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.
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.
Brasier
Allan R
AR
Division of Endocrinology, MRB 8.138, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1060, USA. arbrasie@utmb.edu.
Spratt
Heidi
H
Wu
Zheng
Z
Boldogh
Istvan
I
Zhang
Yuhong
Y
Garofalo
Roberto P
RP
Casola
Antonella
A
Pashmi
Jawad
J
Haag
Anthony
A
Luxon
Bruce
B
Kurosky
Alexander
A
D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis; D018869:Heat-Shock Response; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D020543:Proteome; D012136:Respiratory Syncytial Viruses / Q000502:physiology*; D014779:Virus Replication
D018840:HSP70 Heat-Shock Proteins; D009687:Nuclear Proteins; D020543:Proteome