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

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.

FIG. 1.

Characterization of A549 cell nuclear preparations. (A to D) Microscopic analysis of sucrose step gradient-purified nuclei. Purified nuclei were diluted in PBS, plated on a microscope coverslip, and stained with DAPI (see Materials and Methods). Panels A and C, phase-contrast microscopy; panels B and D, DAPI staining. Panels A and B, low-resolution images. Panels C and D, high-resolution images. In panel C, nucleoli are visible (arrows). (E) Western immunoblot for nuclear and cytoplasmic markers. Duplicate 75-μg samples of nuclear and cytoplasmic extracts were fractionated by one-dimensional SDS-PAGE, transferred to polyvinylidene difluoride, and stained with lamin B and β-tubulin antibodies. The locations of molecular weight markers (10⁻³) are indicated on the left. The lamin B stain is localized to the nuclear fraction (Nuc), whereas the β-tubulin is localized to the cytoplasmic lysates (Cyto).

FIG. 1.

Characterization of A549 cell nuclear preparations. (A to D) Microscopic analysis of sucrose step gradient-purified nuclei. Purified nuclei were diluted in PBS, plated on a microscope coverslip, and stained with DAPI (see Materials and Methods). Panels A and C, phase-contrast microscopy; panels B and D, DAPI staining. Panels A and B, low-resolution images. Panels C and D, high-resolution images. In panel C, nucleoli are visible (arrows). (E) Western immunoblot for nuclear and cytoplasmic markers. Duplicate 75-μg samples of nuclear and cytoplasmic extracts were fractionated by one-dimensional SDS-PAGE, transferred to polyvinylidene difluoride, and stained with lamin B and β-tubulin antibodies. The locations of molecular weight markers (10⁻³) are indicated on the left. The lamin B stain is localized to the nuclear fraction (Nuc), whereas the β-tubulin is localized to the cytoplasmic lysates (Cyto).

FIG. 2.

2DE of soluble nuclear proteins. (A) SYPRO-Ruby-stained 2DE. High-salt extracts containing the soluble proteins from control or RSV-infected (24 h; MOI, 1.0) A549 cells. Proteins were fractionated over immobilized pH gradients from pH 5 to 8 in the horizontal dimension, followed by fractionation by SDS-PAGE in the vertical dimension. Left, apparent migration of molecular mass standards (in kilodaltons). The numbers indicate the spots identified by tryptic peptide mass fingerprinting in Table 1. (B) Gel-to-gel correlation of control replicates. Log normalized spot volumes for gel 1 were plotted pairwise versus gels 2 to 5, and the Pearson's correlation coefficient (cor) was calculated. (C) Hierarchical clustering. Normalized spot intensities were subjected to hierarchical clustering by treatment condition, and their relationships were plotted as a dendrogram. The y axis is dissimilarity. Gels 1 to 6, control nuclei; gels 7 to 12, RSV infected (one RSV-infected gel was excluded due to poor spot resolution). Note that the gels from similar treatments cluster in the same node.

FIG. 2.

2DE of soluble nuclear proteins. (A) SYPRO-Ruby-stained 2DE. High-salt extracts containing the soluble proteins from control or RSV-infected (24 h; MOI, 1.0) A549 cells. Proteins were fractionated over immobilized pH gradients from pH 5 to 8 in the horizontal dimension, followed by fractionation by SDS-PAGE in the vertical dimension. Left, apparent migration of molecular mass standards (in kilodaltons). The numbers indicate the spots identified by tryptic peptide mass fingerprinting in Table 1. (B) Gel-to-gel correlation of control replicates. Log normalized spot volumes for gel 1 were plotted pairwise versus gels 2 to 5, and the Pearson's correlation coefficient (cor) was calculated. (C) Hierarchical clustering. Normalized spot intensities were subjected to hierarchical clustering by treatment condition, and their relationships were plotted as a dendrogram. The y axis is dissimilarity. Gels 1 to 6, control nuclei; gels 7 to 12, RSV infected (one RSV-infected gel was excluded due to poor spot resolution). Note that the gels from similar treatments cluster in the same node.

FIG. 2.

