RESEARCH ARTICLE Quantitative analysis of cellular proteome alterations in human influenza A virus-infected mammalian cell lines Diana Vester 1 , Erdmann Rapp 2 , Do ¨rte Gade 2 , Yvonne Genzel 2 and Udo Reichl 1,2 1 Bioprocess Engineering, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany 2 Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany Received: November 20, 2008 Revised: February 9, 2009 Accepted: March 11, 2009 Over the last years virus–host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2-D DIGE and nanoHPLC-nanoESI-MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2-D gels of the proteomes of uninfected and influenza-infected host cells, 16 quantitatively altered protein spots (at least 71.7-fold change in relative abundance, p o 0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoske- leton system, and for Mx proteins, interferon-induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome-wide profiling of virus infection can provide insights into complexity and dynamics of virus–host cell inter- actions and may accelerate antiviral research and support optimization of vaccine manu- facturing processes. Keywords: 2-D DIGE / Cell culture engineering / Influenza A virus / Mammalian cells / Virus–host cell interaction 1 Introduction Influenza viruses are enveloped viruses that contain segmented, single-stranded RNA of negative polarity. They are known to efficiently use host cell resources for their replication, inducing significant cellular changes. Studies have shown that influenza viruses have a comprehensive effect on the synthesis of cellular mRNAs [1] and proteins [2]. In addition, infections induce complex signal transduc- tion pathways related to antiviral response of the host cells [3]. Both events have an impact on host cell gene expression patterns [4] and therefore cause alterations in morphology and metabolic state of the cells and induce apoptosis. Whether apoptosis results from the inhibition of host cell gene expression, or is related to the antiviral response of the host cell, or by the virus itself is not completely understood. In the last years great efforts have been undertaken to reveal the mechanisms of influenza–host cell interactions. Studies focused either on pathogenesis in humans [4], Abbreviations: HA, hemagglutinin; hpi, hours post infection; MDCK, Madin–Darby canine kidney cell; MOI, multiplicity of infection; RNP, ribonucleoprotein Correspondence: Diana Vester, Bioprocess Engineering, Otto-von- Guericke-University Magdeburg, Universit . atsplatz 2, D-39106 Magdeburg, Germany E-mail: vester@mpi-magdeburg.mpg.de Fax: 149-931-6110-203 & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com 3316 Proteomics 2009, 9, 3316–3327 DOI 10.1002/pmic.200800893