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• W4311468940 abstract "•RIG-I is activated rapidly by small quantities of viral RNA•Signaling relies exclusively on the resident non-induced pool of RIG-I receptors•RIG-I signaling occurs without mass aggregation at the mitochondrial membrane•IFN-induced RIG-I forms signaling-unrelated cytosolic aggregates RIG-I is essential for host defense against viral pathogens, as it triggers the release of type I interferons upon encounter with viral RNA molecules. In this study, we show that RIG-I is rapidly and efficiently activated by small quantities of incoming viral RNA and that it relies exclusively on the constitutively expressed resident pool of RIG-I receptors for a strong antiviral response. Live-cell imaging of RIG-I following stimulation with viral or synthetic dsRNA reveals that RIG-I signaling occurs without mass aggregation at the mitochondrial membrane. By contrast, interferon-induced RIG-I protein becomes embedded in cytosolic aggregates that are functionally unrelated to signaling. These findings suggest that endogenous RIG-I efficiently recognizes viral RNA and rapidly relays an antiviral signal to MAVS via a transient signaling complex and that cellular aggregates of RIG-I have a function that is distinct from signaling. RIG-I is essential for host defense against viral pathogens, as it triggers the release of type I interferons upon encounter with viral RNA molecules. In this study, we show that RIG-I is rapidly and efficiently activated by small quantities of incoming viral RNA and that it relies exclusively on the constitutively expressed resident pool of RIG-I receptors for a strong antiviral response. Live-cell imaging of RIG-I following stimulation with viral or synthetic dsRNA reveals that RIG-I signaling occurs without mass aggregation at the mitochondrial membrane. By contrast, interferon-induced RIG-I protein becomes embedded in cytosolic aggregates that are functionally unrelated to signaling. These findings suggest that endogenous RIG-I efficiently recognizes viral RNA and rapidly relays an antiviral signal to MAVS via a transient signaling complex and that cellular aggregates of RIG-I have a function that is distinct from signaling. RIG-I is a cytosolic innate immune receptor that plays a central role in our ability to respond rapidly to infections by RNA viruses.1Kawai T. Akira S. Antiviral signaling through pattern recognition receptors.J. Biochem. 2007; 141: 137-145Crossref PubMed Scopus (350) Google Scholar,2Goubau D. Deddouche S. Reis e Sousa C. Cytosolic sensing of viruses.Immunity. 2013; 38: 855-869Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar,3Kato H. Sato S. Yoneyama M. Yamamoto M. Uematsu S. Matsui K. Tsujimura T. Takeda K. Fujita T. Takeuchi O. Akira S. Cell type-specific involvement of RIG-I in antiviral response.Immunity. 2005; 23: 19-28Abstract Full Text Full Text PDF PubMed Scopus (1126) Google Scholar,4Thoresen D. Wang W. Galls D. Guo R. Xu L. Pyle A.M. The molecular mechanism of RIG-I activation and signaling.Immunol. Rev. 2021; 304: 154-168Crossref PubMed Scopus (54) Google Scholar This large, multi-domain protein differentiates viral from host RNA by specifically recognizing blunt-ended, double-stranded RNAs containing 5′ di- or tri-phosphates, which are common features of viral RNA genomes and viral replication intermediates.5Schlee M. Roth A. Hornung V. Hagmann C.A. Wimmenauer V. Barchet W. Coch C. Janke M. Mihailovic A. Wardle G. et al.Recognition of 5′ triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus.Immunity. 2009; 31: 25-34Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar,6Goubau D. Schlee M. Deddouche S. Pruijssers A.J. Zillinger T. Goldeck M. Schuberth C. Van der Veen A.G. Fujimura T. Rehwinkel J. et al.Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5′-diphosphates.Nature. 