also called per os infection.Several ODV envelope proteins,called per os infectivity factors(PIFs),have been shown to be essential for oral infection.At least seven PIFs,P74,PIFs 1-4,PIF6 and P95(Ac83),together with other two ODV-specific proteins,Ac5 and Ac 108,have been reported to associate with a PIF complex on the ODV surface.In this study,in order to understand the interactions of these PIFs,the direct protein-protein interactions of the nine components of the Autographa californica multiple nucleopolyhedrovirus(AcMNPV)PIF complex were investigated by yeast two-hybrid(Y2H)combined with bimolecular fluorescence complementation(BiFC).Furthermore,Y2H analysis of the truncations and deletion mutants of P95 and PIF1 were conducted to determine which domains are essential for interaction.P74 is the first identified PIF and previous studies has shown that it plays definite role in ODV binding to midgut cells.In order to identify the host factors essential for ODV attachment,the cDNA library of the midgut of Bombyx mori was constructed and Y2H screen using P74 as bait was done.The results are summarized as followings:1.The protein-protein interactions of the PIFs in the PIF complexSix interactions were identified in Y2H analysis,comprising of PIF1-PIF2,PIF1-PIF3,PIF1-PIF4,PIF1-P95,PIF2-PIF3 and PIF3-PIF4,among which,PIF1-PIF3,PIF2-PIF3 and PIF1-P95 were strong interactions,while others appeared to be less robust.For P74,PIF6,Ac5 and Ac108,no direct interaction was identified.Consistent with the results of Y2H,interactions were present in combinations of PIF 1 and PIF2,PIF1 and PIF3,PIF1 and PIF4,PIF2 and PIF4,PIF3 and PIF4,as well as PIF1 and P95 in BiFC studies,but not other pairs.2.The characterization of interaction of PIF 1 and P95Y2H assays for C-and N-terminal truncations of P95 and PIF1 indicated that truncation of 200-300aa of P95,where the predicted CBD(224-282aa)was located,abrogating its ability to interact with PIF1.Consistent with the truncation assay,deletion of CBD could efficiently block the interaction between P95 and PIF1,highlighting the importance of this region for their interaction.For PIF1,the similar truncations and deletion were conducted to show that the 100-200aa of PIF1 is responsible for the interaction between PIF1 and P95.3.Identification of the P74-interacted proteins in the midgut of Bombyx moriThe fifth-instar larvae of Bombyx mori were dissected and the midguts were isolated for extraction of RNA.Then,the Y2H library of the midgut of Bombyx mori was constructed and screened by using P74 as bait.U3 small nucleolar ribonucleoprotein protein IMP3,60S ribosomal protein L5,cysteine and histidine-rich protein 1-B,JAB-MPN domain protein,eukaryotic translation initiation factor 1A,Actin,cytoplasmic A3 and 35kDa protease were identified in Y2H analysis.Three proteins of them were further identified to interact with P74,including JAB-MPN domain protein,Actin,cytoplasmic A3 and 35kDa protease.Furthermore,co-immunoprecipitation was used to confirm the interactions between P74 and JAB-MPN domain protein.Our results indicated that JAB-MPN domain protein may be the host factor essential for ODV attachment.In conclusion,our study elucidated the character of the protein-protein interaction in baculovirus PIF complex,especially the essential domains for the interaction of P95 and PIF1,and identified the host factor interacted with P74.This studies will help us for well understanding the molecular mechanisms of the per os infection of baculoviruses.