5'-triphosphate RNA requires base-paired structures to activate antiviral signaling via RIG-I.
The ATPase retinoid acid-inducible gene (RIG)-I senses viral RNA in the cytoplasm of infected cells and subsequently activates cellular antiviral defense mechanisms. RIG-I recognizes molecular structures that discriminate viral from host RNA. Here, we show that RIG-I ligands require base-paired structures in conjunction with a free 5'-triphosphate to trigger antiviral signaling. Hitherto unavailable chemically synthesized 5'-triphosphate RNA ligands do not trigger RIG-I-dependent IFN production in cells, and they are unable to trigger the ATPase activity of RIG-I without a base-paired stretch. Consistently, immunostimulatory RNA from cells infected with a virus recognized by RIG-I is sensitive to double-strand, but not single-strand, specific RNases. In vitro, base-paired stretches and the 5'-triphosphate bind to distinct sites of RIG-I and synergize to trigger the induction of signaling competent RIG-I multimers. Strengthening our model of a bipartite molecular pattern for RIG-I activation, we show that the activity of supposedly "single-stranded" 5'-triphosphate RNAs generated by in vitro transcription depends on extended and base-paired by-products inadvertently, but commonly, produced by this method. Together, our findings accurately define a minimal molecular pattern sufficient to activate RIG-I that can be found in viral genomes or transcripts.
Schmidt
Andreas
A
Division of Clinical Pharmacology, Department of Medicine, Ludwig-Maximilian University Munich, 80336 Munich, Germany.
Schwerd
Tobias
T
Hamm
Wolfgang
W
Hellmuth
Johannes C
JC
Cui
Sheng
S
Wenzel
Michael
M
Hoffmann
Franziska S
FS
Michallet
Marie-Cecile
MC
Besch
Robert
R
Hopfner
Karl-Peter
KP
Endres
Stefan
S
Rothenfusser
Simon
S
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D020029:Base Pairing; D001665:Binding Sites; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D006801:Humans; D008024:Ligands; D051379:Mice; D011485:Protein Binding; D055503:Protein Multimerization; D012313:RNA / Q000737:chemistry* / Q000276:immunology*; D051192:Receptors, Pattern Recognition / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D015398:Signal Transduction / Q000276:immunology*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000378:metabolism; D014780:Viruses / Q000276:immunology*
D008024:Ligands; C437968:PLAAT4 protein, human; D051192:Receptors, Pattern Recognition; D018168:Receptors, Retinoic Acid; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D000251:Adenosine Triphosphatases