Comparative Proteomic Analysis Of Infectious Bursal Disease Virus-infected Cells

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) in young chickens, and causes severe immunosuppression. IBDV has been intensively studied as the model for dsRNA virus, since its small genomes only encode several viral proteins. To date, the biological function of viral proteins had been well characterized, but the interactions between IBDV-encoded viral proteins, and the interactions between virus and host cells are still unclear, and all these questions are essential for the potential molecular mechanism of IBDV replication and pathogenesis. In this study, we analyzed the subcellular location relationship between viral proteins, and explored the protein expression profiles of IBDV-infected cells using comparative proteomic analysis, and then analyzed the function of the differentially expressed proteins during IBDV infection.VP1, the putative viral RNA-dependent RNA polymerase encoded by segment B of IBDV, has been suggested to play essential roles in the replication of viral genomic RNAs and the assembly of viral particles. In this study, the full-length cDNA of segment B of cell-adapted IBDV-NB isolate was amplified by long RT-PCR, and then VP1 gene was subcloned into the prokaryotic expression vector pET-28a(+). After induced with IPTG, the His-tagged recombinant VP1 protein (rVP1), with an approximately molecular weight of 97 kDa, was expressed mainly as inclusion body, and the protein tends to degrade in vitro. Using the purified rVP1 by nickel-column chromatography as immunogen, polyclonal antibodies and four monoclonal antibodies (mAbs) specifically recognizing IBDV-VP1 were successfully produced, these antibodies provide useful tools for further study of the molecular mechanism of IBDV replication. Using the special antibodies against VP1, we studied the subcellular location of VP1 protein in cells infected with IBDV or transfected with eukaryotic expression plamid of VP1 (pEGFP-VP1 or pCI-neo-VPl). The results showed that VP1 diffused within the cytoplasm of transfected cells, but distributed by diffusing and forming irregularly shaped particles within the cytoplasm of IBDV-infected cells. These data implied that the formation of granule-like VP1 in infected cells may be involved in IBDV infection.Dual fluorescent staining were carried out to examine the location relationship between VP1 and capsid proteins VP2/VP3, serine protease VP4 and nonstructural protein VP5 within IBDV-infected chicken embryo fibroblasts (CEFs) and Vero cells. The results showed that the expression patterns of VP1, VP2, VP3 and VP5 in IBDV-infected CEFs and Vero cells were fairly similar, but distinct signals of VP4 were observed in two kinds of cells. VP2 located in the cytoplasm by diffusing pattern and granule-like particles, the size of VP2 particles are greater than VP1, but the numbers of VP2 particels are smaller than VP1, the VP2 particles partically colocalize with VP1, suggesting that the region of partical colocalization may be the place for viral assembly. The expression and distribution of VP3 is very similar to VP1, and VP3 co-localizes precisely with VP1, these results were consistent with the previous reports that VP3 and VP1 form a stable complex to be involved in IBDV assembly. VP5 distributes throughout the plasma membrane of IBDV-infected cells, have no colocalization with cytoplasmic VP1, suggesting that VP5 and VP1 plays distinct roles during IBDV infection. Compared with VP1, VP2, VP3 and VP5, protease VP4 distributes both in the nucleus and cytoplasm of IBDV-infected cells. In IBDV-infected CEFs, VP4 forms needle-like signal both in the nucleus and cytoplasm, while forms needle-like signal in the nucleus and agglomerate-like signal within the cytoplasm of IBDV-infected Vero cells. VP1 locates in the interspace of cytoplasmic VP4.Further, the subcellular location relationships of VP2, VP3 and VP4 in IBDV-infected DF-1 chicken fibroblast cell line were determined by dual fluorescent staining. The results showed that the expression pattern of VP2, VP3 and VP4 had gradually changed. Cytoplasmic VP2 changed from diffuse to granule-like or needle-like, cytoplasmic VP3 changed from diffuse to granule-like or hollow agglomerate. Cytoplasmic and intranuclear VP4 gradually changed from diffuse, to dot-like needle, to short needle, to long needle and granule-like particles, and the granule-like particles were only found within the cytoplasm. Granule-like and needle-like VP4 was co-localized with VP2 in the cytoplasm, indicating that VP4 may interact with VP2 during late phase, and be involved in the cleaving and maturation of precursor VP2. The particles of VP2 and VP4 mainly distribute in the center of VP3, suggesting that IBDV replication occurs in the outer region of "viral factories", and assembly and maturation of IBDV particles occurs in the inter region of "viral factories". The