55 Amino acid linker between helicase and carboxyl terminal domains of RIG-I functions as a critical repression domain and determines inter-domain conformation.
In virus-infected cells, viral RNA with non-self structural pattern is recognized by DExD/Hbox RNA helicase, RIG-I. Once RIG-I senses viral RNA, it triggers a signaling cascade, resulting in the activation of genes including type I interferon, which activates antiviral responses. Overexpression of N-terminal caspase activation and recruitment domain (CARD) is sufficient to activate signaling; however basal activity of full-length RIG-I is undetectable. The repressor domain (RD), initially identified as a.a. 735-925, is responsible for diminished basal activity; therefore, it is suggested that RIG-I is under auto-repression in uninfected cells and the repression is reversed upon its encounter with viral RNA. In this report, we further delimited RD to a.a. 747-801, which corresponds to a linker connecting the helicase and the C-terminal domain (CTD). Alanine substitutions of the conserved residues in the linker conferred constitutive activity to full-length RIG-I. We found that the constitutive active mutants do not exhibit ATPase activity, suggesting that ATPase is required for de-repression but not signaling itself. Furthermore, trypsin digestion of recombinant RIG-I revealed that the wild-type, but not linker mutant conforms to the trypsin-resistant structure, containing CARD and helicase domain. The result strongly suggests that the linker is responsible for maintaining RIG-I in a "closed" structure to minimize unwanted production of interferon in uninfected cells. These findings shed light on the structural regulation of RIG-I function.
Laboratory of Molecular Genetics, Institute for Virus Research, Kyoto University, Kyoto, Japan.
Maiko
M
Kageyama
Kiyohiro
K
Takahasi
Ryo
R
Narita
Reiko
R
Hirai
Mitsutoshi
M
Yoneyama
Hiroki
H
Kato
Takashi
T
Fujita
D000595:Amino Acid Sequence; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary / Q000235:genetics; D012313:RNA / Q000737:chemistry; D011971:Receptors, Immunologic
D011971:Receptors, Immunologic; D012313:RNA; C452820:RIGI protein, human; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases