A Conserved Histidine in the RNA Sensor RIG-I Controls Immune Tolerance to N(1)-2′O-Methylated Self RNA

Schuberth-Wagner, Christine; Ludwig, Janos; Bruder, Ann Kristin; Herzner, Anna-Maria; Zillinger, Thomas; Goldeck, Marion; Schmidt, Tobias; Schmid-Burgk, Jonathan L.; Kerber, Romy; Wolter, Steven; Stümpel, Jan-Philip; Roth, Andreas; Bartok, Eva; Drosten, Christian; Coch, Christoph; Hornung, Veit; Barchet, Winfried; Kümmerer, Beate M.; Hartmann, Gunther; Schlee, Martin

The cytosolic helicase retinoic acid-inducible gene-I (RIG-I) initiates immune responses to most RNA viruses by detecting viral 5′-triphosphorylated RNA (pppRNA). Although endogenous mRNA is also 5′-triphosphorylated, backbone modifications and the 5′-ppp-linked methylguanosine ((m7)G) cap prevent immunorecognition. Here we show that the methylation status of endogenous capped mRNA at the 5′-terminal nucleotide (N(1)) was crucial to prevent RIG-I activation. Moreover, we identified a single conserved amino acid (H830) in the RIG-I RNA binding pocket as the mediator of steric exclusion of N(1)-2′O-methylated RNA. H830A alteration (RIG-I(H830A)) restored binding of N(1)-2′O-methylated pppRNA. Consequently, endogenous mRNA activated the RIG-I(H830A) mutant but not wild-type RIG-I. Similarly, knockdown of the endogenous N(1)-2′O-methyltransferase led to considerable RIG-I stimulation in the absence of exogenous stimuli. Studies involving yellow-fever-virus-encoded 2′O-methyltransferase and RIG-I(H830A) revealed that viruses exploit this mechanism to escape RIG-I. Our data reveal a new role for cap N(1)-2′O-methylation in RIG-I tolerance of self-RNA.