Spatiotemporal Dynamics of Innate Immune Signaling Via RIG-I–Like Receptors

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

Katharina Esser-Nobisa, Lauren D. Hatfielda, and Michael Gale Jra,1

aCenter for Innate Immunity and Immune Disease, Department of Immunology, University of Washington School of Medicine, Seattle, WA 98109

Edited by Adolfo Garcia-Sastre, Icahn School of Medicine at Mount Sinai, New York, NY, and approved May 21, 2020 (received for review December 16, 2019) RIG-I, MDA5, and LGP2 comprise the RIG-I–like receptors (RLRs). interaction leading to complex formation with MAVS that facili- RIG-I and MDA5 are essential pathogen recognition receptors sens- tates activation of downstream transcription factors, including IFN ing viral infections while LGP2 has been described as both RLR regulatory factor (IRF) 3 and NF-κB (2, 3). This process induces cofactor and negative regulator. After sensing and binding to viral the expression of target genes, including type 1 and 3 IFNs. IFNs RNA, including double-stranded RNA (dsRNA), RIG-I and MDA5 un- are secreted cytokines that signal through their cognate recep- dergo cytosol-to-membrane relocalization to bind and signal tor both locally and systemically to direct the expression of IFN- through the MAVS adaptor protein on intracellular membranes, stimulated genes (ISGs) whose products restrict viral replication thus directing downstream activation of IRF3 and innate immu- and spread, and also mediate immune modulatory actions leading nity. Here, we report examination of the dynamic subcellular lo- to immune polarization and systemic immune activation (2, 5). calization of all three RLRs within the intracellular response to Governance of this RLR signaling program that underlies immune dsRNA and RNA virus infection. Observations from high resolution activation is critical to protect against immune pathology and biochemical fractionation and electron microscopy, coupled with autoimmunity while suppressing virus infection (6, 7). analysis of protein interactions and IRF3 activation, show that, in Biochemical and in vitro studies have shown that LGP2 me- resting cells, microsome but not mitochondrial fractions harbor the diates suppression of RIG-I signaling (8–11) while it can also serve central components to initiate innate immune signaling. LGP2 in- as a cofactor that facilitates MDA5 signaling (12–15). Moreover, teracts with MAVS in microsomes, blocking the RIG-I/MAVS inter- studies of mice lacking LGP2 expression or LGP2 RNA binding action. Remarkably, in response to dsRNA treatment or RNA virus function have diametrically shown that LGP2 functions as an es- infection, LGP2 is rapidly released from MAVS and redistributed to

