Mosquito and Drosophila entomobirnaviruses suppress dsRNA- and siRNA-induced RNAi.
Bottom Line: We found that the Culex RNAi machinery processes CYV double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNAs).VP3 was found to bind long dsRNA as well as siRNAs and interfered with Dicer-2-mediated cleavage of long dsRNA into siRNAs.Slicing of target RNAs by pre-assembled RNA-induced silencing complexes was not affected by VP3.
Affiliation: Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Radboud Institute for Molecular Life Sciences, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.Show MeSH
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Mentions: Since CYV is a target of the antiviral RNAi machinery in Culex, we deemed it likely that the virus would encode a VSR. We therefore used well-established reporter assays to determine whether CYV counteracts RNAi (41). In these assays, the effect of virus infection or expression of individual viral proteins on RNAi-mediated silencing of a firefly luciferase (FLuc) reporter is monitored. We first determined whether RNAi is suppressed in cells that are infected with CYV. To this end, we measured luciferase activities in mock- and CYV-infected cells that were co-transfected with the FLuc reporter plasmid and 113-nt in vitro transcribed FLuc dsRNA. A Renilla luciferase (RLuc) reporter plasmid was included as a normalization control. As expected, in mock-infected Drosophila S2 cells, the FLuc reporter was efficiently silenced (∼600-fold) by dsRNA treatment (Figure 2A, left panel). However, dsRNA-mediated silencing of the FLuc reporter was strongly suppressed (to ∼6-fold; P = 0.002) in CYV-infected S2 cells (Figure 2A, left panel). A comparable reduction of FLuc silencing (from ∼300-fold to ∼20-fold; P < 0.001) was observed in CYV-infected Culex CT cells (Figure 2A, right panel). CYV infection also suppressed silencing of the FLuc reporter (from ∼15-fold to ∼3-fold; P = 0.025) when we induced RNAi with 21-nt synthetic siRNA duplexes (Figure 2B). These data show that CYV infection inhibits RNAi induced by dsRNA as well as siRNAs.
Affiliation: Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Radboud Institute for Molecular Life Sciences, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.