Error-prone replication of West Nile virus caused by ribavirin.

Ribavirin has been reported to cause error-prone replication and viral extinction in RNA viruses. The antiviral activity of ribavirin against West Nile virus (WNV) was evaluated in various cell lines to select a model in which mutagenic effects could be studied. The antiviral activity was greatest in HeLa cells as compared to CV-1, L929, Vero, or MA-104 cells. WNV was also passaged sequentially in cell monolayers treated with ribavirin to determine whether cumulative mutations could lead to viral extinction in these cell lines. The virus was abrogated in HeLa cells after 4 passages, while high viral titers persisted after many passages in other cells. A molecular clone of WNV was propagated in HeLa cells treated with 15 microg/mL ribavirin, and sequencing of viral genome segments revealed significant increases in transition mutations, demonstrating that ribavirin induced error-prone replication. The relative infectivity of viral RNA synthesized in the presence of ribavirin was shown to be reduced compared with untreated controls. These data support the hypothesis that error catastrophe is one of the modes of action for ribavirin against WNV.

Differential contribution of IL-1Ra isoforms to allele-specific IL-1Ra mRNA accumulation.

The interleukin-1 (IL-1) receptor antagonist (IL-1Ra) gene produces two isoforms of IL-1Ra, intracellular (icIL-1Ra) and secreted (sIL-1Ra). Distinct promoter regions control synthesis of each isoform. Five alleles of this gene, defined by sIL-1Ra intron 2 polymorphism, have been described. Although differences in IL-1Ra protein production have been demonstrated in various tissues and cells obtained from individuals carrying allele 1 vs. allele 2, the underlying mechanisms of this discrepancy remain poorly understood. We hypothesize that one mechanism contributing to differences in protein levels may be allele-specific accumulation of icIL-1Ra or sIL-1Ra mRNA. Quantification of allele-specific differences in mRNA accumulation in colonic biopsies and peripheral blood mononuclear cells (PBMC) of heterozygous individuals shows that the amount of allele 1-specific icIL-1Ra mRNA averaged four times higher relative to allele 2. In transfection assays, gene expression directed by the allele 1-specific icIL-1Ra promoter fragment was found to be greater than that directed by the allele 2 promoter, suggesting that icIL-1Ra promoter activity contributes to the disparity in the allele-specific icIL-1Ra mRNA accumulation. Our data show that differences in the transcriptional regulation of icIL-1Ra alleles 1 and 2 may be involved in the production of icIL-1Ra protein. Disregulated icIL-1Ra production may play a role in chronic inflammatory diseases where the balance between IL-1 and IL-1Ra has been implicated as a key pathogenic mechanism.

A possible role of IL-1ra 3'-untranslated region in modulation of protein production.

Human interleukin 1 (IL-1) receptor antagonist (hIL-1ra), an anti-inflammatory cytokine and naturally occurring antagonist of IL-1, may be regulated at both the transcriptional and post-transcriptional levels. The aim of this study was to evaluate post-transcriptional regulation of hIL-1ra with specific focus on the 3'-untranslated region (3'-UTR) of hIL-1ra mRNA using the luciferase reporter gene system. Constructs were created containing the luciferase reporter gene followed by the hIL-1ra 3'-UTR or its modified variants. In monocyte/macrophage cell lines RAW264.7 and U937, the presence of the hIL-1ra 3'-UTR resulted in a 5.7-fold (n=6, P<0.001) and a 3.9-fold (n=7, P<0.001) decrease in transient reporter gene expression, respectively, with only a less than 2-fold mean difference in steady-state mRNA levels in the former case. In a cell-free translation system, the presence of the middle segment of the 3'-UTR caused a 5.2-fold (n=5, P<0.001) decrease in the amount of luciferase synthesized. In contrast to synthetic 3'-UTR and that derived from bovine growth hormone RNA, the presence of hIL-1ra 3'-UTR resulted in accumulation of unprocessed transcripts in transfected cells. We conclude that hIL-1ra synthesis may be regulated at the post-transcriptional level through mechanisms involving the 3'-UTR of IL-1ra transcripts.