sential cofactor of RLR signaling or as a negative regulator of IMMUNOLOGY AND INFLAMMATION mitochondria, temporally correlating with IRF3 activation. We re- RLR actions in vivo in response to various RNA virus infections veal that IRF3 activation does not take place on mitochondria but (16, 17). While the RLRs are cytosolic proteins, they undergo instead occurs at endoplasmic reticulum (ER)-derived membranes. redistribution among intracellular membranes to mediate innate Our observations suggest ER-derived membranes as key RLR sig- immune signaling after PAMP binding (18–20). Notably, most naling platforms controlled through inhibitory actions of LGP2 RNA viruses replicate their genome within the cytoplasm in col- binding to MAVS wherein LGP2 translocation to mitochondria re- laboration with intracellular membranes, often derived from the leases MAVS inhibition to facilitate RLR-mediated signaling of endoplasmic reticulum (ER), to build up the replication com- innate immunity. partments that harbor viral RNA and proteins (21). Viral PAMP recognition and initiation of innate immune signaling by the RLRs MAVS | LGP2 | RIG-I | IRF3 | innate immunity thus relies on their dynamic redistribution in virus-infected cells (18–20, 22). As such, the spatiotemporal dynamics of intracellular nnate immunity provides our first line of antiviral defense. redistribution across virus infection likely could explain the IDuring acute virus infection, the innate immune response is triggered first within the infected cell when viral macromolecules Significance known as pathogen associated molecular patterns (PAMPs) are recognized as nonself by host cell pathogen recognition receptors RIG-I–like receptors (RLRs) direct innate immunity as our first (PRRs). Viral nucleic acid represents a major PAMP wherein line of defense against RNA virus infections. Mitochondria cytosolic viral DNA, or viral RNA marked with nonself motifs, were previously established as the platform for RLR-mediated are recognized by specific PRRs. PAMP recognition and binding innate immune signaling, based upon crude mitochondrial by PRRs trigger intracellular signaling that directs innate immune fractionation studies. By using high resolution subcellular frac- activation and induction of antiviral defenses (1, 2). – tionation separating mitochondria from other organelles, elec- The retinoic acid inducible gene-I like receptor (RLR) family tron microscopy, and analysis of spatiotemporal RLR dynamics, of RNA helicases are cytosolic PRRs. Retinoic acid inducible we show that activation and regulation of innate immune sig- gene-I (RIG-I) is the charter member of the RLR family that naling do not take place on mitochondria but instead occur at also includes the melanoma differentiation antigen 5 (MDA5) membranes derived from the endoplasmic reticulum. Our data and laboratory of genetics and physiology 2 (LGP2) proteins. argue against the classical view of the spatial organization of RIG-I and MDA5 function as PRRs to recognize most RNA innate immune signaling to reveal insights into RLR signaling viruses, thus serving to induce innate immunity for protection dynamics and regulation by LGP2. against infection (2, 3). In particular, RNA viruses represent the largest class of contemporary and emerging human pathogens Author contributions: K.E.-N. and M.G. designed research; K.E.-N. performed research; (4). The RLR proteins are expressed at low basal levels in most L.D.H. contributed new reagents/analytic tools; K.E.-N. analyzed data; and K.E.-N., L.D.H., nucleated cells to facilitate surveillance for viral PAMPs and are and M.G. wrote the paper. induced to higher level expression in response to interferon The authors declare no competing interest. (IFN). Whereas RIG-I and MDA5 contain tandem amino-terminal This article is a PNAS Direct Submission. caspase activation and recruitment domains (CARDs) that mediate Published under the PNAS license. signaling interaction with the adaptor protein mitochondrial anti- 1To whom correspondence may be addressed. Email: [email protected]. viral signaling (MAVS), LGP2 lacks CARDs (2). In response to This article contains supporting information online at https://www.pnas.org/lookup/suppl/ binding to PAMP RNA produced during viral RNA replication, doi:10.1073/pnas.1921861117/-/DCSupplemental. RIG-I and MDA5 undergo conformation change and cofactor