2DE of soluble nuclear proteins. (A) SYPRO-Ruby-stained 2DE. High-salt extracts containing the soluble proteins from control or RSV-infected (24 h; MOI, 1.0) A549 cells. Proteins were fractionated over immobilized pH gradients from pH 5 to 8 in the horizontal dimension, followed by fractionation by SDS-PAGE in the vertical dimension. Left, apparent migration of molecular mass standards (in kilodaltons). The numbers indicate the spots identified by tryptic peptide mass fingerprinting in Table 1. (B) Gel-to-gel correlation of control replicates. Log normalized spot volumes for gel 1 were plotted pairwise versus gels 2 to 5, and the Pearson's correlation coefficient (cor) was calculated. (C) Hierarchical clustering. Normalized spot intensities were subjected to hierarchical clustering by treatment condition, and their relationships were plotted as a dendrogram. The y axis is dissimilarity. Gels 1 to 6, control nuclei; gels 7 to 12, RSV infected (one RSV-infected gel was excluded due to poor spot resolution). Note that the gels from similar treatments cluster in the same node.

FIG. 3.

Validation of nuclear Hsp70 expression. (A) Total ion chromatogram of tryptic digest corresponding to spot 16 (Fig. 1A and Table 1). Both parent ions, with m/z of 541.78 and 627.30, were selected for MS/MS analysis. (B) MS/MS spectrum of parent ion 627.30. Fragment ions produced by collision induced dissociation in tandem MS. The deduced sequence of the peptide, from the NH₂ terminus, is shown in single-letter amino acid code (red lettering, top), and that from the COOH-terminus is shown beneath (blue). The NH₂-terminal sequence is FEELNADLFR, matching the coding sequence of Hsp70 (amino acids 305 to 314). MS/MS analysis of parent ion 541.78 also exactly matched Hsp70.

FIG. 3.

Validation of nuclear Hsp70 expression. (A) Total ion chromatogram of tryptic digest corresponding to spot 16 (Fig. 1A and Table 1). Both parent ions, with m/z of 541.78 and 627.30, were selected for MS/MS analysis. (B) MS/MS spectrum of parent ion 627.30. Fragment ions produced by collision induced dissociation in tandem MS. The deduced sequence of the peptide, from the NH₂ terminus, is shown in single-letter amino acid code (red lettering, top), and that from the COOH-terminus is shown beneath (blue). The NH₂-terminal sequence is FEELNADLFR, matching the coding sequence of Hsp70 (amino acids 305 to 314). MS/MS analysis of parent ion 541.78 also exactly matched Hsp70.

FIG.4.

RSV-induced Hsp70 redistribution. (A) Immunofluorescence microscopy. Control (mock-infected; 0 h) or RSV-infected (24 h; MOI, 1.0) cells were fixed and stained with anti-Hsp70 (top), or nuclei were stained with DAPI (bottom). Shown is a single confocal slice of the stained cells. Hsp70 was detected in a finely granular distribution throughout the cytoplasm in uninfected cells. Conversely, in RSV-infected cells, the distribution of cytoplasmic Hsp70 was more punctate, and some apparent nuclear redistribution could be observed. (B) Nuclear accumulation of Hsp70. Control or RSV-infected cells were fixed, and the cytoplasm was permeabilized and digested with pepsin prior to being stained with Hsp70 (top) or DAPI (bottom). Hsp70 was strongly associated with the nucleus after RSV infection.

FIG. 5.

RSV induces Hsp family gene expression in distinct profiles. (A) Hierarchical clustering of mRNA profiles. A previously reported database of RSV-inducible gene expression in A549 cells was mined for RSV-induced expression changes in all Hsp genes (74). The following Hsp family members were represented on the chip, and their profiles were extracted: Hsp40B1 (GenBank accession no. D85429), -70 (GenBank accession no, M11717), -70-2 (GenBank accession no. M59830), -90 (GenBank accession no. J04988), -70p2 (GenBank accession no. L26336), and -40 (GenBank accession no. U40992); Hsc70 (GenBank accession no. L12723), -40 (DnaJ homolog; GenBank accession no. U40992), -70p9B (GenBank accession no. L15189), -40-3 (GenBank accession no. AF088982), -27 (IFN inducible; GenBank accession no. X67325), and -B3 (GenBank accession no. U15590). Because of the variable level of expression of the individual genes, the average signal intensity (SI) was normalized by the Z score, where deviation from the mean is measured in standard-deviation units. The Z score is determined for any cell i by the formula Z = (SI_i − SI_row)/SD, where SI_row is the average signal intensity for the gene (across the row) and SD is the standard deviation. The data are represented as a heat map, where each value is the colored representation of the calculated Z score for each time point. The scale is represented by red (Z > +1.2), green (Z < −1.2), and black (Z = 0). At the left is a dendrogram indicating the mathematical dissimilarity of the expression profiles. Genes with similar expression profiles are grouped together and are connected by a short line that connects the two nodes. Two major clusters are seen; the first group are genes expressed at time zero, transiently induced by RSV 6 to 12 h after infection and later falling (representing Hsp40B to Hsp90, indicated at the right), and the second group are genes not expressed at time zero and induced 24 and 36 h after RSV infection (Hsp 40-3, Hsp 27, and Hsp B3). (B) Profile of responses for Hsp subgroups. The average signal intensity changes from three independent microarrays are plotted as a function of time. Top, the profile of the 12-h induced Hsp genes, including Hsp70. Hsp70 mRNA abundance is significantly influenced by RSV infection [analysis of variance with replicates, Pr(F) = 0.0143]. Middle, the profile of Hsc70 and coclustering genes. Bottom, induction profile of Hsp27 and associated genes.

