Dicer-2 senses a 5' secondary structural element of picorna-like viruses for priming antiviral RNA interference

Introduction: Insects as well as plants base their innate antiviral immunity on RNA interference (RNAi). In the model organism Drosophila melanogaster the replicating virus is sensed by the RNase III enzyme Dicer-2, which processes double stranded (ds) viral RNA into 21 nt long small interfering (si) RNAs. A distinctive advantage of the fly antiviral immune system is that these siRNAs provide a footprint of its action. Methods: We use a combination of high throughput sequencing and genetics to unravel the mechanisms involved in viral RNA sensing in Drosophila. Particularly, we cloned and sequenced small RNAs from virus-infected flies. The sequencing was performed by Illumina HiSeq2000. Sequence strategy was 1 x 50 base pairs (bp). Results: We identify a conserved RNA structure in the virus that serves as entry point for Dicer-2 to initiate RNA interference. The recognition of this structure is independent to the helicase activity of the protein. However, the hydrolysis of ATP is essential to mantain the enzyme fully processive. Overall design: Flies were infected with Drosophila C Virus (DCV) and Cricket Paralysis Virus (CrPV). Total RNA was extracted from 12 flies (males and females in equal number) following Trizol (Ambion) datasheet. Small RNAs under 30 nucleotides in size were electrophoretically purified using 15% polyacrylamide gels and subsequently cloned following the protocol described by Sorefan et al., 2012.