2014; 514: 372-375Crossref PubMed Scopus (396) Google Scholar,7Kohlway A. Luo D. Rawling D.C. Ding S.C. Pyle A.M. Defining the functional determinants for RNA surveillance by RIG-I.EMBO Rep. 2013; 14: 772-779Crossref PubMed Scopus (90) Google Scholar,8Linehan M.M. Dickey T.H. Molinari E.S. Fitzgerald M.E. Potapova O. Iwasaki A. Pyle A.M. A minimal RNA ligand for potent RIG-I activation in living mice.Sci. Adv. 2018; 4: e1701854Crossref PubMed Scopus (67) Google Scholar,9Luo D. Ding S.C. Vela A. Kohlway A. Lindenbach B.D. Pyle A.M. Structural insights into RNA recognition by RIG-I.Cell. 2011; 147: 409-422Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar The binding of RNA viral ligands causes RIG-I to extend its N-terminal caspase activation and recruitment domains (CARDs)10Kowalinski E. Lunardi T. McCarthy A.A. Louber J. Brunel J. Grigorov B. Gerlier D. Cusack S. Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA.Cell. 2011; 147: 423-435Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar into solution, where they are then free to interact with homologous CARDs on the mitochondrial antiviral signaling protein (MAVS).11Seth R.B. Sun L. Ea C.-K. Chen Z.J. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-κB and IRF3.Cell. 2005; 122: 669-682Abstract Full Text Full Text PDF PubMed Scopus (2548) Google Scholar,12Peisley A. Wu B. Xu H. Chen Z.J. Hur S. Structural basis for ubiquitin-mediated antiviral signal activation by RIG-I.Nature. 2014; 509: 110-114Crossref PubMed Scopus (243) Google Scholar The interaction between RIG-I and MAVS is thought to initiate the assembly of a MAVS oligomeric complex13Hou F. Sun L. Zheng H. Skaug B. Jiang Q.X. Chen Z.J. MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response.Cell. 2011; 146: 448-461Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar that stimulates translocation of transcription factors IRF3 and NF-κB14Fitzgerald K.A. McWhirter S.M. Faia K.L. Rowe D.C. Latz E. Golenbock D.T. Coyle A.J. Liao S.M. Maniatis T. IKKε and TBK1 are essential components of the IRF3 signaling pathway.Nat. Immunol. 2003; 4: 491-496Crossref PubMed Scopus (2119) Google Scholar,15Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. Ishii K.J. Takeuchi O. Akira S. IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction.Nat. Immunol. 2005; 6: 981-988Crossref PubMed Scopus (2047) Google Scholar,16Zeng W. Sun L. Jiang X. Chen X. Hou F. Adhikari A. Xu M. Chen Z.J. Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity.Cell. 2010; 141: 315-330Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar into the nucleus, resulting in the production of type I interferons and interferon-stimulated genes (ISGs). Two critical events are needed to initiate the process of RIG-I signaling upon infection: (1) binding of viral RNA to RIG-I, with concomitant release of the CARDs, and (2) formation of CARD-CARD interaction between RIG-I and MAVS. Given the immediate danger posed by viral threats, RIG-I signaling must have the following characteristics: First, it must be highly efficient in sensing the first viral RNAs that have infected the cell. Second, it must be extremely rapid to initiate the cascade of signaling reactions and transcriptional events required for alerting the immune system as quickly as possible. Consistent with the need for speed and efficiency, studies of RIG-I signaling in cellulo have previously shown interferon response to viral infection within 2 h,17Weber M. Gawanbacht A. Habjan M. Rang A. Borner C. Schmidt A.M. Veitinger S. Jacob R. Devignot S. Kochs G. et al.Incoming RNA virus nucleocapsids containing a 5′-triphosphorylated genome activate RIG-I and antiviral signaling.Cell Host Microbe. 2013; 13: 336-346Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar,18Liedmann S. Hrincius E.R. Guy C. Anhlan D. Dierkes R. Carter R. Wu G. Staeheli P. Green D.R. Wolff T. et al.Viral suppressors of the RIG-I-mediated interferon response are pre-packaged in influenza virions.Nat. Commun. 2014; 5: 5645Crossref PubMed Scopus (51) Google Scholar and active RIG-I signaling in vivo has been observed within 2 h after injection of RIG-I-specific RNAs.8Linehan M.M. Dickey T.H. Molinari E.S. Fitzgerald M.E. Potapova O. Iwasaki A. Pyle A.M. A minimal RNA ligand for potent RIG-I activation in living mice.Sci. Adv. 2018; 4: e1701854Crossref PubMed Scopus (67) Google Scholar Unfortunately, previous studies on subcellular behavior of RIG-I and its responses to RNA activation, including available studies on RIG-I localization, have been conducted at very late time points, ranging from 10–40 h after introduction of viral RNA into cells.19Onomoto K. Jogi M. Yoo J.S. Narita R. Morimoto S. Takemura A. Sambhara S. Kawaguchi A. Osari S. Nagata K. et al.Critical role of an antiviral stress granule containing RIG-I and PKR in viral detection and innate immunity.PLoS One. 2012; 7: e43031Crossref PubMed Scopus (268) Google Scholar,20Liu H.M. Loo Y.M. Horner S.M. Zornetzer G.A. Katze M.G. Gale M. The mitochondrial targeting chaperone 14-3-3ε; regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity.Cell Host Microbe. 