This study investigates the antiviral response in Drosophila by examining the role of the Dicer-2 protein in processing viral RNA. The research highlights the mechanisms of RNA interference and the subsequent immune response triggered by viral infections.
Take anesthetized wild-type and Dicer-2 mutant male Drosophila on an injection dish under a stereomicroscope. Ensure the flies are free of any symbiotic bacteria that suppress RNA virus replication.
Inoculate the flies intra-thoracically with a nano-injector containing a positive-sense, single-stranded RNA virus suspension.
The virus enters the host cell via receptor-mediated endocytosis and replicates producing double-stranded intermediate RNA. The host pattern recognition receptor Dicer-2 processes the viral double-stranded RNA into small interfering RNAs, siRNAs.
The siRNA binds to the RNA-induced silencing complex, RISC, where its complementary strand is removed. The RISC-loaded siRNAs bind to other viral RNAs via complementary base pairing, causing viral RNA degradation and gene silencing.
Dicer-2 and viral RNA interaction activates downstream antiviral signaling cascades, producing antiviral proteins. These antiviral proteins interact with neighboring host cells' Janus kinase, or JAK, protein-bound cytokine receptors, initiating receptor dimerization and JAK phosphorylation.
Activated JAKs phosphorylate and activate STAT proteins, a signal transducer and transcription activator. Activated STATs translocate to the nucleus and bind to specific DNA sequences, initiating the transcription of antiviral response genes.
Grow the flies for a desired period.
Downstream analysis indicates decreased survival rate and increased viral load in Dicer-2 mutant flies.
For viral infection, first, use a stereomicroscope and thin forceps to break the tip of a glass capillary needle to the appropriate diameter for nano-injection. To assemble the injector, place the sealing O-ring and a white spacer onto the metal plunger of the injector with the large dimple facing outwards.
Use a syringe equipped with a 30-gauge needle to fill the glass needle with mineral oil and place the needle through the collar. Place the larger O-ring around the base of the collar, about 1 millimeter from the blunt end of the needle, and mount the needle onto the plunger of the injector.
Secure the needle onto the plunger, and press the empty button to extend the plunger of the microinjector until an audible signal is heard. Now press Fill to retract the plunger 5 millimeters, and dip the needle into a 100 plaque-forming unit viral suspension. Gently shake one or at least three vials of 20 Wolbachia-free male Drosophila onto the injection dish.
Male-endowed flies are preferred as the hormonal variation during mating and reproduction may influence the readout with females.
Then, inject the thorax of each fly with 50.6 nanoliters of virus solution at the slightly lighter colored region between the mesopleura and the tetrapleura and measure the DCV load by the cytopathic effect assay and quantitative RT-PCR from ground flies as just demonstrated. After the injection, carefully transfer the flies to a fresh vial, and place the vial in a horizontal position to prevent the flies from sticking to the medium while recovering from the anesthesia.
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