Ribavirin treatment up-regulates antiviral gene expression via the interferon-stimulated response element in respiratory syncytial virus-infected epithelial cells.
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.
Zhang
Yuhong
Y
Department of Medicine, The University of Texas Medical Branch, Galveston, Texas 77555-1060, USA.
Jamaluddin
Mohammad
M
Wang
Shaofei
S
Tian
Bing
B
Garofalo
Roberto P
RP
Casola
Antonella
A
Brasier
Allan R
AR
D000998:Antiviral Agents / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000276:immunology / Q000821:virology; D020869:Gene Expression Profiling; D006801:Humans; D007372:Interferons / Q000494:pharmacology*; D020411:Oligonucleotide Array Sequence Analysis; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D018113:Respiratory Syncytial Virus, Human / Q000187:drug effects* / Q000276:immunology / Q000472:pathogenicity; D012137:Respiratory System / Q000276:immunology* / Q000821:virology; D020218:Response Elements / Q000502:physiology*; D012254:Ribavirin / Q000494:pharmacology*; D015854:Up-Regulation
D000998:Antiviral Agents; D011506:Proteins; D012254:Ribavirin; D007372:Interferons