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Zusammenfassung:
Production of human influenza virus vaccines in mammalian cell lines, like MDCK cells, is gaining increasing significance within the vaccine industry. For the optimization of influenza virus pro-duction in MDCK cells it is of utmost importance to understand cellular mechanisms induced upon viral infections impairing influenza virus production. Detailed knowledge of virus-host cell interactions, such as viral induced apoptosis, anti-viral cellular immune response, or cellular requirement for viral replication could give valuable insights, helping to prolong and to increase influenza virus production and vaccine yields in MDCK cells, respectively.
Employing two-dimensional differential gel electrophoresis (2-D DIGE) Vester et al. provided first insights into virus-host cell interactions of influenza A infected adherent MDCK cells on pro-tein level (Vester et al., 2009; Vester et al., 2010). But due to known limitations of 2-D PAGE, low-abundant proteins are mostly absent in such studies. Over the year several methods, like subcel-lular prefractionation were developed, enabling enrichment of low-abundant proteins. Within this present work, three different subcellular prefractionation methods (sequential centrifuga-tion, SQC; differential detergent fractionation, DDF; sequential protein extraction, SQX) were evaluated referring their suitability for the enrichment of low abundant proteins of influenza infected adherent MDCK cells for subsequent 2-D DIGE analysis. After comparing characteristics like yield, or degree of enrichment, DDF turned out to be the most suitable of the three tested prefractionation methods.
Proteome analysis of DDF prefractionated MDCK cells (cytosolic, membrane, and nuclear fraction) revealed several altered proteins in influenza virus infected MDCK cells compared to mock-infected cells (p=0.05, 1.3-fold-change ratio). Altered proteins were mostly upregulated and most frequent alterations were observed within the cytosolic fraction. Protein identification by mass spectrometry (nanoHPLC-nanoESI-MS/MS) revealed differentially expressed proteins of viral origin, like the NS1 protein, or NP protein, and proteins of host cell origin, like stress response proteins (e.g., HSP70), or cytoskeleton proteins (e.g., actin-depolymerizing factor). Altogether, the present work identified more differentially expressed proteins compared to the study of Vester et al. (Vester et al., 2009).
