Double-stranded RNA induces antiviral transcriptional response through the Dicer-2/Ampk/FoxO axis in an arthropod

Abstract

Invertebrates mainly rely on sequence-specific RNA interference (RNAi) to resist viral infections. Increasing studies show that double-stranded RNA (dsRNA) can induce sequence-independent protection and that Dicer-2, the key RNAi player that cleaves long dsRNA into small interfering RNA (siRNA), is necessary for this protection. However, how this protection occurs remains unknown. Herein, we report that it is caused by adenosine triphosphate (ATP)-hydrolysis accompanying the dsRNA-cleavage. Dicer-2 helicase domain is ATP-dependent; therefore, the cleavage consumes ATP. ATP depletion activates adenosine monophosphate-activated protein kinase (Ampk) and induces nuclear localization of Fork head box O (FoxO), a key transcriptional factor for dsRNA-induced genes. siRNAs that do not require processing cannot activate the transcriptional response. This study reveals a unique nonspecific antiviral mechanism other than the specific RNAi in shrimp. This mechanism is functionally similar to, but mechanistically different from, the dsRNA-activated antiviral response in vertebrates and suggests an interesting evolution of innate antiviral immunity.

Keywords: Dicer-2; RNA-interference; double-stranded RNA.

Fig. 1.

DsRNA induces FoxO-dependent antiviral transcriptional response. (A and B) Schematic illustration of experimental procedures and GFP dsRNAs. (C–E) Antiviral protective effect of arbitrary long dsRNAs. Infection was performed 24 h after dsRNA/siRNA stimulation. Viral load was determined another 24 h later. Mean ± SD, n = 4 biological replicates. One-way ANOVA. RFP, red fluorescent protein; hIgG, human immunoglobulin. (F) DEGs between dsGFP and siGFP groups, n = 3 biological replicates. (G) Damage of virions by AlfB1. TEM image of WSSV virions treated with 20 μM of AlfB1 peptide. Scale bar, 200 nm. (H) Affection of hemocytic phagocytosis of virions by Draper-knockdown. Flow-cytometry analysis of hemocytes (10,000) at 2 h after FITC-labeled virions (10⁶) being injected into Draper-pre-silenced shrimp hemocoel. (I–K) Induction of FoxO nuclear-localization by dsGFP. Immunocytochemical analysis of hemocytes at 6 h after stimulation. Scale bar, 10 μm (I). Digitalization of FoxO-nuclei colocalization from eight randomly selected field of view, n > 100 cells (J). Blotting assay of hemocytes nuclear proteins (K). (L and M) Inhibition of dsGFP-induced transcriptional response by FoxO-knockdown. Presilenced shrimp was stimulated with dsGFP/siGFP to evaluate gene transcription 24 h later (L). Infection was performed at 24 h after stimulation to determine viral load another 24 h later (M), n = 4 biological replicates. ^∗∗∗P < 0.001, ns, not significant, Student’s t test. (N) Presence of FoxO-binding sites in the promoters. (O and P) Interaction between FoxO and FoxO-binding sites. Biotin-labeled probes (5 ng) were incubated with recombinant-FoxO or control-tag (2 μg) (O) or dsGFP-stimulated nuclear extracts (5 μg) (P) for EMSA. Excess unlabeled probes and FoxO antibodies were used for competition and supershift assays. (Q) Induction of FoxO–promoter interactions by dsGFP. Immunoprecipitates from dsGFP-stimulated hemocytes were detected using primers for fragments containing FoxO-binding sites. Blank, Protein-A agarose; control, unrelated antibody not cross-reacting with shrimp proteins. Images are representative of three independent replicates.

Fig. 2.

DsRNA activates Dicer-2/Ampk/FoxO axis for transcriptional response. (A) Effect of RNAi-components knockdown on dsGFP-induced antiviral immunity. Presilenced shrimp were stimulated with dsGFP/siGFP. Infection was performed 24 h later to determine viral load another 24 h later, n = 4 biological replicates. (B–D) Inhibition of dsGFP-induced FoxO-nuclear localization by Dicer-2-knockdown. Presilenced shrimp were stimulated with dsGFP/siGFP. Hemocytes were sampled 6 h later to extract nuclear protein for blotting assay (B), and immunocytochemical assay. Scale bar, 10 μm (C). FoxO-nuclei colocalization was digitalized from eight randomly selected field of view, n > 100 cells (D). (E) Inhibition of dsGFP-induced gene transcription by Dicer-2-knockdown. Gene expression was detected at 24 h after stimulation, n = 4 biological replicates. (F) Induction of ADP/ATP ratio by dsGFP. Hemocytes ADP/ATP ratio was determined at 3 h after dsGFP (1, 2, 5 μg/g) or siGFP (5 μg/g) stimulation, n = 4 biological replicates. (G) Inhibition of dsGFP-induced increase in ADP/ATP ratio by Dicer-2-knockdown. Presilenced shrimp were stimulated with dsGFP/siGFP. ADP/ATP ratio was determined 3 h later, n = 4 biological replicates. (H) Induction of hemocytes Ampkα-phosphorylation by dsGFP. Botting assay was performed at 6 h after stimulation. (I) Inhibition of dsGFP-induced Ampkα-phosphorylation by Dicer-2 knockdown. Presilenced shrimp were stimulated with dsGFP/siGFP. Botting assay was performed 6 h later. (J–M) Inhibition of dsGFP-induced FoxO-nuclear localization and gene transcription by Ampkα-knockdown. Experimental procedures are similar to (B–E). (N) Working model. Energy consumption caused by Dicer-2-mediated dsRNA-cleavage activates Ampk/FoxO axis to induce RNAi components and antiviral effectors. Images are representative of three independent replicates. ^∗∗∗P < 0.001, ns, not significant, Student’s t test.