www.pnas.org/cgi/doi/10.1073/pnas.1921861117 PNAS Latest Articles | 1of11 Downloaded by guest on September 30, 2021 variable functions ascribed to LGP2 (8–11, 13, 16, 17, 23–25), binding to MAVS, blocking an interaction between RIG-I and as well as RIG-I and MDA5, for differential control of RLR MAVS, and thereby preventing IRF3 activation. We show that actions. activated IRF3 itself is absent from mitochondria but enriched in A classical model of RLR-mediated innate immune signaling MIC and cytosolic fractions, showing that IRF3 activation does by RIG-I and MDA5 implies that, in response to PAMP rec- not take place on the OMM but instead occurs at microsomal ognition, the RLRs translocate from the cytosol to bind to the membranes including ER, and MAM, reflecting the location of central adaptor MAVS on the outer mitochondrial membrane virus replication and high RNA PAMP concentration. (OMM) (2). In cell-free models, this interaction triggers prion- like oligomerization of MAVS (26). RIG-I or MDA5 binding to Results MAVS imparts the formation of a high mass MAVS signal- Subcellular Fractionation Provides Improved Resolution of Protein osome, which involves TNF receptor-associated factor (TRAF) Localization in Relation to Mitochondria and Their Associated proteins and MAVS phosphorylation by TANK-binding kinase 1 Membranes. The classical view of innate immune signaling via (TBK1) or inhibitor of kappa-B kinase (IKK) to allow binding of RLRs and MAVS (illustration depicted in SI Appendix, Fig. the transcription factor IRF3 and other signaling proteins with S1A), positioning mitochondria at the center of IRF3 activation, MAVS (26–28). In unstimulated cells, IRF3 exists as non- is based on analyses by confocal microscopy or crude mito- phosphorylated and hypophosphorylated monomeric protein. chondrial fractionation (39, 40). These techniques do not pro- Upon binding to MAVS, TBK1- or IKKe-mediated phosphory- vide a sufficient resolution to distinguish between mitochondria lation of IRF3 occurs on Serine residues (S386, S396) and en- and MAMs or other organelles, such as peroxisomes that have ables its dimerization, nuclear translocation, and induction of more recently been implicated in RLR signaling (22, 33). We antiviral target gene expression (29–32). MAVS possesses a first evaluated the distribution of MAVS, mitochondria, and ER membrane anchor motif that places it on the OMM while it has by confocal microscopy, analyzing MAVS, TOM20 (mitochon- also been shown to localize to peroxisomes and mitochondria- drial outer membrane marker) and PDIA3 (ER marker) in A549 associated membranes (MAMs), the latter of which represent human lung epithelial cells and PH5CH8 immortalized human ER membranes tethered to the OMM (22, 33). In addition to hepatocytes (Fig. 1A and SI Appendix, Fig. S1B). We found a the OMM, peroxisomes and MAMs have been shown to play a strong overlap of the protein patterns in both cell lines. This role as RLR innate immune signaling platforms via MAVS (22, spatial overlap in fluorescent signals was confirmed by analysis of 33). However, the dynamics of LGP2 and its actions in RLR line profiles and zooming into selected areas of the cytoplasm signaling among these and other cellular compartments is not (Fig. 1A and SI Appendix, Fig. S1B, yellow arrow and enlarged known. views of boxed area). Thus, a classical confocal microscopy ap- The intracellular landscape of mammalian cells is character- proach cannot fully resolve the subcellular structures implicated ized as an organellar mesh in which organelles are apposed and in RLR signaling. To study the spatiotemporal dynamics of in- their membranes connected via protein bridges, with a typical nate immune signaling, we examined the immunocompetent cell distance of 10 to 30 nm between the membrane interfaces (34, line A549 that demonstrates intact RLR-MAVS signaling in 35). The ER is connected this way to most organelles and is response to RNA virus infection as an epithelial cell experi- heavily involved in the regulation of interorganelle communica- mental model (42). Using transmission electron microscopy tion, morphology, and trafficking (34). Biochemically, the ER (TEM), we first examined the distribution of ER and mito- can be isolated as part of the microsome (MIC) fraction which chondria in A549 cells. TEM micrographs show the close ap- contains vesicles derived from intracellular membranes (36). position of ER and OMM, with an approximate distance of Recent work has shown that mitochondria are also in contact 20 nm between these two subcellular compartments (43) (Fig. with most organelles, including endosomes, lysosomes, peroxi- 1B). A part of the ER appears to be linked to the OMM via somes, and the ER (35). As noted above, the ER membranes membrane connections (Fig. 1 B, Bottom), further defining the that are connected to the OMM constitute the MAM, which can MAM as a subdomain of the ER (38). These results confirm the be visualized by electron microscopy (EM) and advanced mi- close apposition of mitochondria and ER with MAM–OMM croscopy techniques or studied by subcellular fractionation (34, linkage. We therefore employed a high resolution subcellular 37, 38). However, conventional confocal microscopy (resolution fractionation scheme to separate mitochondria from their asso- limit 200 nm) or crude mitochondrial fractions that contain mi- ciated membranes and isolate mitochondria (Mito), MAMs, tochondria and other organelles do not provide the resolution microsomes (MICs), and cytosol (Cyto) from cultured cells (Fig. required to distinguish between mitochondria and MAMs or 1C) (22, 41). A549 cells were subjected to this subcellular frac- other organelles. Earlier studies reported that the OMM was the tionation, and the obtained fractions were analyzed for various central platform of RLR-MAVS signaling, but these observa- cellular markers to control for their enrichment (Fig. 1D and SI tions were based on low resolution biochemical separation and Appendix, Fig. S1 C and D). Immunoblot analyses demonstrate imaging of RLR-MAVS localization, nor did these studies con- that the MIC fraction was enriched for ER (Calnexin, PDIA3, duct parallel examination of all three RLRs (39, 40). Thus, we and FACL4) and ribosomes (S6 ribo) and additionally contained sought to assess RLR dynamics, including LGP2 function, using Golgi (Golgin97), endosomes (EEA1), and lysosomes (Lamp1) a combination of high resolution subcellular fractionation (41), while the MAM fraction primarily consisted of ER (Calnexin, electron microscopy, and RLR expression with LGP2 mutational PDIA3, and FACL4) but also contained peroxisomes (Catalase), analyses to assess the spatiotemporal events taking place during in agreement with an interaction between the OMM and per- innate immune signaling across organelles. oxisomes (35). Importantly, the Mito fraction proved to be highly Here, we report that all major components required for in- enriched for mitochondrial markers (SOD2; CoxIV [inner mi- nate immune signaling, including RLRs, MAVS, TBK1, TRAF, tochondrial membrane]; CytC [intermembrane space]; VDAC TRIM25, and IRF3, were present in the MIC fraction in absence [OMM]) while the Cyto fraction was marked with α-tubulin. of stimulation. We found that LGP2 was constitutively bound to Catalase, Golgin97, and EEA1, proteins linked with peroxi- MAVS in the MIC fraction but that activation of RLR signal- somes, golgi, and endosomes, were also found in the cytosol. ing by treatment of cells with double-stranded RNA (dsRNA) Mitofusin 1 (MFN1) was found to be enriched in the mito- resulted in release of LGP2 from MAVS coincident with IRF3 chondrial fraction. Both MAM and mitochondrial fractions activation and LGP2 translocation to mitochondria. Functional contained MFN2 in line with its known localization as a MAM analysis of wild-type and mutant LGP2 indicates that LGP2 acts and OMM-linked protein (22, 38) while Caveolin-1 was primarily as a negative feedback regulator of RLR signaling, possibly by detected in MAM as reported earlier (44). Importantly, MAVS

