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.