Antiviral immunity in Drosophila requires systemic RNA interference spread

Abstract

Multicellular organisms evolved sophisticated defence systems to confer protection against pathogens. An important characteristic of these immune systems is their ability to act both locally at the site of infection and at distal uninfected locations. In insects, such as Drosophila melanogaster, RNA interference (RNAi) mediates antiviral immunity. However, the antiviral RNAi defence in flies seems to be a local, cell-autonomous process, as flies are thought to be unable to generate a systemic RNAi response. Here we show that a recently defined double-stranded RNA (dsRNA) uptake pathway is essential for effective antiviral RNAi immunity in adult flies. Mutant flies defective in this dsRNA uptake pathway were hypersensitive to infection with Drosophila C virus and Sindbis virus. Mortality in dsRNA-uptake-defective flies was accompanied by 100-to 10(5)-fold increases in viral titres and higher levels of viral RNA. Furthermore, inoculating naked dsRNA into flies elicited a sequence-specific antiviral immune response that required an intact dsRNA uptake pathway. These findings suggest that spread of dsRNA to uninfected sites is essential for effective antiviral immunity. Notably, infection with green fluorescent protein (GFP)-tagged Sindbis virus suppressed expression of host-encoded GFP at a distal site. Thus, similar to protein-based immunity in vertebrates, the antiviral RNAi response in flies also relies on the systemic spread of a virus-specific immunity signal.

Figure 1

Model for systemic RNAi viral immunity in Drosophila melanogaster. Upon viral infection, virus-specific dsRNAs (eg., replication intermediates) are generated during the initial rounds of virus replication. Following cell death or lysis, dsRNAs are taken up and processed by uninfected cells to protect them from subsequent infection, thereby preventing virus spread.

Figure 2

in vivo dsRNA immunization provides sequence-specific antiviral protection in D. melanogaster. a, Immunization protocol. b and c, Wild type flies infected with Sindbis-GFP virus two days after intrathoracical injection of dsRNA against Drosophila C virus (DCV, 442 base-pair in length, corresponding to the viral polymerase between nucleotides 5589 to 6030), dsRNA against Sindbis virus non-structural proteins 1 and 2 (dsSin1, 901 base-pairs in length, corresponding to nucleotides 1211 to 2112) or dsRNA Sindbis virus corresponding to the non-structural proteins 3 and 4 (dsSin2, 954 base-pairs long, length, corresponding to nucleotides 5485 to 6439). Buffer: control injection. d.p.i.: days post infection. Sindbis-GFP virus replication was monitored by GFP production. b, Fluorescence images. c, Western blot with an anti-GFP antibody. d, Sindbis-GFP virus challenge in wild type, homozygous Dcr2^L811fsX (Dcr2^-/-) and homozygous Ago⁴¹⁴ (Ago2^-/-) flies. e, dsRNA immunization protects in a dose-dependent manner. Flies were inoculated with dsRNA, dsSin2, directed against Sindbis-GFP (5ng, 0.5 ng, 50 pg, and 5 pg). Virus replication over time (d.p.i.: 2 to 5) was monitored by westernblotting using and an anti-GFP antibody.

Figure 3

Increased viral susceptibility of dsRNA uptake deficient mutants. a, b, Survival of dsRNA uptake mutant flies after virus infection. Homozygous egh^EP804 (egh^-/-), NinaC³ (NinaC^-/-), CG4572^c05963 (CG4572^-/-), and wild type flies were injected with 500 TCID₅₀ DCV (a) or 500 PFU Sindbis-GFP virus (b) and monitored daily for survival. c, DCV replicates at higher levels in dsRNA uptake mutant flies. Flies were injected with 500 TCID₅₀ DCV, and virus production 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. The error bars report the average +/- s.d. for at least 3 independent experiments.d, Sindbis-GFP virus replicates at higher levels in dsRNA uptake mutant flies as shown by increased GFP expression in the fat body after 3 days post-infection when compared to wild type flies.

Figure 4

Core RNAi machinery and antibacterial immunity are intact in dsRNA uptake mutants. a, Schematic to test the core RNAi machinery integrality. b, RNAi processing of an inverted repeat IR [Ecr] induced by the GMR-GAL4 driver prevents the formation of the corneal lens (EM picture). c, Monitoring corneal lens formation and eye color in transgenic flies deficient in the dsRNA uptake pathway. d, Susceptibility of dsRNA uptake mutant flies to infection is specific to the viruses, as the dsRNA uptake mutant flies are able to produce antimicrobial peptides in response to an infection by pathogenic gram + and gram − bacteria.

Figure 4

Core RNAi machinery and antibacterial immunity are intact in dsRNA uptake mutants. a, Schematic to test the core RNAi machinery integrality. b, RNAi processing of an inverted repeat IR [Ecr] induced by the GMR-GAL4 driver prevents the formation of the corneal lens (EM picture). c, Monitoring corneal lens formation and eye color in transgenic flies deficient in the dsRNA uptake pathway. d, Susceptibility of dsRNA uptake mutant flies to infection is specific to the viruses, as the dsRNA uptake mutant flies are able to produce antimicrobial peptides in response to an infection by pathogenic gram + and gram − bacteria.

Figure 5

Systemic spread of dsRNA follows virus infection and it is essential for effective antiviral immunity. a, Drosophila C virus infection in wild type, and homozygous egh^EP804 (egh^-/-), NinaC³ (NinaC^-/-) and CG4572^c05963 (CG4572^-/-) mutant flies treated with the inoculated with dsRNA. DCV replication was monitored by westernblotting using an antibody directed against DCV capsid protein VP1. (b-d) dsRNA produced during virus replication can spread and silence endogenous GFP expressed at a distal site of infection. Flies expressing eGFP (Tub-eGFP) inoculated with Sindbis-GFP (b, c) or Sindbis:Luciferase virus (d) by intrathoracical inoculation. b, Viral replication monitored by RT-PCR using primers that amplify NSP1-2 virus genes. c, Expression of endogenous GFP monitored by Western blot with an anti-GFP antibody. d, same as (c) except that flies were infected with Sindbis-Luciferase virus.