2of11 | www.pnas.org/cgi/doi/10.1073/pnas.1921861117 Esser-Nobis et al. Downloaded by guest on September 30, 2021 A TOM20 DAPI B MAVS PDIA3 PH5CH8 2500 A549 mmaxmaxIPaxxIPIP 2000 * * 1500 * * 1000 intensity 500 * ER 0 0510 Distance [μm] N MAVS PDIA3 TOM20 *

ER C Cell homogenate D 600xg, 2x 5min A549

Nuclei + Cytosol + unbroken cells Organelles 10,400xg, 10min

crude Cytosol + Mitochondria Membranes WCL MIC MAM Mito Cyto Crude Mito 10.4k Homogenization 10,400xg, 2x 10min MAVS Percoll gradient 95,000xg, 65min 100,000xg, 60min CoxIV

MAM Mitochondria Cytosol Microsomes MFN2 6,300xg, 2x 10min 6,300xg, (Cyto) (MIC) Calnexin 100,000xg, 60min 2x 10min PDIA3 purified purified Catalase IMMUNOLOGY AND INFLAMMATION MAM Mitochondria Golgin97 (MAM) (Mito) EEA1

Fig. 1. Subcellular fractionation provides improved resolution of protein localization in relation to mitochondria and their associated membranes. (A) Pattern of MAVS, ER, and mitochondria in PH5CH8 cells. Cells were stained with antibodies directed against MAVS, PDIA3 (ER-marker), and TOM20 (outer mitochondrial membrane marker) and analyzed on a Nikon Eclipse Ti confocal microscope. Merged images are derived from maximum intensity projectionsof z-stacks. Enlarged views of the boxed areas are shown next to the merge. The graph depicts the intensity profile for the indicated markers along the yellow line shown in the merged image. Depicted are representative images of two independent experiments (n = 2). (Scale bar: 10 μm.) (B) Routine EM imaging of ER and mitochondria in A549 cells at magnifications of 10,000, 25,000, and 50,000. (Scale bars: 200 nm, 100 nm, 50 nm, respectively.) N, nucleus; asterisks, mitochondria; ER, endoplasmic reticulum; arrowheads point to mitochondria-associated ER membrane (MAM). (C) Schematic presentation of subcellular fractionation protocol used to isolate MAM, mitochondria (Mito), microsomes (MIC), and cytosol (Cyto). (D) A549 cells were subjected to subcellular fractionation. The distribution of various compartmental marker proteins was analyzed by Western blot. CoxIV, inner mitochondria; MFN2, MAM + mitochondria; Calnexin/PDIA3, ER; Catalase, peroxisomes; Golgin97, Golgi; EEA1, α-tubulin, cytosol. WCL, whole cell lysate.

