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. 2003 May;77(10):5933-47.
doi: 10.1128/jvi.77.10.5933-5947.2003.

Ribavirin treatment up-regulates antiviral gene expression via the interferon-stimulated response element in respiratory syncytial virus-infected epithelial cells

Yuhong Zhang  1 , Mohammad Jamaluddin, Shaofei Wang, Bing Tian, Roberto P Garofalo, Antonella Casola, Allan R Brasier
Affiliations

Ribavirin treatment up-regulates antiviral gene expression via the interferon-stimulated response element in respiratory syncytial virus-infected epithelial cells

Yuhong Zhang et al. J Virol. 2003 May.

Abstract

Respiratory syncytial virus (RSV) is a mucosa-restricted virus that is a leading cause of epidemic respiratory tract infections in children. RSV replication is a potent activator of the epithelial-cell genomic response, influencing the expression of a spectrum of cellular pathways, including proinflammatory chemokines of the CC, CXC, and CX(3)C subclasses. Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a nontoxic antiviral agent currently licensed for the treatment of severe RSV lower respiratory tract infections. Because ribavirin treatment reduces the cytopathic effect in infected cells, we used high-density microarrays to investigate the hypothesis that ribavirin modifies the virus-induced epithelial genomic response to replicating virus. Ribavirin treatment administered in concentrations of 10 to 100 micro g/ml potently inhibited RSV transcription, thereby reducing the level of RSV N transcripts to approximately 13% of levels in nontreated cells. We observed that in both the absence and the presence of ribavirin, RSV infection induced global alterations in the host epithelial cell, affecting approximately 49% of the approximately 6,650 expressed genes detectable by the microarray. Ribavirin influences the expression of only 7.5% of the RSV-inducible genes (total number of genes, 272), suggesting that the epithelial-cell genetic program initiated by viral infection is independent of high-level RSV replication. Hierarchical clustering of the ribavirin-regulated genes identified four expression patterns. In one group, ribavirin inhibited the expression of the RSV-inducible CC chemokines MIP-1 alpha and -1 beta, which are important in RSV-induced pulmonary pathology, and interferon (IFN), a cytokine important in the mucosal immune response. In a second group, ribavirin further up-regulated a set of RSV- and IFN-stimulated response genes (ISGs) encoding antiviral proteins (MxA and p56), complement products, acute-phase response factors, and the STAT and IRF transcription factors. Because IFN-beta expression itself was reduced in the ribavirin-treated cells, we further investigated the mechanism for up-regulation of the IFN-signaling pathway. Enhanced expression of IFI 6-16, IFI 9-27, MxA/p78, STAT-1 alpha, STAT-1 beta, IRF-7B, and TAP-1-LMP2 transcripts were independently reproduced by Northern blot analysis. Ribavirin-enhanced TAP-1-LMP2 expression was a transcriptional event where site mutations of the IFN-stimulated response element (ISRE) blocked RSV and ribavirin-inducible promoter activity. Furthermore, ribavirin up-regulated the transcriptional activity of a reporter gene selectively driven by the ISRE. In specific DNA pull-down assays, we observed that ribavirin enhanced RSV-induced STAT-1 binding to the ISRE. We conclude that ribavirin potentiates virus-induced ISRE signaling to enhance the expression of antiviral ISGs, suggesting a mechanism for the efficacy of combined treatment with ribavirin and IFN in other chronic viral diseases.

