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Spatiotemporal dynamics of innate immune signaling via RIG-I-like receptors.

ABSTRACT: RIG-I, MDA5, and LGP2 comprise the RIG-I-like receptors (RLRs). RIG-I and MDA5 are essential pathogen recognition receptors sensing viral infections while LGP2 has been described as both RLR cofactor and negative regulator. After sensing and binding to viral RNA, including double-stranded RNA (dsRNA), RIG-I and MDA5 undergo cytosol-to-membrane relocalization to bind and signal through the MAVS adaptor protein on intracellular membranes, thus directing downstream activation of IRF3 and innate immunity. Here, we report examination of the dynamic subcellular localization of all three RLRs within the intracellular response to dsRNA and RNA virus infection. Observations from high resolution biochemical fractionation and electron microscopy, coupled with analysis of protein interactions and IRF3 activation, show that, in resting cells, microsome but not mitochondrial fractions harbor the central components to initiate innate immune signaling. LGP2 interacts with MAVS in microsomes, blocking the RIG-I/MAVS interaction. Remarkably, in response to dsRNA treatment or RNA virus infection, LGP2 is rapidly released from MAVS and redistributed to mitochondria, temporally correlating with IRF3 activation. We reveal that IRF3 activation does not take place on mitochondria but instead occurs at endoplasmic reticulum (ER)-derived membranes. Our observations suggest ER-derived membranes as key RLR signaling platforms controlled through inhibitory actions of LGP2 binding to MAVS wherein LGP2 translocation to mitochondria releases MAVS inhibition to facilitate RLR-mediated signaling of innate immunity.

SUBMITTER: Esser-Nobis K

PROVIDER: S-EPMC7354926 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Spatiotemporal dynamics of innate immune signaling via RIG-I-like receptors.

Esser-Nobis Katharina K Hatfield Lauren D LD Gale Michael M

Proceedings of the National Academy of Sciences of the United States of America 20200622 27

RIG-I, MDA5, and LGP2 comprise the RIG-I-like receptors (RLRs). RIG-I and MDA5 are essential pathogen recognition receptors sensing viral infections while LGP2 has been described as both RLR cofactor and negative regulator. After sensing and binding to viral RNA, including double-stranded RNA (dsRNA), RIG-I and MDA5 undergo cytosol-to-membrane relocalization to bind and signal through the MAVS adaptor protein on intracellular membranes, thus directing downstream activation of IRF3 and innate imm ...[more]

PMID: 32571931

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Project description:Invading pathogen nucleic acids are recognized and bound by cytoplasmic (retinoic acid-inducible gene I [RIG-I]-like) and membrane-bound (Toll-like) pattern recognition receptors to activate innate immune signaling. Modified nucleotides, when present in RNA molecules, diminish the magnitude of these signaling responses. However, mechanisms explaining the blunted signaling have not been elucidated. In this study, we used several independent biological assays, including inhibition of virus replication, RIG-I:RNA binding assays, and limited trypsin digestion of RIG-I:RNA complexes, to begin to understand how RNAs containing modified nucleotides avoid or suppress innate immune signaling. The experiments were based on a model innate immune activating RNA molecule, the polyU/UC RNA domain of hepatitis C virus, which was transcribed in vitro with canonical nucleotides or with one of eight modified nucleotides. The approach revealed signature assay responses associated with individual modified nucleotides or classes of modified nucleotides. For example, while both N-6-methyladenosine (m6A) and pseudouridine nucleotides correlate with diminished signaling, RNA containing m6A modifications bound RIG-I poorly, while RNA containing pseudouridine bound RIG-I with high affinity but failed to trigger the canonical RIG-I conformational changes associated with robust signaling. These data advance understanding of RNA-mediated innate immune signaling, with additional relevance for applying nucleotide modifications to RNA therapeutics. The innate immune system provides the first response to virus infections and must distinguish between host and pathogen nucleic acids to mount a protective immune response without activating autoimmune responses. While the presence of nucleotide modifications in RNA is known to correlate with diminished innate immune signaling, the underlying mechanisms have not been explored. The data reported here are important for defining mechanistic details to explain signaling suppression by RNAs containing modified nucleotides. The results suggest that RNAs containing modified nucleotides interrupt signaling at early steps of the RIG-I-like innate immune activation pathway and also that nucleotide modifications with similar chemical structures can be organized into classes that suppress or evade innate immune signaling steps. These data contribute to defining the molecular basis for innate immune signaling suppression by RNAs containing modified nucleotides. The results have important implications for designing therapeutic RNAs that evade innate immune detection.

Dataset Information

Spatiotemporal dynamics of innate immune signaling via RIG-I-like receptors.

Publications

Spatiotemporal dynamics of innate immune signaling via RIG-I-like receptors.

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