was detected in all three membrane fractions: MIC, MAM, and nontreated cells and accumulated in the membrane fraction fol- Mito (Fig. 1D). Thus, the following proteins are used as quality lowing dsRNA treatment. Thus, LGP2 has distinct membrane control markers throughout this study: Calnexin, ER marker, association dynamics compared to RIG-I and MDA5. enriched in MIC and MAM; MFN2, mitofusin 2, enriched in To evaluate the dynamics of RLRs during innate immune MAM and Mito; CoxIV, inner mitochondrial membrane, enriched signaling, we subjected A549-RML cells to dsRNA treatment, in Mito; and α-tubulin, enriched in cytosol. followed by subcellular fractionation. IRF3 activation and the distribution of RLRs in MIC, MAM, Mito, and Cyto fractions dsRNA Imparts Dynamic Localization of LGP2 and Regulated were determined through 40 h of dsRNA treatment (Fig. 2 A–E). Interaction with MAVS. To achieve reliable detection of RLR In the absence of treatment, all RLRs were absent from Mito but proteins, we used lentiviral vectors to generate cell lines stably present in MIC and Cyto while LGP2 was also basally present in expressing RIG-I, MDA5, and LGP2 (RML) (SI Appendix, Fig. MAM. Strikingly, LGP2 accumulated in Mito at all time points S2B). To stimulate RLR-MAVS mediated IRF3 activation, following dsRNA treatment while RIG-I accumulated in MAM A549-RML cells were treated with dsRNA via poly inosine:cy- and Mito over the time course (Fig. 2 B–F). Compared to RIG-I, tosine (polyI:C) transfection or were infected with SeV, a neg- the amount of MDA5 increased in MAM and Mito at later time ative sense RNA virus that activates RIG-I (3). When treated points while distribution of MAVS, TRIM25, and the specific with dsRNA or infected with SeV, A549-RML cells responded to organelle markers did not change (SI Appendix, Fig. S3A). A undergo innate immune activation marked by IRF3 phosphory- dsRNA-induced enrichment of LGP2 in Mito was also revealed lation and expression of the IRF3-target gene, IFIT1 (SI Appendix, in PH5CH8-RML cells, confirming that dsRNA triggers the re- Fig. S2 C–F). A membrane flotation assay and immunoblot distribution of LGP2 from MAM to Mito (SI Appendix,Fig.S3B). analyses of the recovered fractions from A549-RML cells revealed To further assess the spatial distribution of endogenous RLRs, that LGP2 was basally abundant in the membrane fraction, even parent PH5CH8 cells were pretreated for 24 h with IFN-β to in- in the absence of stimulation (SI Appendix,Fig.S2G and H), duce RLR expression before dsRNA treatment to thereby increase while RIG-I and MDA5 were present in cytosolic fractions in endogenous RLR abundance. Endogenous LGP2 was found in the

Esser-Nobis et al. PNAS Latest Articles | 3of11 Downloaded by guest on September 30, 2021 ABA549 RML CA549 RML Mock poly(I:C) 5h Mock poly(I:C) 10h A549 RML WCL MIC MAM Mito Cyto WCL MIC MAM Mito Cyto WCL MIC MAM Mito Cyto WCL MIC MAM Mito Cyto

Mock 5 10 15 20 40 poly(I:C) 1ug/ml RIG-I RIG-I IRF3 S386 MDA5 ◄ MDA5 IRF3 LGP2 LGP2 IFIT1 MAVS MAVS MAVS MFN2 MFN2 Actin Calnexin Calnexin CoxIV CoxIV α-tubulin α-tubulin Endogenous RLRs PH5CH8 D A549 RML EGA549 RML Mock poly(I:C) 20h Mock poly(I:C) 40h untreated IFNb IFNb TNFa WCL MIC MAM Mito Cyto WCL MIC MAM Mito Cyto WCL MIC MAM Mito Cyto WCL MIC MAM Mito Cyto RIG-I RIG-I RIG-I MDA5 ◄ MDA5 MDA5 LGP2 LGP2 Actin LGP2 IFNb MAVS MAVS IFNb, 24h poly(I:C) 5h MFN2 MFN2 Calnexin Calnexin

