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Gene expression profiling of Pseudorabies virus (PrV) infected bovine cells by combination of transcript analysis and quantitative proteomic techniques

Skiba, Martin, Glowinski, Frithjof, Koczan, Dirk, Mettenleiter, Thomas C., Karger, Axel
Veterinary microbiology 2010 v.143 no.1 pp. 14-20
cell fractionation, Suid alphaherpesvirus 1, transcriptional activation, proteome, isotope labeling, kidney cells, Aujeszky disease, solid phase extraction, cell physiology, proteomics, gene expression, mass spectrometry, viral proteins, in vitro studies, phosphotransferases (kinases), biochemical mechanisms, cell lines, gel electrophoresis, microbial genetics
Infection of target cells by alphaherpesviruses leads to extensive modulation of host cell gene expression. To gain detailed information on the molecular pathways affected by infection of Madin-Darby bovine kidney (MDBK) cells with PrV, transcript analysis was combined with a stable isotope-based quantitative proteomic approach (SILAC). Four hours after infection cells were harvested and processed in parallel either for transcript analysis, for subcellular fractionation into nuclei and cytosol, for extraction of phosphoproteins, or for affinity extraction with Heparin Sepharose and Cibacron Blue F3G-A-Sepharose. All fractions were further analysed by large format two-dimensional gel electrophoresis in different pH-ranges to maximize the number of proteins to be identified and quantified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Cell fractionation was quick and easy to perform but in comparison to affinity fractionation yielded lower numbers of identified and quantified proteins. After infection with PrV, only two of the 55 proteins with significantly modulated protein levels showed significant changes in transcript levels, indicating that posttranslational modifications may play a major role in the cellular response to PrV infection. Application of isotope labelling to cell cultures infected with wild-type PrV-Ka and a US3 protein kinase negative mutant allowed to monitor pUS3-dependent changes in the expression levels of viral proteins pUL29, pUL39 and pUL42.