nuclear localization of VP4 indicated that serine protease VP4 may play important roles in IBDV pathogenesis by interacting with host cells, in addition to cleave the polyprotein and VP2 precursor.The comparative proteomes of IBDV-infected and mock-infected CEFs at different time points were analyzed using two-dimensional gel electrophoresis (2-DE) followed by MALDI-TOF/TOF protein identification. The analyses of multiple 2-DE gels revealed that a total of 102 protein spots differentially expressed during IBDV infection and the majority of protein expression changes appeared at 48 h and 96 h post infection. MALDI-TOF/TOF mass spectrometry successfully identified 81 protein spots (corresponding to 51 altered cellular proteins) of the 102 differentially expressed protein spots, including 13 up-regulated proteins and 38 down-regulated proteins. These data revealed that IBDV infection induced the overexpression of polyubiquitin, apolipoprotein A-1, heat shock 27 kDa protein 1, actins, tubulins, eukaryotic translation initiation factor 4A isoform 2, acidic ribosomal phosphoprotein, and p40 ribosomal associated protein, while 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 changes between infected and uninfected CEFs. Western blot further confirmed the inhibition of Rho protein GDP dissociation inhibitor and the induction of polyubiquitin during IBDV infection. These data showed that IBDV infection changed the cellular cytoskeleton, stress response, ubiquitin-proteosome pathway, macromolecular biosynthesis, energy metabolism and signal transduction. This work effectively provides useful dynamic protein-related information to facilitate further investigation of the underlying mechanism of IBDV infection and pathogenesis.Comparative proteomic analysis of IBDV-infected CEFs showed that multiple cytoskeleton-associated proteins were up-or down-regulated during viral infection. To further investigate the effects of IBDV infection on host cytoskeletal network, the microfilament, microtubule and intermediate filament in IBDV-infected cells (which were probed with antibodies against VP1) were detected respectively using FITC-phalloidin, antibodies againstβ-tubulin and vimentin. The results showed that IBDV infection induced the structural and distribution changes of microfilaments, microtubules and imtermediate filaments in IBDV-infected cells. Among then, the radial microtubule array anchored at the centrosome was partially or totally disrupted during late phase of infection, and the vimentin network within the cytoplasm also collapsed during IBDV infection. However, the signals of F-actin were greatly enhanced in IBDV-infected cells, with needle-like F-actin in the nucleus, with needle-like structure in the perinuclear region of cytoplasm of IBDV-infected CEFs, and with agglomerate in the cytoplasm of IBDV-infected Vero cells. The similar signal patterns were observed with anti-VP4 antibody and FITC-phalloidin in IBDV-infected cells, suggesting potential interactions between VP4 and F-actin. To further assess this possibility, their subcellular location relationship was characterized using the dual fluorescent staining. The results showed that F-actin and VP4 colocalize in both the nucleus and cytoplasm of IBDV-infected CEFs, Vero and bursal cells, but VP4 distributes within the cytoplasm of VP4-transfected cells, partically colocalize with higher signal F-actin, indicating that VP4 may interact with F-actin, and that the appearance of needle-like VP4 and F-actin in the nucleus were associated with IBDV infection. To further investigate the interaction between F-actin and VP4, IBDV-infected cells were treated with Triton X-100, and the binding activity of VP4 to monomer actin was detected using antibodies against VP4 or beta-actin by immunoprecipitation assay. The results showed that a small quantity of VP4 exists as soluble forms, and has no binding reactivity to monomer actin, while large quantity of VP4 exists as insoluble forms. These results indicated that VP4 may bind to the insoluble F-actin, and exist as insoluble forms. To assess whether F-actin is involved in IBDV infection, infected cells were treated with F-actin depolymerizing drug cytochalasin D, and the effects of drug on viral production were examined by determining the TCID50 on CEFs, the results showed that the production of infective virions were greatly decreased by treating with cytochalasin D, revealing that depolymerized F-actin potentially affects IBDV replication and release. Since IBDV replication occurs in the cytoplasm, the appearance of VP4 and F-actin in the nucleus suggested that the biological function of VP4 may largely unknown. Further investigation, including the molecular mechanism of VP4 import into the nucleus, the interaction between VP4 and F-actin, and the biological function of F-actin during IBDV infection, will greatly contribute to the elucidation of IBDV replication and pathogenesis.