Encyclopedia of Experiments: Immunology
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Transcript
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.
