A Conserved Histidine in the RNA Sensor RIG-I Controls Immune Tolerance to N1-2′O-Methylated Self RNA

Schuberth-Wagner, Christine; Ludwig, János; 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

doi:10.1016/j.immuni.2015.06.015

Cited by 236 publications

(239 citation statements)

References 37 publications

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“…4D), this observation suggests that H830A RIG-I is activated by cellular RNAs. A recent study (23) similarly showed that cellular RNAs activate H830A and that the activity of Cap-1 methyltansferase enzyme, MTr1, suppresses WT RIG-I activation by endogenous RNAs, but showed no such effect on H830A. Taken together, the biochemical, structural, and cell-based studies indicate that the H830 residue is crucial for discriminating between Cap-0 and Cap-1 RNAs.…”

Section: Rig-i Accommodates the M7g Cap Without Perturbation Of The Pppmentioning

confidence: 86%

“…In summary, before this and another study (23), it was hypothesized that discrimination of self from nonself RNA was due to the presence of the m7G moiety, which was thought to sterically hinder binding to RIG-I. This presumption is not true because dsRNAs with m7G show comparable binding affinity, ATPase, and cell signaling activity as dsRNAs with a 5′ triphosphate.…”

Section: Rig-i Accommodates the M7g Cap Without Perturbation Of The Pppmentioning

confidence: 97%

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Structural basis for m7G recognition and 2′-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I

Devarkar

Wang

Miller

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

276

341

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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...

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Section: Rig-i Accommodates the M7g Cap Without Perturbation Of The Pppmentioning

confidence: 86%

Section: Rig-i Accommodates the M7g Cap Without Perturbation Of The Pppmentioning

confidence: 97%

Structural basis for m7G recognition and 2′-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I

Devarkar

Wang

Miller

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

276

341

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“…The N-7-methylation of the viral RNA cap by ZIKV NS5-MTase allows recognition by the translation initiation factor eIF4E and stimulation of translation into viral proteins (8). On the other hand, 2=-O-methylation of the cap is expected to mask the presence of exogenous viral RNAs from host cell sensors such as RIG-I and MDA5 (9)(10)(11), which induce the production of interferons. In addition, 2=-O-methylation of viral RNA is thought to prevent translation restriction by interferon-stimulated genes (ISGs) such as the IFIT-1 gene (12,13).…”

Section: Discussionmentioning

confidence: 99%

“…The N-7 methylation of the cap structure is essential for RNA stability and stimulates their translation into viral protein by recognizing the translation initiation factor eIF4E (8). The 2=-O-methylation of the cap structure was demonstrated to protect viral RNA from being recognized by host cell sensors such as RIG-I and MDA5 that stimulate the production of interferons (9)(10)(11). In turn, interferon-stimulated genes, such as IFIT-1, can detect miscapped RNA and restrict their translation into proteins (12,13).…”

mentioning

confidence: 99%

Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives

Coutard

Barral

Lichière

et al. 2017

J Virol

118

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The Flavivirus Zika virus (ZIKV) is the causal agent of neurological disorders like microcephaly in newborns or Guillain-Barre syndrome. Its NS5 protein embeds a methyltransferase (MTase) domain involved in the formation of the viral mRNA cap. We investigated the structural and functional properties of the ZIKV MTase. We show that the ZIKV MTase can methylate RNA cap structures at the N-7 position of the cap, and at the 2=-O position on the ribose of the first nucleotide, yielding a cap-1 structure. In addition, the ZIKV MTase methylates the ribose 2=-O position of internal adenosines of RNA substrates. The crystal structure of the ZIKV MTase determined at a 2.01-Å resolution reveals a crystallographic homodimer. One chain is bound to the methyl donor (S-adenosyl-L-methionine [SAM]) and shows a high structural similarity to the dengue virus (DENV) MTase. The second chain lacks SAM and displays conformational changes in the ␣X ␣-helix contributing to the SAM and RNA binding. These conformational modifications reveal a possible molecular mechanism of the enzymatic turnover involving a conserved Ser/Arg motif. In the second chain, the SAM binding site accommodates a sulfate close to a glycerol that could serve as a basis for structure-based drug design. In addition, compounds known to inhibit the DENV MTase show similar inhibition potency on the ZIKV MTase. Altogether these results contribute to a better understanding of the ZIKV MTase, a central player in viral replication and host innate immune response, and lay the basis for the development of potential antiviral drugs. IMPORTANCE The Zika virus (ZIKV) is associated with microcephaly in newborns,and other neurological disorders such as Guillain-Barre syndrome. It is urgent to develop antiviral strategies inhibiting the viral replication. The ZIKV NS5 embeds a methyltransferase involved in the viral mRNA capping process, which is essential for viral replication and control of virus detection by innate immune mechanisms. We demonstrate that the ZIKV methyltransferase methylates the mRNA cap and adenosines located in RNA sequences. The structure of ZIKV methyltransferase shows high structural similarities to the dengue virus methyltransferase, but conformational specificities highlight the role of a conserved Ser/Arg motif, which participates in RNA and SAM recognition during the reaction turnover. In addition, the SAM binding site accommodates a sulfate and a glycerol, offering structural information to initiate structure-based drug design. Altogether, these results contribute to a better understanding of the Flavivirus methyltransferases, which are central players in the virus replication.KEYWORDS methyltransferase, RNA processing, Zika virus, flavivirus, protein structure-function

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“…In this issue of Immunity, Schuberth-Wagner et al (2015) show that a histidine residue in the RNA binding pocket of RIG-I sterically excludes the cap1 structure of self RNA, thereby preventing downstream activation.…”

mentioning

confidence: 98%

A Sense of Self: RIG-I’s Tolerance to Host RNA

Schoggins

2015

Immunity

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A Conserved Histidine in the RNA Sensor RIG-I Controls Immune Tolerance to N1-2′O-Methylated Self RNA

Cited by 236 publications

References 37 publications

Structural basis for m7G recognition and 2′-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I

Structural basis for m7G recognition and 2′-O-methyl discrimination in capped RNAs by the innate immune receptor RIG-I

Zika Virus Methyltransferase: Structure and Functions for Drug Design Perspectives

A Sense of Self: RIG-I’s Tolerance to Host RNA

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