Nuclear Heat Shock Response and Novel Nuclear Domain 10 Reorganization in Respiratory Syncytial Virus-Infected A549 Cells Identified by High-Resolution Two-Dimensional Gel Electrophoresis

• DOI: 10.1128/jvi.78.21.11461-11476.2004
• Publication Date: 2004-10-12T17:40:21Z
• Authors: Allan R. Brasier; Heidi Spratt; Zheng Wu; Istvan Boldogh; Yuhong Zhang; Roberto P. Garofalo; Antonella Casola; Jawad Pashmi; Anthony Haag; Bruce Luxon; Alexander Kurosky
• Keywords: Nuclear matrix; HSP60; Proteome

The pneumovirus respiratory syncytial virus (RSV) is a leading cause of epidemic tract infection. Upon entry, RSV replicates in the epithelial cytoplasm, initiating compensatory changes cellular gene expression. In this study, we have investigated RSV-induced nuclear proteome A549 alveolar type II-like cells by high-resolution two-dimensional gel electrophoresis (2DE). Replicate 2D gels from uninfected and RSV-infected nuclei were compared for protein We identified 24 different proteins peptide mass fingerprinting after matrix-assisted laser desorption ionization-time flight spectrometry (MS), whose average normalized spot intensity was statistically significant differed +/-2-fold. Notable among cytoskeletal cytokeratins, RNA helicases, oxidant-antioxidant enzymes, TAR DNA binding (a that associates with domain 10 [ND10] structures), heat shock 70- 60-kDa isoforms (Hsp70 Hsp60, respectively). identification Hsp70 also validated liquid chromatography quadropole-TOF tandem MS (LC-MS/MS). Separate experiments using immunofluorescence microscopy revealed induced cytoplasmic aggregation accumulation. Data mining genomic database showed replication coordinate Hsp family proteins, including 70, 70-2, 90, 40, 40-3 isoforms. Because ND10s, examined effect infection on ND10 organization. striking dissolution structures redistribution component promyelocytic leukemia (PML) speckled 100-kDa (Sp100) into as well inducing their synthesis. Our findings suggest induces response, causes disruption, redistributes PML Sp100 to cytoplasm. Thus, proteomics approach, combined localization coupled response data, yielded unexpected novel insights responses