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.
Division of Endocrinology, MRB 8.138, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1060, USA. arbrasie@utmb.edu.
Allan R
AR
Brasier
Heidi
H
Spratt
Zheng
Z
Wu
Istvan
I
Boldogh
Yuhong
Y
Zhang
Roberto P
RP
Garofalo
Antonella
A
Casola
Jawad
J
Pashmi
Anthony
A
Haag
Bruce
B
Luxon
Alexander
A
Kurosky
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