Noncoding flavivirus RNA displays RNA interference suppressoractivity in insect and mammalian cells

West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) thatcause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounterdifferent host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA virusesin insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteinsto block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in osquitoes depleted forRNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whetheror not flaviviruses counteract RNAi via expression of an RSS. First, we established thaflaviviral RNA replication suppressedsiRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encodedproteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast,we found that the 3=-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressedsiRNA-and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibitsin vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as anucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses.