Proteomics Analysis of Host Cells Infected with Infectious Bursal Disease Virus*□S

The effect of infectious bursal disease virus (IBDV) infection on cellular protein expression is essential for viral pathogenesis. To characterize the cellular response to IBDV infection, the differential proteomes of chicken embryo fibroblasts, with and without IBDV infection, were analyzed at different time points with two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF/TOF identification. Comparative analysis of multiple 2-DE gels revealed that the majority of protein expression changes appeared at 48 and 96 h after IBDV infection. Mass spectrometry identified 51 altered cellular proteins, including 13 up-regulated proteins and 38 down-regulated proteins 12–96 h after infection. Notably 2-DE analysis revealed that IBDV infection induced the increased expression of polyubiquitin, apolipoprotein A-I, heat shock 27-kDa protein 1, actins, tubulins, eukaryotic translation initiation factor 4A isoform 2, acidic ribosomal phosphoprotein, and ribosomal protein SA isoform 2. In addition, IBDV infection considerably suppressed those cellular proteins involved in ubiquitin-mediated protein degradation, energy metabolism, intermediate filaments, host translational apparatus, and signal transduction. Moreover 38 corresponding genes of the differentially expressed proteins were quantitated by real time RT-PCR to examine the transcriptional profiles between infected and uninfected chicken embryo fibroblasts. Western blot further confirmed the inhibition of Rho protein GDP dissociation inhibitor expression and the induction of polyubiquitin during IBDV infection. Subcellular distribution analysis of the cytoskeletal proteins vimentin and -tubulin clearly demonstrated that IBDV infection induced the disruption of the vimentin network and microtubules late in IBDV infection. Thus, this work effectively provides useful dynamic protein-related information to facilitate further investigation of the underlying mechanism of IBDV infection and pathogenesis. Molecular & Cellular Proteomics 7:612–625, 2008. Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a pathogenic agent that damages the precursors of antibody-producing B lymphocytes in the bursa of Fabricius (central humoral immune organ) and causes severe immunodeficiency and mortality in young chickens (1). Its bisegmented double-stranded RNA genome encodes an RNAdependent RNA polymerase, VP1; two major structural proteins, namely VP2 and VP3; a viral protease, VP4; and a nonstructural protein, VP5. IBDV replicates within the cytoplasm of infected cells, leading to cytopathic effect or cytolytic infection. IBDV-induced apoptosis has been found in chicken peripheral blood lymphocytes (2), thymus (3), bursal lymphoid cells of chicken embryos in vivo (4), and cultured CEFs and Vero cells in vitro (5–7). The viral protein VP2 was shown to contribute greatly to the induction of apoptosis (8). The early protein VP5 plays a crucial role in IBDV infection by inhibiting apoptosis in the early stage of viral infection (9) and accumulates within the host plasma membrane, thus contributing to cell lysis in the late stage of viral replication (10). Recently the differentially expressed transcripts of IBDV-infected CEFs were analyzed by cDNA microarrays, which provide an overview of the mRNA expression profiles of infected cells (11). The mRNA abundance is not always consistent with the protein level (12), and viral infection results in post-translational modifications, such as ubiquitination (13), phosphorylation, and glycosylation (14) without affecting their transcription rates. Therefore, the proteomics analysis of host cellular responses to virus infection is more likely to probe potential cellular factors involved directly or indirectly in viral infection and to identify potential drug targets of antiviral treatment. To date, a small but increasing number of studies have