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Figures

FIG. 1.
FIG. 1.
Treatment of epithelial cells with ribavirin. Northern blot of the RSV N transcript from uninfected (lane 1) or pRSV-infected (MOI, 1) (lanes 2 to 8) A549 cells. Cells were pretreated with ribavirin (Riba; 100 μg/ml for 2 h) prior to viral adsorption (lanes 3 to 5) or posttreated with ribavirin (1 h) after viral adsorption. Concentrations of ribavirin used were 0 μg/ml (lanes 1 and 2), 10 μg/ml (lanes 3 and 6), 32 μg/ml (lanes 4 and 7), and 100 μg/ml (lanes 5 and 8). Bottom panel, rehybridization with the 18S probe.
FIG. 2.
FIG. 2.
Analysis of GeneChip data set. (A) Reproducibility of hybridization intensity. As a measure of experimental reproducibility, the fluorescence intensity (normalized average difference value) for each probe set present on the gene chip was plotted in one experiment to its value in the second. Least-squares linear-regression analysis was used to determine the relationship between the two data sets. The data could be described by a straight line with a slope of 1.04 (r2 = 0.99). Further, of the 12,625 probe sets present on the chip, 6,651 ± 290 had hybridization signals that were significantly above background and were considered present. This value did not deviate by more than 4% across the different treatment groups, indicating that neither the drug treatment nor viral infection had global effects on the performance of the gene chip. (B) Agglomerative hierarchical-clustering analysis for each treatment condition was performed by using the unweighted-pair group method with arithmetic means technique (as described in Materials and Methods). Note that the results for each treatment condition most closely cluster with those for its independent replicate. C1 and C2, first and second controls, respectively; Rib1 and Rib2, first and second treatments with ribavirin, respectively; RSV1 and RSV2, first and second treatments with RSV, respectively.
FIG. 3.
FIG. 3.
Relationships between sets of genes regulated by RSV infection or ribavirin treatment. (A) Venn diagram showing the intersections between RSV and ribavirin-regulated genes in A549 cells. Two hundred forty-one genes were common to both data sets. (B) Hierarchical clustering and heat map of the 241 genes common to both treatment groups. The left section is the dendrogram produced by gene expression profiles. The middle section is the heat map, based on hybridization intensity (green is 100, black is 5,000, and red is 10,000 fluorescence intensity units). The right portion is vertical lines that indicate the four distinct gene expression patterns, labeled I to IV. Group I genes are those that are RSV inducible but inhibited by ribavirin (Table 2). Group II genes are those that are RSV inducible and further enhanced by ribavirin (Table 3). C1 and C2, first and second controls, respectively; Rib1 and Rib2, first and second treatments with ribavirin, respectively; RSV1 and RSV2, first and second treatments with RSV, respectively; R+R1 and R+R2, first and second treatments with ribavirin and RSV, respectively; R+R1 and R+R2, first and second treatments with ribavirin and RSV, respectively.
FIG. 3.
FIG. 3.
Relationships between sets of genes regulated by RSV infection or ribavirin treatment. (A) Venn diagram showing the intersections between RSV and ribavirin-regulated genes in A549 cells. Two hundred forty-one genes were common to both data sets. (B) Hierarchical clustering and heat map of the 241 genes common to both treatment groups. The left section is the dendrogram produced by gene expression profiles. The middle section is the heat map, based on hybridization intensity (green is 100, black is 5,000, and red is 10,000 fluorescence intensity units). The right portion is vertical lines that indicate the four distinct gene expression patterns, labeled I to IV. Group I genes are those that are RSV inducible but inhibited by ribavirin (Table 2). Group II genes are those that are RSV inducible and further enhanced by ribavirin (Table 3). C1 and C2, first and second controls, respectively; Rib1 and Rib2, first and second treatments with ribavirin, respectively; RSV1 and RSV2, first and second treatments with RSV, respectively; R+R1 and R+R2, first and second treatments with ribavirin and RSV, respectively; R+R1 and R+R2, first and second treatments with ribavirin and RSV, respectively.
FIG. 4.
FIG. 4.
Hierarchical clustering and heat map of the RSV-inducible chemokines. The gene expression data for 16 chemokines previously shown to be inducible by RSV were retrieved from the original data set (67). The data are as described in the legend to Fig. 3, with a cluster dendrogram at left and a heat map based on hybridization intensity (green is 100, black is 5,000, and red is 10,000 fluorescence intensity units) in the middle. Common names are indicated at right. Fract., fractalkine.
FIG. 5.
FIG. 5.
Ribavirin's effect on the expression of TAP-1-LMP2 genes. (A) Potentiating effect of ribavirin on ISGs as shown by Northern blot analysis. Equal amounts of RNA samples (20 μg) from untreated control (Cont) and ribavirin (Riba)-, RSV-, or ribavirin- and RSV-treated (Riba + RSV) A549 cells were separated and hybridized with specific cDNA probes for IFI 6-16, IFI 9-27, MxA/p78, STAT-1, and IRF-7B; transcripts are indicated at right. The hybridization signal for 18S RNA is shown as the loading control. (B) Expression of the TAP-1-LMP2 locus. A549 cells were treated as described for Fig. 5A, and total RNA was isolated. Shown are the results of Northern blot hybridization for TAP-1 and LMP2 as indicated at right. The bottom panel shows the results of RNA hybridization for 18S RNA as a recovery marker.
FIG. 6.
FIG. 6.
Ribavirin treatment potentiates RSV-induced TAP-1-LMP2 transcriptional activity. (A) A549 cells transiently transfected with either pWT.LMP2-TAP-1.Luc (WT) or pMut.ISRE.Luc (Mut). Following transfection, cells were pretreated with ribavirin (100 μg/ml) for 2 h or not treated and infected with RSV (MOI, 1) for 15 h or not treated. Luciferase activity was normalized to β-Gal and expressed as the multiple of the level of induction over that of the control (uninfected). ∗, P < 0.001 compared to results with RSV alone. (B) Cells were transfected with the multimeric ISRE Luc reporter plasmid. Treatment and assay were as described for panel A.
FIG. 7.
FIG. 7.
Enhanced binding of STAT-1 to the TAP-1-LMP2 ISRE. Biotinylated DNA (corresponding to the TAP-1-LMP2 ISRE site) was used to pull down nuclear proteins from control (Cont), ribavirin-treated (Riba), RSV-infected (RSV), or RSV- and ribavirin-treated (Riba + RSV) cells as described for Fig. 4B. Shown is a Western blot of the affinity-isolated proteins stained with antibody to STAT-1. Ribavirin increases the binding of STAT-1α and -1β to the ISRE compared to that induced by RSV infection alone.
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