2012; 11: 528-537Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar,21Esser-Nobis K. Hatfield L.D. Gale M. Spatiotemporal dynamics of innate immune signaling via RIG-I–like receptors.Proc. Natl. Acad. Sci. USA. 2020; 117: 15778-15788Crossref PubMed Scopus (37) Google Scholar These late snapshots of RIG-I signaling, together with ex vivo biochemical and biophysical studies performed with transfected or recombinant protein, have led to a “massive oligomer” model for RIG-I-MAVS signal transmission.22Peisley A. Wu B. Yao H. Walz T. Hur S. RIG-I forms signaling-competent filaments in an ATP-dependent, ubiquitin-independent manner.Mol. Cell. 2013; 51: 573-583Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar According to this model, RNA-stimulated populations of RIG-I migrate en masse to the mitochondrial network or to the mitochondria-associated membrane (MAM) of the ER,20Liu H.M. Loo Y.M. Horner S.M. Zornetzer G.A. Katze M.G. Gale M. The mitochondrial targeting chaperone 14-3-3ε; regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity.Cell Host Microbe. 2012; 11: 528-537Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar,23Horner S.M. Liu H.M. Park H.S. Briley J. Gale Jr., M. Mitochondrial-associated endoplasmic reticulum membranes (MAM) form innate immune synapses and are targeted by hepatitis C virus.Proc. Natl. Acad. Sci. USA. 2011; 108: 14590-14595Crossref PubMed Scopus (364) Google Scholar where RIG-I becomes enmeshed within a large prion-like MAVS aggregate that is required for signal transduction.13Hou F. Sun L. Zheng H. Skaug B. Jiang Q.X. Chen Z.J. MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response.Cell. 2011; 146: 448-461Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar This subcellular structure is thought to be so large that it must ultimately be cleared by autophagy.24Jounai N. Takeshita F. Kobiyama K. Sawano A. Miyawaki A. Xin K.Q. Ishii K.J. Kawai T. Akira S. Suzuki K. Okuda K. The Atg5 Atg12 conjugate associates with innate antiviral immune responses.Proc. Natl. Acad. Sci. USA. 2007; 104: 14050-14055Crossref PubMed Scopus (476) Google Scholar However, unlike other innate immune signaling complexes, such as the inflammasome25Fernandes-Alnemri T. Wu J. Yu J.W. Datta P. Miller B. Jankowski W. Rosenberg S. Zhang J. Alnemri E.S. The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation.Cell Death Differ. 2007; 14: 1590-1604Crossref PubMed Scopus (754) Google Scholar and myddosome,26Latty S.L. Sakai J. Hopkins L. Verstak B. Paramo T. Berglund N.A. Cammarota E. Cicuta P. Gay N.J. Bond P.J. et al.Activation of toll-like receptors nucleates assembly of the MyDDosome signaling hub.eLife. 2018; 7: e31377Crossref PubMed Scopus (65) Google Scholar direct visualization of functional RIG-I signaling complexes has been elusive in cells. Indeed, RIG-I signaling complexes have never been characterized or studied in cellulo within the short time frame that is suggested by in vivo studies of RIG-I function. Not only does RIG-I trigger the initial interferon response upon infection by RNA viruses, it is itself an ISG. Given this behavior, it has been suggested that interferon-induced RIG-I expression can boost the antiviral response by supplying additional receptors that bind viral RNA and amplify RIG-I signaling, creating a late-stage second wave of interferon induction and a positive feedback loop. Indeed, this “priming” of RIG-I signaling by interferon has been proposed to play a central role in antiviral signaling.27Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses.Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3153) Google Scholar,28Sumpter R. Loo Y.M. Foy E. Li K. Yoneyama M. Fujita T. Lemon S.M. Gale M. Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I.J. Virol. 2005; 79: 2689-2699Crossref PubMed Scopus (741) Google Scholar However, naive and unprimed cells must be able to respond to viral infection and trigger a robust immune response or an initial infection would escape detection.17Weber M. Gawanbacht A. Habjan M. Rang A. Borner C. Schmidt A.M. Veitinger S. Jacob R. Devignot S. Kochs G. et al.Incoming RNA virus nucleocapsids containing a 5′-triphosphorylated genome activate RIG-I and antiviral signaling.Cell Host Microbe. 2013; 13: 336-346Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar,29Fay E.J. Aron S.L. Macchietto M.G. Markman M.W. Esser-Nobis K. Gale M. Shen S. Langlois R.A. Cell type- and replication stage-specific influenza virus responses in vivo.PLoS Pathog. 2020; 16: e1008760Crossref PubMed Google Scholar Therefore, the role of downstream, interferon-induced RIG-I expression remains unclear. To address these gaps in our understanding and reveal the dynamic behavior of functional RIG-I molecules as they actively engage in signaling, we measured endogenous RIG-I signaling kinetics and efficiency in response to both synthetic and viral pathogen-associated molecular patterns (PAMPs). We then linked these measurements with direct observation of endogenous RIG-I via fluorescence confocal microscopy in order to observe the receptor distribution during active signaling. Here, we demonstrate that not only is RIG-I signaling both rapid and efficient in cellulo but that the active RIG-I signal is transmitted to MAVS without visible RIG-I accumulation on the mitochondrial network. We observe two separate phases of RIG-I behavior upon activation by viral RNA ligands. The “early phase” involves a burst of RIG-I signaling and IFN induction that is initiated within an hour and which is not accompanied by increases in RIG-I protein expression. The “late phase” of RIG-I induction involves dramatic increases in RIG-I expression but coincides with a decline in signaling and the production of type I interferon. Rather than accumulating at the mitochondria at long time points after RNA induction, RIG-I forms subcellular bodies at other positions within the cytosol, none of which are involved in RNA-stimulated RIG-I signaling. Importantly, we show that blocking interferon-induced RIG-I protein expression does not disrupt robust RNA-induced RIG-I signaling, thereby demonstrating that the resident cytosolic pool of RIG-I is sufficient for a robust antiviral response. Finally, by carefully quantitating the stoichiometry of stimulatory ligands, we show that the RIG-I response requires only a few cytosolic dsRNA molecules to enter the cell in order to initiate strong interferon induction. Collectively, these findings suggest that the RIG-I signaling pathway is a rapid relay that enables a sensitive antiviral response to be efficiently transmitted by a small number of activated RIG-I receptors. We first sought to comprehensively measure endogenous RIG-I signaling kinetics over 24 h in lung epithelial (A549) cells. Cells were stimulated either via transfection of 1 μg/mL stem-loop RNA-14 (SLR14), a RIG-I-specific RNA ligand containing a single RIG-I-binding site, or with 100 haemagglutination units (HA)/mL Sendai virus (SeV), and the completion of RIG-I signaling was determined via type I interferon mRNA transcription with RT-qPCR. In all cases, regardless of RNA ligand identity or delivery method, overall signaling kinetics were strikingly rapid. SLR14 stimulated IFN-β transcription within 1 h (Figure 1A), indicating that RIG-I signaling had already been triggered before this time frame. IFN-β mRNA transcription peaked 3–6 h post-transfection and by 24 h after transfection IFNB mRNA was declining (Figure 1A). With SeV, IFN-β transcription was again initiated in less than 1 h, peaking by 3 h, and declining by 12–24 h after infection (Figure 1A). The relative decline in IFNB mRNA transcription observed at 24 h was almost 8-fold lower for SLR14-induced RIG-I signaling (fold increase of 11,500 at 6 h versus 1,500 at 24 h) and 100-fold lower for SeV-induced response than what was observed at the 3 h peak (fold increase of 29,400 at 3 h versus 343 at 24 h). These data