CoxIV WCL MIC MAM Mito Cyto WCL MIC MAM Mito Cyto CoxIV α-tubulin RIG-I* α-tubulin RIG-I F LGP2 RIG-I MDA5 MDA5 MIC enriched LGP2 medium Calnexin MAM low CoxIV Mito α-tubulin 0 5 10 20 40 0 5 10 20 40 0 5 10 20 40 h p.t. I H A549 A549 RML MAVS IP, A549 RML -+-+-+ - + poly(I:C), 20h CHX poly(I:C)/CHX

0 0 10 10 25 25 50 50 CHX ug/ml, 19h

RIG-I Input Eluate WCL MIC MAM Mito Cyto WCL MIC MAM Mito Cyto Input Eluate IRF3 S386 RIG-I IRF3 -+ -+poly(I:C) 5h IFIT1 LGP2 Actin MAVS IgG ctrl MIC IgG ctrl MIC MIC MAM MIC MAM MIC MAM MIC MAM MFN2 LGP2 Calnexin MAVS CoxIV Calnexin α-tubulin

Fig. 2. Spatiotemporal dynamics of RLRs upon dsRNA stimulation. (A) Kinetics of innate immune signaling after poly(I:C) transfection of A549-RML cells. Cells were lysed at the indicated time points and analyzed by Western blot. Depicted is one representative blot out of two independent experiments (n = 2). (B–E) Subcellular fractionation of A549-RML cells after 5 h (B), 10 h (C), 20 h (D), and 40 h (E) of poly(I:C) transfection. Fractions were analyzed by Western blot using antibodies directed against RLRs, MAVS, or marker proteins (MFN2, MAM + mitochondria; Calnexin, ER; CoxIV, inner mitochondria; α-tubulin, Cytosol). A total of eight independent fractionations comprise this set of experiments; per each time point, two fractionations were performed. (F) Graphical summary of observations made in B–E.(G) Analysis of endogenous RLRs. (Upper) PH5CH8 cells were treated with 500 IU/mL IFN-β or IFN-β and 10 ng/mL TNF-α to detect expression of endogenous RLRs. (Lower) PH5CH8 cells were treated with IFN-β (500 IU/mL, 24 h) and either mock transfected or transfected with 1 μg/mL poly(I:C) to study dynamics of endogenous RLRs. Note that IFN treatment seems to affect separation of MAM and mitochondria. Depicted is one representative out of two independent fractionations. (H, Left) A549 cells were transfected with poly(I:C) followed by treatment with the indicated concentrations of Cycloheximide (CHX) 1 h posttransfection (p.t.). Then, 20 h after poly(I:C) stimulation, cell lysates were harvested and analyzed by Western blot to monitor induction of RIG-I protein expression. (H, Right) A549-RML cells were mock transfected or transfected with 1 μg/mL poly(I:C) and treated with 10 mg/mL CHX at 1 h p.t. Then, 16 h after poly(I:C) stimulation, cells were subjected to subcellular fractionation. Fractions were analyzed by Western blot. (I) Immuno- precipitation of endogenous MAVS from MIC and MAM subcellular fractions after mock treatment or 5 h of stimulation with poly(I:C). An equal amount of IgG control antibody was incubated with the MIC fraction (mock). For Western blot analysis, 1/20 of the Input and total eluate were loaded onto an SDS gel. Eluates were analyzed for coimmunoprecipitation of LGP2 or Calnexin (loading control). One representative dataset out of two independent experiments is shown. Arrowheads point to specific protein band. Cyto, cytosol; MAM, mitochondria-associated membranes; MIC, microsomes; Mito, mitochondria; WCL, whole cell lysate.

