Mosquito andDrosophilaentomobirnaviruses suppress dsRNA- and siRNA-induced RNAi

Abstract

RNA interference (RNAi) is a crucial antiviral defense mechanism in insects, including the major mosquito species that transmit important human viruses. To counteract the potent antiviral RNAi pathway, insect viruses encode RNAi suppressors. However, whether mosquito-specific viruses suppress RNAi remains unclear. We therefore set out to study RNAi suppression by Culex Y virus (CYV), a mosquito-specific virus of the Birnaviridae family that was recently isolated from Culex pipiens mosquitoes. We found that the Culex RNAi machinery processes CYV double-stranded RNA (dsRNA) into viral small interfering RNAs (vsiRNAs). Furthermore, we show that RNAi is suppressed in CYV-infected cells and that the viral VP3 protein is responsible for RNAi antagonism. We demonstrate that VP3 can functionally replace B2, the well-characterized RNAi suppressor of Flock House virus. 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. Finally, we show that the RNAi-suppressive activity of VP3 is conserved in Drosophila X virus, a birnavirus that persistently infects Drosophila cell cultures. Together, our data indicate that mosquito-specific viruses may encode RNAi antagonists to suppress antiviral RNAi.

A Unique Nodavirus with Novel Features: Mosinovirus Expresses Two Subgenomic RNAs, a Capsid Gene of Unknown Origin, and a Suppressor of the Antiviral RNA Interference Pathway

“…The following plasmids were described previously: pMTLuc and pMT-Ren (48), pAWH-CrPV1A (32,58), and pAc-DXV VP3 (57). To clone MoNV B2 into an insect expression plasmid, total RNA was isolated from MoNV-infected C6/36 cells using Isol-RNA lysis reagent (5 Prime) and reverse transcribed into cDNA using TaqMan reverse transcription reagents (Roche) and random hexamers (Roche).…”

“…RNAi reporter assays were performed as described earlier (47,57,70). Briefly, for double-stranded-RNA (dsRNA) feeding assays, S2 Rϩ cells were seeded, and pMT-Luc, pMT-Ren, and VSR expression plasmids were transfected the next day.…”

“…Expression of the reporters was induced with 0.5 mM CuSO 4 , and luciferase reporter activity was quantified using the dual-luciferase reporter system (Promega). Sequential transfection of siRNAs in S2 cells was described previously (57). S2 cells were seeded at a density of 2.5 ϫ 10 5 cells per well in a 24-well plate, and 1 day later, 300 ng of VSR expression plasmid or control vector along with 100 ng of pCo-BLAST (Life technologies) was transfected using the transfection reagent Effectene (Qiagen).…”

“…Dicer assays were performed as described before (47,57). Briefly, U4.4 cell lysates were prepared using 30 mM HEPES-KOH (pH 7,4), 100 mM potassium acetate (KOAc), 2 mM Mg(OAc) 2 , 5 mM DTT, 1ϫ protease inhibitor (Roche).…”

“…Members of other insect virus families also encode VSRs. For example, Drosophila C virus 1A inhibits RNAi by binding long dsRNA (48), Drosophila X virus and Culex Y virus VP3 have dsRNA and siRNA binding activity (57), whereas Nora virus VP1 and cricket paralysis virus (CrPV) 1A suppress RNAi by antagonizing the catalytic activity of AGO2 (32,47,58,59).…”