The RNA silencing endonuclease Argonaute 2 mediates specific antiviral immunity in Drosophila melanogaster

Abstract

Most organisms have evolved defense mechanisms to protect themselves from viruses and other pathogens. Arthropods lack the protein-based adaptive immune response found in vertebrates. Here we show that the central catalytic component of the RNA-induced silencing complex (RISC), the nuclease Argonaute 2 (Ago-2), is essential for antiviral defense in adult Drosophila melanogaster. Ago-2-defective flies are hypersensitive to infection with a major fruit fly pathogen, Drosophila C virus (DCV), and with Cricket Paralysis virus (CrPV). Increased mortality in ago-2 mutant flies was associated with a dramatic increase in viral RNA accumulation and virus titers. The physiological significance of this antiviral mechanism is underscored by our finding that DCV encodes a potent suppressor of RNA interference (RNAi). This suppressor binds long double-stranded RNA (dsRNA) and inhibits Dicer-2-mediated processing of dsRNA into short interfering RNA (siRNA), but does not bind short siRNAs or disrupt the microRNA (miRNA) pathway. Based on these results we propose that RNAi is a major antiviral immune defense mechanism in Drosophila.

Figure 1.

D. melanogaster RNAi mutant flies are hypersensitive to virus infection. (A) Transgenic expression of the flock house virus B2 protein, an inhibitor of Dicer function, increases sensitivity to DCV infection. Flies with the indicated genotypes were injected in the abdomen with 350 TCID₅₀ DCV, and survival was monitored daily. B2 expression (under control of the UASp sequence) was induced by crossing UAS-B2 flies with flies expressing the Gal4 transcription factor from the Daughterless promoter. The F1 progeny from this cross (B2/+; Gal4/+, open squares) expressed B2, whereas B2 expression was not detectable in the control crosses (data not shown). (B,C) Survival of Ago-2-null male and female mutant flies after virus infection. Homozygous ago-2^−/−, dcr-2^−/−, and wild-type (wt) flies were injected with 350 TCID₅₀ DCV (B) or CrPV (C) and monitored daily for survival.

Figure 2.

DCV replicates at higher levels in ago-2-null mutant flies. (A) Homozygous ago-2^−/− and wild-type flies were injected with 350 TCID₅₀ DCV, and virus production in the flies was monitored over time. At each time point, three pools of five flies were homogenized, and the viral titer in the homogenate was determined by end-point dilution. Titers represent averages and standard deviations of three independent pools of five flies each. (B,C) DCV RNA accumulation is more efficient in ago-2^−/−-null mutant flies. ago-2^−/− and wild-type flies were injected with 350 TCID₅₀ DCV, and 25 flies were harvested daily for detection of viral RNA. (B) Ethidium bromide-stained agarose gel of total RNA preparation. (C) Northern blot analysis of viral RNA. As a loading control, the blots were stripped and rehybridized with a probe specific for actin 42A. Numbers at the top of the panels indicate days post-infection.

Figure 3.

Drosophila C virus encodes a suppressor of long dsRNA-initiated RNAi. (A) Uninfected or DCV-infected Drosophila S2 cells were transfected with expression plasmids encoding firefly and Renilla luciferase and either a 541-bp dsRNA targeting Renilla luciferase or unrelated control dsRNA. Renilla luciferase counts were normalized using firefly luciferase counts, and data are expressed as fold silencing compared with control dsRNA. (B) Experiment was performed as described in A, but 21-bp synthetic siRNA targeting firefly luciferase or unrelated control siRNA was cotransfected to initiate RNAi. (C) Experiment was performed as described in A. RNAi was initiated with dsRNAs of 21–592 nt in length, as indicated. Silencing is expressed as percent silencing in DCV-infected over uninfected S2 cells. Data in A–C indicate averages and standard deviations of four independent experiments. (D) Processing of dsRNA in Drosophila S2 cell extract. S2 cells were mock-infected, or infected with DCV or CrPV 20 h before preparation of cell extracts, at an MOI of 1. Processing of a 116-bp dsRNA into 21-bp siRNA was analyzed on a 12% denaturing polyacrylamide gel. An end-labeled 21-bp synthetic siRNA was used as a size marker.

Figure 4.

