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

Allan R. Brasier, Heidi Spratt, Zheng Wu, Istvan Boldogh, Yuhong Zhang, Roberto Garofalo, Antonella Casola, Jawad Pashmi, Anthony Haag, Bruce Luxon, Alexander Kurosky

Research output: Contribution to journalArticle

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Abstract

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

Original languageEnglish (US)
Pages (from-to)11461-11476
Number of pages16
JournalJournal of Virology
Volume78
Issue number21
DOIs
StatePublished - Nov 2004

Fingerprint

Heat-Shock Response
Respiratory Syncytial Viruses
two-dimensional gel electrophoresis
Electrophoresis, Gel, Two-Dimensional
viruses
cells
Respiratory Syncytial Virus Infections
Cytoplasm
Proteins
DNA-binding proteins
cytoplasm
DNA-Binding Proteins
Virus Replication
virus replication
Tandem Mass Spectrometry
leukemia
proteins
Pneumovirus
Protein Isoforms
Leukemia

ASJC Scopus subject areas

  • Immunology

Cite this

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. / Brasier, Allan R.; Spratt, Heidi; Wu, Zheng; Boldogh, Istvan; Zhang, Yuhong; Garofalo, Roberto; Casola, Antonella; Pashmi, Jawad; Haag, Anthony; Luxon, Bruce; Kurosky, Alexander.

In: Journal of Virology, Vol. 78, No. 21, 11.2004, p. 11461-11476.

Research output: Contribution to journalArticle

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AU - Brasier, Allan R.

AU - Spratt, Heidi

AU - Wu, Zheng

AU - Boldogh, Istvan

AU - Zhang, Yuhong

AU - Garofalo, Roberto

AU - Casola, Antonella

AU - Pashmi, Jawad

AU - Haag, Anthony

AU - Luxon, Bruce

AU - Kurosky, Alexander

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