FIG. 5.

RSV induces Hsp family gene expression in distinct profiles. (A) Hierarchical clustering of mRNA profiles. A previously reported database of RSV-inducible gene expression in A549 cells was mined for RSV-induced expression changes in all Hsp genes (74). The following Hsp family members were represented on the chip, and their profiles were extracted: Hsp40B1 (GenBank accession no. D85429), -70 (GenBank accession no, M11717), -70-2 (GenBank accession no. M59830), -90 (GenBank accession no. J04988), -70p2 (GenBank accession no. L26336), and -40 (GenBank accession no. U40992); Hsc70 (GenBank accession no. L12723), -40 (DnaJ homolog; GenBank accession no. U40992), -70p9B (GenBank accession no. L15189), -40-3 (GenBank accession no. AF088982), -27 (IFN inducible; GenBank accession no. X67325), and -B3 (GenBank accession no. U15590). Because of the variable level of expression of the individual genes, the average signal intensity (SI) was normalized by the Z score, where deviation from the mean is measured in standard-deviation units. The Z score is determined for any cell i by the formula Z = (SI_i − SI_row)/SD, where SI_row is the average signal intensity for the gene (across the row) and SD is the standard deviation. The data are represented as a heat map, where each value is the colored representation of the calculated Z score for each time point. The scale is represented by red (Z > +1.2), green (Z < −1.2), and black (Z = 0). At the left is a dendrogram indicating the mathematical dissimilarity of the expression profiles. Genes with similar expression profiles are grouped together and are connected by a short line that connects the two nodes. Two major clusters are seen; the first group are genes expressed at time zero, transiently induced by RSV 6 to 12 h after infection and later falling (representing Hsp40B to Hsp90, indicated at the right), and the second group are genes not expressed at time zero and induced 24 and 36 h after RSV infection (Hsp 40-3, Hsp 27, and Hsp B3). (B) Profile of responses for Hsp subgroups. The average signal intensity changes from three independent microarrays are plotted as a function of time. Top, the profile of the 12-h induced Hsp genes, including Hsp70. Hsp70 mRNA abundance is significantly influenced by RSV infection [analysis of variance with replicates, Pr(F) = 0.0143]. Middle, the profile of Hsc70 and coclustering genes. Bottom, induction profile of Hsp27 and associated genes.

FIG. 6.

ND10 redistribution as a function of RSV infection. (A) Redistribution of PML. Immunofluorescence microscopy was performed using anti-PML antibody (top). The bottom images are DAPI-stained nuclei. Left, control cells; right, RSV infected. In control cellular nuclei, PML is present in distinct ND10 structures. In RSV-infected cells, strong PML immunofluorescence is redistributed into the cytoplasm. (B) Redistribution of Sp100. Immunofluorescence using anti-Sp100. The bottom images are as in Fig. 6A. (C) Effect of RSV in expression of ND10 major structural proteins. Hierarchical clustering and heat map of ND10 structural proteins represented in the A549 genomic database, including Sp100 (GenBank accession no. M60618), Daxx (GenBank accession no. AB015051), NDp52 (GenBank accession no. U22897), Sp100B (GenBank accession no. U36501), PML (GenBank accession no. M79463), Blooms' helicase (BLM; GenBank accession no. U39817), replication protein A 14 kDa (Rpa; GenBank accession no. L07493), and replication protein A 70 kDa (RepA; GenBank accession no. M63488). The data are calculated and presented as described for Fig. 5A. RSV induces the coordinate expression of PML, Sp100B, NDp52, Daxx, and Sp100. (D) Western immunoblot analysis of PML and Sp100 protein expression in A549 cells. Cells infected with RSV for 0 (uninfected), 12, 24, and 36 h were fractionated by one-dimensional SDS-PAGE and probed with the indicated antibody. β-Actin was used as a loading control. ND10 redistribution occurs in the setting of increased expression of PML and Sp100.

FIG. 6.