DCV infection does not affect miRNA biogenesis and function. (A) Processed miRNA levels in DCV-infected and uninfected S2 cells. S2 cells were infected with DCV at an MOI of 0.01 and 100, and the presence of miR2b over time was analyzed by Northern blot. As a loading control, the blot was stripped and rehybridized with a probe specific for U6 snRNA. (B) miR2b is assembled into functional RISC complexes. Uninfected or DCV-infected (MOI of 1) S2 cells were transfected with a plasmid expressing luciferase mRNA containing two copies of a sequence that is perfectly complementary to mature miR2b in either the sense or antisense orientation in the 3′UTR. Luciferase activity was normalized to the expression of a cotransfected Renilla luciferase plasmid that does not contains miR2b complementary sites. Luciferase activity is expressed as the ratio of normalized luciferase counts in luciferase-miR-2 antisense over luciferase-miR-2 sense transfections. Bars indicate averages and standard deviations of four independent experiments.

Figure 5.

Drosophila C virus dsRBD protein is a suppressor of RNAi. (A) Schematic representation of the DCV genome. The graphic indicates three dsRBD-containing domains that were cloned and examined for suppression of RNAi. (B) Drosophila S2 cells were transfected with plasmids expressing firefly and Renilla luciferase and the indicated dsRBD-containing DCV ORF-1 domains. RNAi was initiated by adding dsRNA specific for firefly luciferase or control dsRNA to the culture supernatant. Data are expressed as fold silencing compared with control unrelated dsRNA. Note that soaking dsRNA is a less efficient method to induce silencing than cotransfecting dsRNA, as was done in Figure 3. (C) Alignment of dsRBD sequences from DCV and different model organisms. Boxed residues indicate residues that directly interact with dsRNA in Xenopus laevis RNA-binding protein A in X-ray structure (Ryter and Schultz 1998). (D) Suppression of RNAi by wild-type and mutant DCV dsRBD in S2 cells. Experiment was performed as described in B. Data in B and D indicate averages and standard deviations of four independent experiments. (E) DCV-1A suppresses RNAi in vivo. RNA silencing of the white gene by an inverted repeat (IR [W]) in flies that are transgenic for wild-type (wt) or KK73AA DCV-1A, or GFP as a control. Maximum white gene expression in the absence of RNAi was examined in the GMR-Gal4/+; UASp-GFP/+ control flies that do not express the inverted repeat (left panel, control).

Figure 6.

DCV-1A has dsRNA-binding activity and inhibits Dicer processing of dsRNA. (A) RNA mobility shift analyses showing binding of DCV-1A to dsRNA of 211 bp (left panel) and 31 bp (middle panel), and to 21-bp siRNA (right panel). Each probe was incubated with buffer (−) or increasing concentrations of WT-DCV-1A. Concentrations of protein were 0 (lanes 1,9,15), 0.0001 (lanes 2,16), 0.001 (lanes 3,10,17), 0.005 (lanes 4,11,18), 0.01 (lanes 5,12,19), 0.05 (lanes 6,13,20), 0.1 (lanes 7,14,21), and 0.5 μM (lane 8). Position of free probe is indicated by arrows. (B) Binding affinity of WT-DCV-1A to dsRNAs of the indicated lengths. The percentage of radiolabeled probe bound by DCV-1A over total probe in mobility shift assays is plotted against WT-DCV-1A concentrations. (C) RNA mobility shift assay of mutant L28Y-DCV-1A to 211-bp dsRNA (left panel) and of WT-DCV-1A to 211-nt ssRNA (right panel). Each probe was incubated with buffer (−) or increasing concentrations of wild-type or mutant DCV-1A. A mobility shift assay of WT-DCV-1A with a 211-bp dsRNA probe was run in parallel as a positive control (lanes 1,2). Concentrations of protein were 0 (lanes 1,3,11), 0.0001 (lanes 4,12), 0.001 (lanes 5,13), 0.005 (lanes 6,14), 0.01 (lanes 7,15), 0.05 (lanes 8,16), 0.1 (lanes 9,17), and 0.5 μM (lanes 2,10,18). (D) DCV-1A inhibits Dicer processing in S2 cell extracts. Radiolabeled 116-bp dsRNA was processed into siRNA in an S2 cell extract in the presence of increasing concentrations of wild-type and L28Y-DCV-1A or buffer (−). Reaction products were analyzed on a 12% denaturing polyacrylamide gel. An end-labeled 21-bp synthetic siRNA was used as a size marker (Mkr). Concentrations of DCV-1A protein were 0 (lane 2), 0.5 (lane 3), 0.125 (lanes 4,8), 0.05 (lanes 5,9), 0.005 (lanes 6,10), and 0.00125 μM (lanes 7,11).