Essential role of the N-terminal domain in the regulation of RIG-I ATPase activity.
Retinoic acid-inducible gene I (RIG-I) is a cytosolic receptor that recognizes viral RNA and activates the interferon-mediated innate antiviral response. To understand the mechanism of signal activation at the receptor level, we cloned, expressed, and purified human RIG-I containing the two caspase activation and recruitment domains (CARDs) followed by the C-terminal helicase domain. We found that recombinant RIG-I is a functional protein that interacts with double-stranded RNA with substantially higher affinity as compared with single-stranded RNA structures unless they contain a 5'-triphosphate group. Viral RNA binding to RIG-I stimulates the velocity of ATP hydrolysis by 33-fold, which at the cellular level translates into a 43-fold increase of interferon-beta expression. In contrast, the isolated ATPase/helicase domain is constitutively activated while also retaining its RNA ligand binding properties. These results support the recent model by which RIG-I signaling is autoinhibited in the absence of RNA by intra-molecular interactions between the CARDs and the C terminus. Based on pH profile and metal ion dependence experiments, we propose that the active site of RIG-I cannot efficiently accommodate divalent cations under the RNA-free repressed conformation. Overall, these results show a direct correlation between RNA binding and ATPase enzymatic function leading to signal transduction and suggest that a tight control of ATPase activity by the CARDs prevents RIG-I signaling in the absence of viral RNA.
Gee
Peter
P
Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA.
Chua
Pong Kian
PK
Gevorkyan
Jirair
J
Klumpp
Klaus
K
Najera
Isabel
I
Swinney
David C
DC
Deval
Jerome
J
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020169:Caspases / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002413:Cations, Divalent / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D007113:Immunity, Innate / Q000502:physiology; D016899:Interferon-beta / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008670:Metals / Q000276:immunology / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014777:Virus Diseases / Q000201:enzymology / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D002413:Cations, Divalent; D008670:Metals; C437968:PLAAT4 protein, human; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; D016899:Interferon-beta; D020169:Caspases; D000251:Adenosine Triphosphatases