Viruses induce dramatic changes in target tissue during pathogenesis, including host cellular responses that either limit or support the pathogen. The infectious bursal disease virus (IBDV) targets primarily the bursa of Fabricius (BF) of chickens, causing severe immunodeficiency. Here, we characterized the cellular proteome changes of the BF caused by IBDV replication in vivo using 2DE followed MALDI-TOF MS identification. Comparative analysis of multiple 2DE gels revealed that the majority of protein expression changes appeared between 24 and 96 h after IBDV infection. MS identified 54 altered cell proteins, 12 of which were notably upregulated by IBDV infection. Meanwhile, the other 42 cellular proteins were considerably suppressed by IBDV infection and are involved in protein degradation, energy metabolism, stress response, host macromolecular biosynthesis, and transport process. The upregulation of β-actin and downregulation of dynamin during IBDV infection were also confirmed by Western blot and immunofluorescence analysis. These altered protein expressions provide a response profile of chicken BF to virulent IBDV infection. Further functional study on these altered proteins may lead to better understanding of pathogenic mechanisms of virulent IBDV infection and to new potential therapeutic targets.
"Although the functional significance of some individual ARV proteins has been examined, global changes in cell function during different stages of the ARV life cycle have not been explored. Recently, proteomic approaches incorporating two-dimensional differential-image gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been extensively utilized to monitor protein expression in response to viral infection –. These proteomic methods provide a comprehensive view of protein profiles of virus-infected host cells as well as insight into the molecular pathogenesis of viral infection. "
[Show abstract][Hide abstract] ABSTRACT: Avian reovirus (ARV) is a member of the Orthoreovirus genus in the Reoviridae family. It is the etiological agent of several diseases, among which viral arthritis and malabsorption syndrome are the most commercially important, causing considerable economic losses in the poultry industry. Although a small but increasing number of reports have characterized some aspects of ARV infection, global changes in protein expression in ARV-infected host cells have not been examined. The current study used a proteomics approach to obtain a comprehensive view of changes in protein levels in host cells upon infection by ARV.
The proteomics profiles of DF-1 chicken fibroblast cells infected with ARV strain S1133 were analyzed by two-dimensional differential-image gel electrophoresis. The majority of protein expression changes (≥1.5 fold, p<0.05) occurred at 72 h post-infection. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified 51 proteins with differential expression levels, including 25 that were upregulated during ARV infection and 26 that were downregulated. These proteins were divided into eight groups according to biological function: signal transduction, stress response, RNA processing, the ubiquitin-proteasome pathway, lipid metabolism, carbohydrate metabolism, energy metabolism, and cytoskeleton organization. They were further examined by immunoblotting to validate the observed alterations in protein expression.
This is the first report of a time-course proteomic analysis of ARV-infected host cells. Notably, all identified proteins involved in signal transduction, RNA processing, and the ubiquitin-proteasome pathway were downregulated in infected cells, whereas proteins involved in DNA synthesis, apoptosis, and energy production pathways were upregulated. In addition, other differentially expressed proteins were linked with the cytoskeleton, metabolism, redox regulation, and stress response. These proteomics data provide valuable information about host cell responses to ARV infection and will facilitate further studies of the molecular mechanisms underlying ARV pathogenesis.
PLoS ONE 03/2014; 9(3):e92154. DOI:10.1371/journal.pone.0092154 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Infectious bursal disease virus (IBDV) infection causes immunodeficiency in chickens. To understand cell-mediated immunity during IBDV infection, this study perform a detailed analysis of chicken γc chain (chCD132) and γc family cytokines, including interleukins 2, 4, 7, 9, and 15. The mouse anti-chCD132 monoclonal antibody (mAb) was first generated by the E.coli-expressed γc protein. Immunofluorescence assay further showed that γc was a protein located with the anti-chCD132 mAb on the surface of chicken's splenic mononuclear cells. Real-time quantitative RT-PCR revealed that the chCD132 mRNA transcript was persistently downregulated in embryo fibroblasts, spleen and thymus of chickens infected with IBDV. Correspondingly during IBDV infection, the transcription of five γc family cytokines was downregulated in the thymus and presented an imbalance in the spleen. Fluorescence-activated cell sorting analyses also indicated that the percentage of CD132(+)CD8(+) T cells linearly decreased in the bursa of IBDV-infected chickens. These results confirmed that IBDV infection disturbed the in vivo balance of CD132 and γc family cytokine expression and that IBDV-induced immunodeficiency involved cellular networks related to the γc family.
PLoS ONE 01/2014; 9(1):e84503. DOI:10.1371/journal.pone.0084503 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rabies, as the oldest known infectious disease, remains a serious threat to public health worldwide. The eukaryotic cytosolic chaperonin TRiC/CCT complex facilitates the folding of proteins through ATP hydrolysis. Here we investigated expression, cellular localization and function of neuronal CCTγ during neurotropic rabies virus (RABV) infection using mouse N2a cells as a model. Following RABV infection, 24 altered proteins were identified using two-dimensional electrophoresis and mass spectrometry, including 20 up-regulated proteins and 4 down-regulated proteins. In mouse N2a cells infected with RABV or co-transfected with RABV genes encoding nucleoprotein (N) and phosphoprotein (P), confocal microscopy demonstrated that up-regulated cellular CCTγ was co-localized with viral proteins N and P, which formed a hollow cricoid inclusion within the region around the nucleus. These inclusions, which correspond to Negri Bodies (NBs), did not form in mouse N2a cells only expressing viral proteins N or P. Knockdown of CCTγ by lentivirus-mediated RNA interference led to a significant inhibition of RABV replication. These results demonstrate that the complex consisting of viral proteins N and P recruits CCTγ to NBs and identify the chaperonin CCTγ as a host factor facilitates intracellular RABV replication. This work illustrates how viruses can utilize cellular chaperonins and compartmentalization for their own benefit.
Journal of Virology 05/2013; 87(13). DOI:10.1128/JVI.03186-12 · 4.44 Impact Factor
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