ND10 redistribution as a function of RSV infection. (A) Redistribution of PML. Immunofluorescence microscopy was performed using anti-PML antibody (top). The bottom images are DAPI-stained nuclei. Left, control cells; right, RSV infected. In control cellular nuclei, PML is present in distinct ND10 structures. In RSV-infected cells, strong PML immunofluorescence is redistributed into the cytoplasm. (B) Redistribution of Sp100. Immunofluorescence using anti-Sp100. The bottom images are as in Fig. 6A. (C) Effect of RSV in expression of ND10 major structural proteins. Hierarchical clustering and heat map of ND10 structural proteins represented in the A549 genomic database, including Sp100 (GenBank accession no. M60618), Daxx (GenBank accession no. AB015051), NDp52 (GenBank accession no. U22897), Sp100B (GenBank accession no. U36501), PML (GenBank accession no. M79463), Blooms' helicase (BLM; GenBank accession no. U39817), replication protein A 14 kDa (Rpa; GenBank accession no. L07493), and replication protein A 70 kDa (RepA; GenBank accession no. M63488). The data are calculated and presented as described for Fig. 5A. RSV induces the coordinate expression of PML, Sp100B, NDp52, Daxx, and Sp100. (D) Western immunoblot analysis of PML and Sp100 protein expression in A549 cells. Cells infected with RSV for 0 (uninfected), 12, 24, and 36 h were fractionated by one-dimensional SDS-PAGE and probed with the indicated antibody. β-Actin was used as a loading control. ND10 redistribution occurs in the setting of increased expression of PML and Sp100.

FIG. 6.

ND10 redistribution as a function of RSV infection. (A) Redistribution of PML. Immunofluorescence microscopy was performed using anti-PML antibody (top). The bottom images are DAPI-stained nuclei. Left, control cells; right, RSV infected. In control cellular nuclei, PML is present in distinct ND10 structures. In RSV-infected cells, strong PML immunofluorescence is redistributed into the cytoplasm. (B) Redistribution of Sp100. Immunofluorescence using anti-Sp100. The bottom images are as in Fig. 6A. (C) Effect of RSV in expression of ND10 major structural proteins. Hierarchical clustering and heat map of ND10 structural proteins represented in the A549 genomic database, including Sp100 (GenBank accession no. M60618), Daxx (GenBank accession no. AB015051), NDp52 (GenBank accession no. U22897), Sp100B (GenBank accession no. U36501), PML (GenBank accession no. M79463), Blooms' helicase (BLM; GenBank accession no. U39817), replication protein A 14 kDa (Rpa; GenBank accession no. L07493), and replication protein A 70 kDa (RepA; GenBank accession no. M63488). The data are calculated and presented as described for Fig. 5A. RSV induces the coordinate expression of PML, Sp100B, NDp52, Daxx, and Sp100. (D) Western immunoblot analysis of PML and Sp100 protein expression in A549 cells. Cells infected with RSV for 0 (uninfected), 12, 24, and 36 h were fractionated by one-dimensional SDS-PAGE and probed with the indicated antibody. β-Actin was used as a loading control. ND10 redistribution occurs in the setting of increased expression of PML and Sp100.

FIG. 6.

ND10 redistribution as a function of RSV infection. (A) Redistribution of PML. Immunofluorescence microscopy was performed using anti-PML antibody (top). The bottom images are DAPI-stained nuclei. Left, control cells; right, RSV infected. In control cellular nuclei, PML is present in distinct ND10 structures. In RSV-infected cells, strong PML immunofluorescence is redistributed into the cytoplasm. (B) Redistribution of Sp100. Immunofluorescence using anti-Sp100. The bottom images are as in Fig. 6A. (C) Effect of RSV in expression of ND10 major structural proteins. Hierarchical clustering and heat map of ND10 structural proteins represented in the A549 genomic database, including Sp100 (GenBank accession no. M60618), Daxx (GenBank accession no. AB015051), NDp52 (GenBank accession no. U22897), Sp100B (GenBank accession no. U36501), PML (GenBank accession no. M79463), Blooms' helicase (BLM; GenBank accession no. U39817), replication protein A 14 kDa (Rpa; GenBank accession no. L07493), and replication protein A 70 kDa (RepA; GenBank accession no. M63488). The data are calculated and presented as described for Fig. 5A. RSV induces the coordinate expression of PML, Sp100B, NDp52, Daxx, and Sp100. (D) Western immunoblot analysis of PML and Sp100 protein expression in A549 cells. Cells infected with RSV for 0 (uninfected), 12, 24, and 36 h were fractionated by one-dimensional SDS-PAGE and probed with the indicated antibody. β-Actin was used as a loading control. ND10 redistribution occurs in the setting of increased expression of PML and Sp100.