RNA interference directs innate immunity against viruses in adult Drosophila

Abstract

Innate immunity against bacterial and fungal pathogens is mediated by Toll and immune deficiency (Imd) pathways, but little is known about the antiviral response in Drosophila. Here, we demonstrate that an RNA interference pathway protects adult flies from infection by two evolutionarily diverse viruses. Our work also describes a molecular framework for the viral immunity, in which viral double-stranded RNA produced during infection acts as the pathogen trigger whereas Drosophila Dicer-2 and Argonaute-2 act as host sensor and effector, respectively. These findings establish a Drosophila model for studying the innate immunity against viruses in animals.

Fig. 1

Antiviral silencing in Drosophila embryos requires Dcr-2 and Ago-2. Northern blot detection of FHV RNA accumulation in wt, dcr-2, and ago-2 embryos micro-injected with in vitro synthesized transcripts R1, R1fs and R1ΔB2 as shown in Fig. s1.

Fig. 2

The siRNA pathway controls the innate immunity against FHV in adult flies. (A) Survival of wt, dcr-2 and r2d2 flies after FHV infection. Data shown represented mean of triplicates and the error bars indicate standard derivation. (B, C) Detection of viral RNAs by a probe specific for RNAs 1 and 3, and viral coat protein (CP) by a rabbit anti-FHV serum (18) in wt, dcr-2, and r2d2 flies injected with FHV. (D) Detection of FHV siRNAs in the infected adult flies as in C (d2, dcr-2; r2, r2d2). The membrane was also probed for miR-8 and U6 RNA.

Fig. 3

dcr-2 and r2d2 flies exhibit enhanced disease susceptibility to CrPV. (A) Survival of wt, dcr-2 and r2d2 flies after CrPV infection. Data shown represented mean of triplicates and the error bars indicate standard derivation. (B) Accumulation of CrPV genomic RNA in wt, dcr-2, and r2d2 adults injected with CrPV.

Fig. 4

Induction and suppression of antiviral silencing by CrPV. (A) Accumulation of the genomic RNA and siRNAs of CrPV in infected S2 cells. (B) pFR1gfp directed transcription of a recombinant FHV RNA1 in which the coding sequence for B2 was replaced by that of GFP. S2 cells were transfected with pFR1gfp alone (middle) or pFR1gfp plus either dsRNA-targeting Ago-2 (right) or CrPV superinfection (left). (C) Identification of CrPV RNAi suppressor. Cells were co-transfected with pFR1gfp and a plasmid as indicated (see Fig. s2), and total RNA analyzed for the accumulation of pFR1gfp-encoded RNA1 and RNA3. P, the empty plasmid; VP, the virion protein precursor; V1, VP1; V2, VP2; V3, VP3; V4, VP4; V0, VP0 (precursor for VP3 and VP4); A, the first 140 codons of the upstream ORF; ΔA, a frameshift mutant of A; and b, the first 107 codons of the upstream ORF. NS1 is an RNAi suppressor of Influenza A virus as described previously (11).