Screening And Identification Of Host Cellular Proteins Interacting With Autographa Californica Multiple Nucleopolyhedrovirus39K

Baculoviridae is a family of insect DNA viruses. Protein-protein interactions are common in process of DNA replication, transcription, translation as well as signal transduction of baculoviruses. Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the representative species of the Baculoviridae, is the first baculovirus whose genome is completely sequenced and is studied most extensively and deeply. AcMNPV39k was initially studied as a prototype "delayed early gene" which was expressed both at the early stage and late stage of infection.39K is distributed in the nucleus of AcMNPV-infected cells, being associated with the virogenic stroma. It was shown that AcMNPV39K was related with the regulation of late genes expression. In this study, the AcMNPV39k was used as a bait in yeast two-hybrid GAL4sysyem screening the Sf9cells, to identify host proteins that interacted with the39K. The resultes produced include:(1). Screening the Sf9host proteins interacted with AcMNPV39K.We constructed the bait vector pGBKT7-39d and used the two-hybrid GAL4system to have screened17positive clones interacting with39K. After the genes were classified and sequenced, they could be confirmed as5kinds of gene, respectively encoding X、 Muscle-specific protein300, GPI-anchor transamidase%Exocyst complex84-kDa subunit、translation initiation factor2. Among all of them, X, localized in the nuclear and related with endoplasmic reticulum stress, drew our great attention.(2). Subcellular colocalization analysis of AcMNPV39K and X.We generated the recombinant bacmid, expressing the39K-EGFP fusion protein and X-RFP fusion protein by use of the Bac-to-Bac system. Then, Sf9cells were transfected and infected with the recombinant bacmid. Confocal microscope analysis indicated that both of them are localized in the nuclear of host cell. That is,39K and X are shown to have colocalization phenomenon.(3). Bimolecular fluorescence complementation analysis of interaction between39K and X.We first made39k (used to the N-coding sequence of YFP, meanwhile, making the x fused to the C-coding sequence of YFP. Then, the fusion sequences were transposed to the AcMNPV to generate the recombinant bacmid. After the Sf9were infected by the above recombinant bacmid, yellow fluorescence appeared in Sf9cells, which was consistented with the result of subcellular colocalization analysis.(4). Co-Immunoprecipitation analysis of interaction between39K and X. AcMNPV39K, which was expressed and purified in E.coli, was uesd as antigen to immunize mice in order to get the polyclonal antibody. Recombinant bacmid, expressing the X-His fusion protein, was constructed through Bac-to-Bac system. After the Sf9cells were infected by the above recombinant bacmid, we used anti-39K to deal with infected cells. Co-Immunoprecipitation analysis indicated that there is real interaction between39K and X.