RNAs with 5′-triphosphate (ppp) are detected in the cytoplasm principally by the innate immune receptor Retinoic Acid Inducible Gene-I (RIG-I), whose activation triggers a Type I IFN response. It is thought that self RNAs like mRNAs are not recognized by RIG-I because 5′ppp is capped by the addition of a 7-methyl guanosine (m7G) (Cap-0) and a 2′-O-methyl (2′-OMe) group to the 5′-end nucleotide ribose (Cap-1). Here we provide structural and mechanistic basis for exact roles of capping and 2′-O-methylation in evading RIG-I recognition. Surprisingly, Cap-0 and 5′ppp doublestranded (ds) RNAs bind to RIG-I with nearly identical K d values and activate RIG-I's ATPase and cellular signaling response to similar extents. On the other hand, Cap-0 and 5′ppp single-stranded RNAs did not bind RIG-I and are signaling inactive. Three crystal structures of RIG-I complexes with dsRNAs bearing 5′OH, 5′ppp, and Cap-0 show that RIG-I can accommodate the m7G cap in a cavity created through conformational changes in the helicase-motif IVa without perturbing the ppp interactions. In contrast, Cap-1 modifications abrogate RIG-I signaling through a mechanism involving the H830 residue, which we show is crucial for discriminating between Cap-0 and Cap-1 RNAs. Furthermore, m7G capping works synergistically with 2′-O-methylation to weaken RNA affinity by 200-fold and lower ATPase activity. Interestingly, a single H830A mutation restores both high-affinity binding and signaling activity with 2′-Omethylated dsRNAs. Our work provides new structural insights into the mechanisms of host and viral immune evasion from RIG-I, explaining the complexity of cap structures over evolution.etinoic Acid Inducible Gene-I (RIG-I) is a cytosolic innate immune receptor with the remarkable ability of distinguishing cellular self RNAs from pathogenic nonself RNAs (1, 2). RIG-I belongs to the DExH/D-box family of RNA helicases and has a multidomain architecture with three helicase domains (Hel1, Hel2, and Hel2i) located centrally, flanked by a C-terminal repressor domain (RD) and two N-terminal Caspase Activation and Recruitment Domains (CARDs) (3-7). The helicase and RD are involved in RNA recognition and binding whereas the N-terminal CARDs relay the signal to downstream factors. RIG-I is present in an inactive autoinhibited state in the absence of pathogen associated molecular pattern (PAMP) RNA ligand, but upon PAMP RNA binding, RIG-I gets activated to initiate a cell signaling response ultimately leading to Type I IFN production.RNAs carrying a 5′-triphosphate (5′ppp) moiety and bluntended double-stranded (ds)RNAs are the best characterized PAMP ligands of RIG-I, showing high-affinity binding and robust stimulation of the ATP hydrolysis activity (8-10). Although RIG-I is surrounded by cellular RNAs, they do not activate RIG-I due to posttranscriptional modification of the RNA 5′ ends. The 5′ end of many cellular RNAs like mRNAs is modified with the addition of a 7-methyl guanosine (m7G) connected by a 5′-to-5′ triphosphate bridge to the first nucleotide (C...

The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome. Our cryo-EM structure of the Nsp1-40S ribosome complex shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the structure of the 48S preinitiation complex formed by Nsp1, 40S, and the cricket paralysis virus internal ribosome entry site (IRES) RNA, which shows that it is nonfunctional because of the incorrect position of the mRNA 3′ region. Our results elucidate the mechanism of host translation inhibition by SARS-CoV-2 and advance understanding of the impacts from a major pathogenicity factor of SARS-CoV-2.

Summary RIG-I has a remarkable ability to specifically select viral 5’ppp dsRNAs for activation from a pool of cytosolic self-RNAs. The ATPase activity of RIG-I plays a role in RNA discrimination and activation, but the underlying mechanism was unclear. Using transient state kinetics, we have elucidated the ATPase-driven ‘kinetic proofreading’ mechanism of RIG-I activation and RNA discrimination, akin to DNA polymerases, ribosomes, and T-cell receptors. Even in the autoinhibited state of RIG-I, the C-terminal domain kinetically discriminates against self-RNAs by fast off-rates. ATP-binding facilitates dsRNA engagement, but interestingly makes RIG-I promiscuous, explaining the constitutive signaling by Singleton-Merton syndrome-linked mutants that bind ATP without hydrolysis. ATP hydrolysis dissociates self-RNAs faster than 5’ppp dsRNA, but more importantly, drives RIG-I oligomerization through translocation, that we show is regulated by helicase motif-IVa. RIG-I translocates directionally from dsRNA-end into stem-region and 5’ppp-end “throttles” translocation to provide a mechanism for threading and building a signaling-active oligomeric complex.