ITRAQ-based Subcellular Proteomic Analysis Of DF-1 Cells Infected With Infectious Bursal Disease Virus

Infectious bursal disease virus (IBDV) is an Avibirnavirus belonging to the Birnaviridae family. It is a pathogenic agent that damages the precursors of antibody-producing B lymphocytes in the bursa of Fabricius and causes severe immunodeficiency and mortality in young chickens. IBDV has been intensively studied as the model for dsRNA virus, since its small genomes only encode several viral proteins. Genome of IBDV consists of two segments (A and B), and encodes the viral RNA-denpendent RNA polymerase VP1, capsid protein VP2 and VP3, virus peotease VP4 and nonstructural protein VP5. The pathogenic mechanism of IBDV is still unclear, and the research on interaction of the host cell and virus is relatively limited.In this study, infectious bursal disease virus infection of DF-1 cells was used as a infection model, the application of the mothod of subcellular combined with isobaric tags for relative and absolute quantitation (iTRAQ) proteomics protein expression in IBDV-infected cells were explored.In order to explore the interaction of virus-host cell, this study uncover DF-1 cellular dynamics response to IBDV, the subcellular proteome profiles were analyzed utilizing iTRAQ followed by LC-MS/MS identification. Comparative analysis of results by Mass spectrometry revealed that in cytoplasmic proteoms identified alterd cellular proteins, including 31 up-regulated proteins and 8 down-regulated proteins; in nuclear proteomes identified 49 alterd cellular proteins, including 21 up-regulated proteins and 28 down-regulated proteins. Bioinformatics analyses were used to generate IBDV-regulated host response networks consisting of regulated proteins. These differentially expressed proteins was found to be coordinated primarily through the immune response process, signal transduction, RNA processing, macromolecular biosynthesis, energy metabolism, biological processes of virus binding, and cells apoptotic, etc., and their interactions. The mass spectrometry quantitation data for selected proteins were validated by Western blot and immunofluorescence confocal microscopy. By using immunofluorescence assay (IFA) and immunoblotting(WB), we found the hnRNPH1, NF45, API5, RhoGDI, NPL4 and DDX42 can alter and tranlocate into cytoplasm after IBDV infection. In this study, combined with LC-MS/MS iTRAQ technology to quickly and effectively subcellular proteomics information IBDV infection quantitative analysis, the basis for the pathogenesis and identify potential drug targets of IBDV.In order to study the function of Rho protein GDP dissociation inhibitor (RhoGDI) and related regulatory factor signaling pathways of Rho proteins in infectious bursal disease virus infection, elucidate the relationship of them, this study detected RhoGDI and related transcripts and protein expression levels by quantitative PCR and Western bolt after IBDV infection. The levels of transcriptional and expression Rho proteins all down-regulated. Analysis of subcellular localization of RhoA which the effector proteins of RhoGDI in IBDV infected cells, Rho A was found upregulated and co-localizated with viral VP3 protein in IBDV infected cells by immunofluorescence. After overexpression RhoGDI, viral transcription level of the gene encoding decreased, while the expression levels have inhibited. Simultaneously, we found that viral titer declined significantly. We speculated that RhoGDI can affect the replication of IBDV in the host cell.