indicate that not only is RIG-I signaling rapidly induced but also is shut off almost as quickly. In addition to stimulating an early interferon response to intracellular dsRNA, RIG-I is itself an ISG, and this induced population of RIG-I molecules has the potential to influence the course of signaling. To monitor the correlation between kinetics of RIG-I gene expression and the kinetics of signaling, we measured both RIG-I mRNA (Figure 1B) and protein expression (Figure 1C) over time. In contrast to the kinetics displayed by RIG-I induction of type I interferon, prior to 3 h post-stimulation, there was almost no change in the levels of RIG-I mRNA or RIG-I protein during this time frame. RIG-I mRNA expression began to increase at 3 h and then rose dramatically at 12–24 h (Figure 1B) to over 100-fold higher than baseline (fold increase of 180 for SLR14 and 110 for SeV). RIG-I protein expression increased more slowly and less dramatically than mRNA expression (Figure 1C). However, even in that case, RIG-I expression continued to increase by 24 h, displaying approximately a 50-fold increase in total RIG-I protein at 24 h after challenge with SLR14. These findings have two important interpretations: first, that the rapid early burst of RIG-I signaling requires no additional RIG-I expression beyond the initial pool of receptors that is circulating in the cytosol prior to infection, and second, that the dramatic increase in RIG-I expression observed at long time points correlates with a decrease in RIG-I signaling, rather than the positive influence that was expected. To examine whether RIG-I signaling was enhanced by interferon-induced RIG-I expression, we pretreated cells with 100 μg/mL cycloheximide for 2 h prior to SLR14 transfection to block translation and ensure that no additional RIG-I protein was expressed during the 24 h time course. Despite the inhibition of induced RIG-I translation, RIG-I signaling kinetics, as reflected in IFNB mRNA expression, did not differ in either speed or magnitude relative to untreated A549 cells during early time points (1–6 h, Figure 1D). This indicates that the resident pool of RIG-I that is present prior to infection is sufficient to produce a strong, rapid antiviral response. Interestingly, at long time points (12–24 h), cycloheximide treatment prevents IFNB mRNA expression from being turned off, as it blocks the translation of enzymes that inhibit IRF3, which is a process independent of RIG-I function. Given the novel finding that RIG-I signaling appears to be disconnected from interferon-stimulated RIG-I expression, it was important to evaluate whether the interferon stimulation we are tracking in this study is entirely reliant on RIG-I signaling. Simultaneous treatment of wild type (WT) and RIG-I−/− A549 cells with SLR14, SeV and OH-SLR14, a stem loop RNA lacking triphosphate, for 6 h demonstrated that RIG-I−/− cells failed to produce any type I interferon (Figure 1E). This confirms that the signaling observed here is RIG-I-dependent and that the kinetics of this process derive entirely from action of endogenous RIG-I molecules. We next sought to directly observe changes in RIG-I subcellular distribution following stimulation with RIG-I-specific ligands using fluorescence confocal microscopy. First, we set out to determine whether the fluorescent tags used in this study will perturb RIG-I signaling. Attachment of a mNeonGreen or HaloTag fluorescent tag to the RIG-I amino-terminus via a flexible linker (Figure S1A) had no effect on RIG-I signaling using the IFN-β-luciferase reporter system (Figure S1B) in HEK293T cells. Specifically, HEK293T cells ectopically expressing either WT-RIG-I, mNG-RIG-I, or Halo-RIG-I produced equivalently strong interferon responses when stimulated with SLR14 containing a 5′ triphosphate and produced almost no interferon when mock-stimulated. Cells lacking the transfected RIG-I construct failed to signal in response to SLR14 treatment, indicating that the stimulated response was generated entirely by the ectopically expressed constructs within these cells. Attachment of an AlexaFluor647 fluorophore to the tetraloop region of SLR14 (Figure S1C), which is located far from the RIG-I binding site, has no