Abstract
BackgroundCells are exposed to multiple stressors that induce significant alterations in signaling pathways and in the cellular state. As obligate parasites, all viruses require host cell material and machinery for replication. Virus infection is a major stressor leading to numerous induced modifications. Previous gene array studies have measured infected cellular transcriptomes. More recently, mass spectrometry-based quantitative and comparative assays have been used to complement such studies by examining virus-induced alterations in the cellular proteome.MethodsWe used SILAC (stable isotope labeling with amino acids in cell culture), a non-biased quantitative proteomic labeling technique, combined with 2-D HPLC/mass spectrometry and reciprocal labeling to identify and measure relative quantitative differences in HeLa cell proteins in purified cytosolic and nuclear fractions after reovirus serotype 3 Dearing infection. Protein regulation was determined by z-score analysis of each protein’s label distribution.ResultsA total of 2856 cellular proteins were identified in cytosolic fractions by 2 or more peptides at >99% confidence and 884 proteins were identified in nuclear fractions. Gene ontology analyses indicated up-regulated host proteins were associated with defense responses, immune responses, macromolecular binding, regulation of immune effector processes, and responses to virus, whereas down-regulated proteins were involved in cell death, macromolecular catabolic processes, and tissue development.ConclusionsThese analyses identified numerous host proteins significantly affected by reovirus T3D infection. These proteins map to numerous inflammatory and innate immune pathways, and provide the starting point for more detailed kinetic studies and delineation of virus-modulated host signaling pathways.
Highlights
Cells are exposed to multiple stressors that induce significant alterations in signaling pathways and in the cellular state
We have shown, using SILAC and 2D-HPLC/MS, that proteins involved in cell death, cell growth and proliferation, molecular transport, gene expression, and inflammatory response pathways are affected in mammalian reoviruses (MRV) T1L-infected HEK-293 cells [21]
We evaluated the time course and confirmed the capacity of Type 3 Dearing (T3D) to enter and replicate in HeLa cells, and to cause cytopathic effect (CPE), under various infection conditions (Figure 1)
Summary
We used SILAC (stable isotope labeling with amino acids in cell culture), a non-biased quantitative proteomic labeling technique, combined with 2-D HPLC/mass spectrometry and reciprocal labeling to identify and measure relative quantitative differences in HeLa cell proteins in purified cytosolic and nuclear fractions after reovirus serotype 3 Dearing infection. Protein regulation was determined by z-score analysis of each protein’s label distribution
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