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Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells
Published on: June 3, 2012
Subcellular proteomic analysis of human host cells infected with H3N2 swine influenza virus
Xiaopeng Wu1, Sanying Wang, Yang Yu
1Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou, P. R. China; State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou, P. R. China.
Abstract:
Cross-species transmissions of swine influenza viruses (SIVs) raise great public health concerns. In this study, subcellular proteomic profiles of human A549 cells inoculated with H3N2 subtype SIV were used to characterize dynamic cellular responses to infection. By 2DE and MS, 27 differentially expressed (13 upregulated, 14 downregulated) cytoplasmic proteins and 20 differentially expressed (13 upregulated, 7 downregulated) nuclear proteins were identified. Gene ontology analysis suggested that these differentially expressed proteins were mainly involved in cell death, stress response, lipid metabolism, cell signaling, and RNA PTMs. Moreover, 25 corresponding genes of the differentially expressed proteins were quantitated by real time RT-PCR to examine the transcriptional profiles between mock- and virus-infected A549 cells. Western blot analysis confirmed that changes in abundance of identified cellular proteins heterogeneous nuclear ribonucleoprotein (hnRNP) U, hnRNP C, ALDH1A1, tryptophanyl-tRNA synthetase, IFI35, and HSPB1 in H3N2 SIV-infected cells were consistent with results of 2DE analysis. By confocal microscopy, nucleus-to-cytoplasm translocation of hnRNP C and colocalization between the viral nonstructural protein 1 and hnRNP C as well as N-myc (and STAT) interactor were observed upon infection. Ingenuity Pathway Analysis revealed that cellular proteins altered during infection were grouped mainly into NFκB and interferon signaling networks. Collectively, these identified subcellular constituents provide an important framework for understanding host/SIV interactions and underlying mechanisms of SIV cross-species infection and pathogenesis.
Insights
Swine influenza virus (SIV) infection alters cellular protein expression in human cells, impacting pathways like NFκB signaling. Understanding these host-pathogen interactions is crucial for addressing cross-species transmission risks.
Area of Science:
- Virology
- Cellular Proteomics
- Molecular Biology
Background:
- Cross-species transmission of swine influenza viruses (SIVs) poses significant public health risks.
- Understanding cellular responses to SIV infection is vital for predicting and mitigating disease spread.
Purpose of the Study:
- To characterize dynamic cellular responses in human A549 cells infected with H3N2 subtype SIV using subcellular proteomic profiling.
- To identify differentially expressed proteins and genes involved in host-pathogen interactions.
Main Methods:
- Two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) for protein identification.
- Real-time RT-PCR for gene expression analysis.
- Western blot and confocal microscopy for protein validation and localization.
Main Results:
- Identified 27 cytoplasmic and 20 nuclear differentially expressed proteins in SIV-infected cells.
- Proteins involved in cell death, stress response, lipid metabolism, and signaling pathways were altered.
- Observed nucleus-to-cytoplasm translocation of hnRNP C and its colocalization with viral protein NS1.
Conclusions:
- SIV infection induces significant changes in host cell proteomes and transcriptomes.
- Altered proteins are primarily involved in NFκB and interferon signaling pathways.
- Findings provide a framework for understanding SIV cross-species infection mechanisms and pathogenesis.
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