Mito fraction after dsRNA treatment but not after IFN-β treat- 10 mg/mL CHX to A549 cell cultures was sufficient to block ment alone, demonstrating that the LGP2 translocation events IRF3 target gene and ISG expression (RIG-I, IFIT1) otherwise observed in the RML cell lines are triggered by dsRNA and also induced by dsRNA (Fig. 2 H, Left). Subcellular fractionation of account for endogenous receptors (Fig. 2G). While α-tubulin levels A549-RML cells treated with dsRNA followed by CHX treat- were qualitatively variable after dsRNA treatment of cells ment, but not CHX treatment alone, showed enrichment of LGP2 – (Fig. 2 B E and G), immunofluorescent staining of cells shows and RIG-I in the mitochondrial fraction. These results indicate that Actin (Phalloidin Alexa Fluor 488) and α-tubulin patterns that dsRNA induces the translocation of preexisting RLRs rather remained unaltered in A549-RML cells after dsRNA treatment than de novo RLRs produced in response to dsRNA signaling (SI Appendix,Fig.S3C). To determine whether the enrichment of RLRs in different (Fig. 2 H, Right). Based on qualitative comparison of LGP2 protein fractions resulted from translocation of already existing protein in different fractions obtained from mock and dsRNA treated cells, or from transport of newly expressed protein, we subjected A549 we found that, in response to dsRNA, LGP2 became enriched in cells to dsRNA treatment, followed by cycloheximide (CHX) to Mito while decreasing in MIC and Cyto. These data indicate that block translation elongation and prevent induction of RLR ex- LGP2 translocates from the ER and cytosol to mitochondria upon pression due to innate immune signaling (Fig. 2H). Addition of stimulation.

4of11 | www.pnas.org/cgi/doi/10.1073/pnas.1921861117 Esser-Nobis et al. Downloaded by guest on September 30, 2021 To determine if LGP2 translocation and membrane associa- A A549 B tion are linked with MAVS interaction, we evaluated LGP2/ A549 LGP2-HA RM MAVS binding in subcellular fractions via coimmunoprecipitation/ Mock poly(I:C) 16h immunoblot analyses. We established MAVS immunoprecipitation WCL MIC MAM Mito Cyto WCL Mito Cyto MIC MAM untransduced LGP2-HA (IP) from MIC and MAM fractions and probed for LGP2 within HA-LGP2 HA (CST) the recovered products. These analyses indeed revealed an in- LGP2 MFN2 HA (Enzo) Calnexin teraction of LGP2 and MAVS primarily in MIC fractions. In ad- HA (CST) CoxIV dition, MIC fractions from dsRNA-treated cells contained less Actin α-tubulin LGP2 (Input), in line with the previously observed LGP2 trans- C A549 LGP2-HA RM D location resulting in a lower amount of LGP2 in complex with 12 ns M M M 10 MAVS (Fig. 2I). Calnexin served as loading control as well as 8 ns control for the nonspecific pulldown of membrane-associated M 6 M 4 proteins. Taken together, these observations indicate that, in the Mock 2 M ►