effect on the stimulation of RIG-I signaling, again measured by IFN-luciferase response in HEK293T cells (Figure S1D). Therefore, for the first time, both RIG-I and a strong stimulatory RNA ligand could be monitored in live cells without disrupting the signaling pathway. Having confirmed that fluorescent labels on both RIG-I and RNA ligand do not disrupt signaling, we sought to visualize RIG-I interactions with, and responses to, a viral RNA mimic in real time with live-cell fluorescent microscopy. Additionally, labeling of the mitochondrial network with potentiometric dyes (MitoTracker-CMXRos and MitoTracker-DeepRed) enabled us to determine whether RIG-I accumulated at the mitochondrial network following RNA activation. Ectopic expression of HaloTag-RIG-I labeled with OregonGreen in HEK293T cells produced robust RIG-I fluorescence which uniformly fills the cytosol in the absence of RNA stimulation (not shown). Following transfection of 1 μg/mL of SLR14-647 for 24 h, the stimulatory RNA concentrated primarily at local cytoplasmic sites that appear as bright puncta (shown in red, Figure 2A). Despite the clear presence of the strong stimulatory RNA ligand within the cell, the subcellular distribution of RIG-I (green) remains uniform (Figure 2A). Most notably, RIG-I did not visibly accumulate at the mitochondrial network region of the cell (shown in yellow, Figure 2A), and line tracing across the cell shows that although the mitochondrial signal is punctate, RIG-I distribution remains uniform across the cell (Figure 2B). Given that HEK293T cells do not produce an interferon response without ectopic expression of RIG-I (Figure S1B), the absence of any changes in mNG-RIG-I localization was unexpected. This result was doubly surprising considering the large quantities of dsRNA delivered: 100-fold more than the minimum dose needed to stimulate an interferon response (vide infra). To investigate whether this unexpected absence of any RIG-I redistribution was specific to the cell type or type of RNA used, we ectopically expressed mNG-RIG-I in Huh7.5 cells, which lack functional endogenous RIG-I expression,28Sumpter R. Loo Y.M. Foy E. Li K. Yoneyama M. Fujita T. Lemon S.M. Gale M. Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I.J. Virol. 2005; 79: 2689-2699Crossref PubMed Scopus (741) Google Scholar and then infected them with 100 HA/mL SeV (Figure 2C). Similar to our observations in HEK293T cells, introduction of viral RNA did not affect the subcellular distribution of RIG-I at 24 h (Figure 2C) in comparison to uninfected cells. In contrast to previous reports,20Liu H.M. Loo Y.M. Horner S.M. Zornetzer G.A. Katze M.G. Gale M. The mitochondrial targeting chaperone 14-3-3ε; regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity.Cell Host Microbe. 2012; 11: 528-537Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar we did not observe mass migration of RIG-I to the perinuclear region of the cell. Instead, we see RIG-I uniformly distributed through the cell, as demonstrated by line tracing (Figure 2D). To test whether the uniform subcellular distribution of RIG-I during signaling was somehow a product of the plasmid transfection system used to express mNG-RIG-I, we incorporated the fluorescently tagged RIG-I into FlpIn HeLa cells, which express mNG-RIG-I under the control of a TetOn promoter. When these cells were stimulated with 0.1 μg/mL tetracycline and then transfected with 1 μg/mL SLR14-647 for 6 h prior to imaging (Figure 2E), the fluorescently labeled RNA (red) was clearly visible within these cells. Although some colocalization between the SLR14 puncta and RIG-I is observed, no redistribution of RIG-I to the perinuclear region of the cell was observed following stimulation. Finally, A549 cells transiently expressing both pUNO-mNG-RIG-I (green) and pUNO-mCherry-IRF3 (yellow) were either mock infected (Figure 2F) or infected with 100 HA/mL SeV for 24 h (Figure 2G). RIG-I+/IRF3+ A549 cells showed a significant increase in IRF3 nuclear localization following SeV infection compared with mock-infected cells, indicating that RIG-I signaling was active. However, the cytosolic distribution of RIG-I remained uniform in these cells, regardless the of signaling status. This