Circulating Immune Cells Mediate a Systemic RNAi-Based Adaptive Antiviral Response in Drosophila

Abstract

Effective antiviral protection in multicellular organisms relies on both cell autonomous and systemic immunity. Systemic immunity mediates the spread of antiviral signals from infection sites to distant uninfected tissues. In arthropods, RNA interference (RNAi) is responsible for antiviral defense. Here we show that flies have a sophisticated systemic RNAi-based immunity mediated by macrophage-like haemocytes. Haemocytes take up dsRNA from infected cells and, through endogenous transposon reverse transcriptases, produce virus-derived complementary DNAs (vDNA). These vDNAs template de novo synthesis of secondary viral siRNAs (vsRNA) which are secreted in exosome-like vesicles. Strikingly, exosomes containing vsRNAs, purified from haemolymph of infected flies, confers passive protection against virus challenge in naïve animals. Thus, similar to vertebrates, insects use immune cells to generate immunological memory, in the form of stable vDNAs, that generate systemic immunity, which is mediated by the vsRNA-containing exosomes.

Molecular mechanisms influencing efficiency of RNA interference in insects

“…Experiments utilizing a reverse transcriptase inhibitor demonstrated that viral RNA is converted into vDNA by endogenous reverse transcriptase which is then used as a template to synthesize siRNAs for immune priming (Table S7). This group used a recently developed sequencing technique that can distinguish primary and secondary siRNAs to show that viral RNA is transcribed into DNA inside hemocytes in an Ago2‐dependent manner, and the DNA is then used as a template to synthesize secondary siRNAs de novo . This is the first time the existence of an RNAi amplification mechanism has been observed in insects .…”

“… This group used a recently developed sequencing technique that can distinguish primary and secondary siRNAs to show that viral RNA is transcribed into DNA inside hemocytes in an Ago2‐dependent manner, and the DNA is then used as a template to synthesize secondary siRNAs de novo . This is the first time the existence of an RNAi amplification mechanism has been observed in insects . The caveat is that this amplification mechanism is stimulated only in response to viral infections, and in the absence of a viral infection, exogenous dsRNA is converted only into primary siRNA, not into secondary siRNAs .…”

Defense Mechanisms against Viral Infection in <em>Drosophila</em>: RNAi versus Non-RNAi

“…The importance of RNAi is most clearly illustrated by the specific generation of a siRNA pathway in somatic tissues of insects that is dedicated to defense against invading nucleic acids, and that is maintained separately from the miRNA pathway that regulates physiological and developmental processes [12,15]. The requirement for base-pairing prior to initiation of degradation provides great specificity and mechanisms for enhancement of efficiency have also evolved such as amplification via DNA forms and production of secondary siRNAs followed by systemic spread via exosomes [74,75]. On the other hand, efficiency of antiviral RNAi can be affected by physiological conditions such as nutritional status and stress [19,168].…”

“…Hemocytes are not efficiently infected with SINV, but are proposed to acquire viral dsRNA through phagocytosis of dying cells from other tissues [28] or direct endocytosis from the hemolymph. Uptake of dsRNA in combination with viral infection subsequently initiates an amplification mechanism in the hemocytes that comprises the generation of viral DNA forms (after reverse transcription by endogenous retrotransposons; [74]) that function as templates for the production of secondary viral siRNAs as an amplification mechanism [75]. Secondary viral siRNAs provide systemic protection after their secretion in exosomal-like vesicles that are formed from multivesicular bodies.…”

Mechanisms and Significance of Phagocytic Elimination of Cells Undergoing Apoptotic Death

“…More recently, Drosophila phagocytes were shown to capture viral RNA to produce and secrete short interfering RNA for the RNA interference of viral genomes or transcripts. 68) This mechanism explains the occurrence of anti-viral RNA interference in cells that are not infected with a virus. The production of virus-specific short interfering RNA appeared to last for a long time, and, thus, was regarded as immunological memory resembling the production of antibodies in evolutionarily higher organisms.…”