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Summary: Antiviral immunity is triggered by immunorecognition of viral nucleic acids. The cytosolic helicase RIG-I is a key sensor of viral infections and is activated by RNA containing a triphosphate at the 5′ end. The exact structure of RNA activating RIG-I remains controversial. Here, we established a chemical approach for 5′ triphosphate oligoribonucleotide synthesis and found that synthetic single-stranded 5′ triphosphate oligoribonucleotides were unable to bind and activate RIG-I. Conversely, the addition of the synthetic complementary strand resulted in optimal binding and activation of RIG-I. Short double-strand conformation with base pairing of the nucleoside carrying the 5′ triphosphate was required. RIG-I activation was impaired by a 3′ overhang at the 5′ triphosphate end. These results define the structure of RNA for full RIG-I activation and explain how RIG-I detects negative-strand RNA viruses that lack long double-stranded RNA but do contain blunt short double-stranded 5′ triphosphate RNA in the panhandle region of their single-stranded genome. [Copyright &y& Elsevier]

Summary 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. [ABSTRACT FROM AUTHOR]