Abstract
RNA interference can be mediated by fully complementary siRNA or partially complementary miRNA. siRNAs are widely used to suppress viral replication and the fully complementary siRNA bound Ago-2 in the RISC is known to degrade the target RNA. Although other argonaute proteins lacking slicer activity can also bind oligonucleotides with both si and miRNA structures, whether they can also contribute to antiviral effects is not entirely clear. We tested si and miRNA structured oligos for target repression in dual luciferase assays as well as for inhibition of Dengue and West Nile virus replication in ES cells expressing individual Ago proteins. In luciferase assays, both fully complementary and partially complementary oligos effectively repressed their targets in all individual Ago expressing cell lines, although the efficacy with fully complementary oligos was higher in Ago-2+ cells. However, partially complementary oligos had no effect on virus replication in any cell line, while fully complementary siRNAs were highly effective in Ago-2 expressing, but not in cells expressing other Ago proteins. This occurred irrespective of whether the target sequences were located in the coding region or 3′UTR of the virus. We conclude that Ago-2 slicer activity is essential for anti-viral efficacy of siRNAs and miRNA-mediated translational repression/transcript destabilization is too weak to suppress the abundantly expressed flaviviral proteins.
Highlights
RNA interference (RNAi) is a phenomenon where small double stranded RNAs mediate sequence-specific regulation of gene expression
Oligonucleotides with both si and miRNA structure can bind to all the Ago proteins and repress targets, the efficacy is highest for siRNAs in Ago-2 expressing cells
Our results suggest that effective in reporter assays, completely complementary siRNAs loaded to Agos1, 3 and 4 and siRNAs mimicking miRNAs with only seed matches to the viral targets in the coding region or 39UTR loaded to any of the Agos fail to suppress an acute flaviviral infection
Summary
RNA interference (RNAi) is a phenomenon where small double stranded RNAs mediate sequence-specific regulation of gene expression. The 21– 23 nucleotide dsRNAs associate in the cytoplasm with the RNAinduced silencing complex (RISC), whereupon one of the two RNA strands (passenger strand) is discarded and the other guide strand guides the RISC to mediate sequence-specific degradation of the corresponding mRNA (in the case of siRNAs) and/or translational repression/transcript destabilization by binding to the 39 untranslated region (UTR) (in the case of miRNAs) [5,6]. While siRNAs primarily degrade the target mRNA, miRNAs lead to translational repression/mRNA destabilization (reviewed in [7]). Both mi and siRNAs use the same RNAi machinery, the exact mechanism of gene silencing is different. Since only mammalian Ago-2 has slicer activity, the role of other Ago proteins in the siRNA pathway is unclear
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