Quantitative Proteomics Using SILAC Coupled to LC−MS/MS Reveals Changes in the Nucleolar Proteome in Influenza A Virus-Infected Cells

• Published in: Journal of proteome research 2010-10, Vol.9 (10), p.5335-5345
• Main Authors: Emmott, Edward, Wise, Helen, Loucaides, Eva M, Matthews, David A, Digard, Paul, Hiscox, Julian A
• Format: Article
• Language: English
• Subjects: Amino Acids - metabolism; Animals; Blotting, Western; Cell Line; Cell Nucleolus - metabolism; Cell Nucleolus - virology; Chromatography, Liquid - methods; Host-Pathogen Interactions; Humans; Influenza A virus - physiology; Influenza A Virus, H1N1 Subtype - physiology; Influenza A Virus, H3N2 Subtype - physiology; Isotope Labeling; Mass Spectrometry - methods; Microscopy, Confocal; Nuclear Proteins - analysis; Proteomics - methods
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/pr100593g

Influenza A virus (IAV) is a major human pathogen whose genotypic diversity results in unpredictable pandemics and epidemics. Interaction with the cell nucleus is essential to IAV infection, allowing recruitment of cellular components to facilitate virus replication. Viral proteins are also targeted to the nucleolus, a subnuclear structure involved in ribosomal biogenesis, RNA maturation, stress response, and control of cell growth, but the functional consequences of this are unclear. We took an unbiased approach to studying IAV−nucleolar interactions by using stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with LC−MS/MS to quantify changes in the nucleolar proteome following infection with A/PR/8/34 (H1N1) and A/Udorn/72 (H3N2) strains of the virus. Only a minority of nucleolar proteins showed significant changes in abundance after infection; these alterations were mostly different between the two strains but could be validated by confocal microscopy of infected cells. Many of the affected proteins comprised functional groupings, including components of ribonuclease P, RNA polymerase I, the MLL1 histone methyltransferase complex, as well as nuclear paraspeckles and the RNA editing apparatus. This, as well as comparison with other viruses that cause changes in the nucleolar proteome, suggests that IAV targets specific nucleolar pathways.