can be clearly seen again via line tracing, in which both unstimulated and actively signaling A549 cells display a uniform distribution of RIG-I throughout the cell (Figure 2H). Throughout the time courses examined in this study (6–24 h), the distribution of overexpressed RIG-I did not change, regardless of the cell type or stimulatory RNA ligand. Despite a clear uptake of the fluorescently labeled dsRNA and direct evidence of active RIG-I signaling via IRF3, there was no large-scale translocation of RIG-I, in contradiction of previously published studies.20Liu H.M. Loo Y.M. Horner S.M. Zornetzer G.A. Katze M.G. Gale M. The mitochondrial targeting chaperone 14-3-3ε; regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity.Cell Host Microbe. 2012; 11: 528-537Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar To determine whether the lack of apparent subcellular RIG-I redistribution was caused by the inherent limitations of ectopic RIG-I expression or whether it represents an actual property of RIG-I signaling, we used CRISPR-Cas9 to fluorescently tag the endogenous RIG-I amino-terminus with mNeonGreen in A549 cells. In this case, we used the same flexible fluorophore linker as we employed in the transiently expressed construct, and we verified that the fluorescently tagged RIG-I was expressed as effectively as WT using western blotting (Figure S2A). In addition to all of the inherent benefits of monitoring endogenously expressed genes, this approach enabled us to remove the overwhelming excess of RIG-I that is produced from transfected constructs, and it enabled us to visualize the distinct phases in endogenous RIG-I signaling observed via qPCR (Figure 1). Addition of the fluorophore on RIG-I had no impact on RIG-I stimulation by SLR14 as measured by qPCR (Figure S2B), indicating that similar to the transfected RIG-I constructs, fluorescent tagging did not disrupt or influence the RIG-I signaling response. Finally, we measured the total mean mNG signal over 24 h following stimulation by both SLR14 and SeV using flow cytometry and noted that total cellular mNG fluorescence increased significantly from 12–24 h, mirroring what was observed of total RIG-I protein expression in unlabeled cells (Figures S2C and S2D). In the absence of dsRNA stimulation (Figure 3A), mNG-RIG-I (green) remained uniformly distributed throughout the cytosol. The relative concentration of RIG-I at the mitochondrial network (labeled with MitoTracker dye, red) was not significantly higher than in regions of the cytosol lacking mitochondria (Figure 3A). Transfected mCherry-IRF3 was completely excluded from the nucleus in the absence of stimulatory RNA (Figure 3A), confirming that signaling is inactive. These results agree with the expectation that, absent stimulation, RIG-I circulates throughout the cytosol, sampling host RNAs and remaining inactive. At 3 h after transfection of mNG-RIG-I A549 cells with 100 ng/mL SLR14-647, the distribution of mNG-RIG-I (green) remained unchanged, with no significant redistribution to the perinuclear region, as seen by line tracing (Figure 3B). In cells ectopically expressing mCherry-IRF3 (yellow), transfection of SLR14 (red) produced a robust translocation of IRF3 to the nucleus (Figure 3B), confirming that cells transfected with SLR14 had fully activated the RIG-I signaling pathway by this early time point and confirming visually what was seen from the qPCR data (Figure 1). Measurement of the distribution of RIG-I from the nucleus to the cell membrane via line traces, depicted in (Figure 3A) and (Figure 3B" @default.
• W4311468940 created "2022-12-26" @default.
• W4311468940 creator A5007909912 @default.
• W4311468940 creator A5028279043 @default.
• W4311468940 creator A5042323609 @default.
• W4311468940 creator A5061985231 @default.
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• W4311468940 date "2023-01-01" @default.
• W4311468940 modified "2023-10-03" @default.
• W4311468940 title "A rapid RIG-I signaling relay mediates efficient antiviral response" @default.
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• W4311468940 doi "https://doi.org/10.1016/j.molcel.2022.11.018" @default.
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