number per image 0 absence of stimulation, LGP2 is associated with membranes of the Mock pIC Mock pIC ER or other organelles present in MIC via direct or indirect in- M Mitochondria gold teraction with MAVS while dsRNA treatment triggers LGP2 re- particles M 4 lease from MAVS and translocation to mitochondria. Fractionation ► * analysis of nontargeting A549 RML cells (gNT RML) in com- 3 ► M 2 parison to MAVS knockout RML cells (gMAVS RML) suggested M 1 that MAVS was not required for LGP2 membrane association or poly(I:C), 16h 0 -1 relocalization, indicating that LGP2 interacts with other factors Mock pIC gold on mito per image independent of MAVS to regulate its membrane association (SI Appendix,Fig.S3D). Fig. 3. Ultrastructural analyses showing LGP2 translocation to mitochondria following dsRNA treatment. (A) Immunoblot analysis of A549 cells stably expressing HA-LGP2 or LGP2-HA (N- or C-terminal HA-tag, respectively) using Ultrastructural Analysis Defines dsRNA-Induced Accumulation of LGP2 antibodies directed against LGP2 or HA. (B) A549 cells stably expressing on Mitochondria. To define the intracellular localization dynamics LGP2-HA (C-terminal HA-tag) together with RIG-I and MDA5 (A549 LGP2-HA of LGP2 on an ultrastructural level, we assessed the membrane- RM) were subjected to subcellular fractionation 16 h after poly(I:C) trans- to-mitochondria translocation of LGP2 by TEM using immu- fection. One representative experiment is shown from two independent nogold labeling. For this purpose, we generated A549 cells stably fractionations. (C) Immunogold-labeling of A549 LGP2-HA RM cells 16 h IMMUNOLOGY AND INFLAMMATION expressing hemagglutinin (HA)-tagged LGP2 (Fig. 3A). Based after poly(I:C) or mock transfection using a monoclonal antibody directed on the homogeneous expression level of LGP2 in the cell pop- against HA. Arrowheads point to immunogold in the cytosol (yellow) or on ulation as determined by immunofluorescent microscopy (SI mitochondria (red). The images in the third column are a magnified view of Appendix, Fig. S4A), we chose A549 cells expressing C-terminally the boxed areas in column 2 and the images in the second column are a tagged LGP2 for further analyses. After confirming the dsRNA- magnified view of the boxed areas in column 1. M, Mitochondria. (Scale bars in second column: 200 nm.) (D, Top) Number of mitochondria or gold par- induced translocation of LGP2-HA in A549 cells expressing ticles per image of mock or poly(I:C) (pIC) treated A549 LGP2-HA RM cells. At RIG-I, MDA5, and LGP2-HA (A549-LGP2-HA-RM cells) (Fig. least 45 images per condition were analyzed. (D, Bottom) Number of gold 3B), we subjected these cells to immunogold labeling using a particles on mitochondria per image. At least 45 images per condition were monoclonal anti-HA antibody and analyzed the samples by TEM. analyzed manually using the ImageJ software (version 1.52; NIH). Each data As seen in Fig. 3C, yellow arrowheads point to nonmitochondrial point represents one image. To test for statistical significance, a one-way gold particles while the red arrowhead indicates gold particles ANOVA was performed, followed by Sidak’s multiple comparison test using associated with mitochondria (Fig. 3 C, Right and SI Appendix,Fig. GraphPad Prism. ns, not significant; *P < 0.05. Enzo, Enzo Life Sciences, Inc.; S4B). Image analyses demonstrated no significant difference be- CST, Cell Signaling Technology. tween the total number of mitochondria per image or the total number of gold particles per image between mock and dsRNA- treated cells (Fig. 3 D, Top Graph). In contrast, we found a sig- mitochondrial translocation of LGP2 which was only detectable at nificant increase in the number of gold particles on mitochondria 2 h after treatment (Fig. 4 E and F), thus linking dynamic reloc- per image in dsRNA-treated cells compared to mock-treated cells alization of LGP2 with the onset of IRF3 activation in response (Fig. 3 D, Bottom Graph), confirming that dsRNA induces trans- to dsRNA. location of LGP2 to mitochondria. LGP2 Is a Negative Regulator of RIG-I Signaling. To assess the impact LGP2 Translocation Induction by dsRNA Correlates with IRF3 of LGP2 on dsRNA-induced IRF3 activation, we examined the Activation. To determine if RLR dynamics are specific for acti- stoichiometric relationship of LGP2 expression and IRF3 activation of the RLR-MAVS pathway or instead represent a gen- eral response to innate immune activation, we assessed the vation in dsRNA-treated cells. We first measured the expression response to calf thymus DNA (ctDNA), which activates IRF3 via of the RLRs across a set of human cell lines and hTert immortalized human foreskin fibroblasts (HFFs) that were mock-treated the cGAS-STING pathway (Fig. 4A) (1). ctDNA treatment of β A549-RML cells induced rapid and robust IRF3 activation (Fig. or treated with IFN- . We found that baseline RIG-I and MDA5 4B) but did not alter the subcellular localization of LGP2 or expression were relatively consistent between the five cell lines RIG-I (Fig. 4C). Thus, RLR spatial dynamics are specifically re- under resting conditions (Fig. 5A). In contrast, baseline LGP2 sponsive to dsRNA. Since the LGP2/MAVS interaction was re- expression was remarkably variable, with lowest basal level of duced upon dsRNA stimulation, we reasoned that LGP2 might LGP2 expressed in A549 cells and highest present in HFFs block or inhibit downstream IRF3 activation in resting cells by (123.55-fold difference, SD ± 49.26 between these two cell types). interaction with MAVS, possibly preventing MAVS interaction In addition, transcript expression of all RLRs was strongly induced with RIG-I or another accessory signaling component required to by IFN-β across all cell lines. We next assessed the response to impart IRF3 activation. We therefore measured IRF3 activation at dsRNA of LGP2 HFF knockout (KO) cells generated by early time points after dsRNA treatment of A549-RML cells, CRISPR/Cas9 gene targeting (HFF-gLGP2_6 cells). Immunoblot identifying 2 h posttreatment as the earliest time point of detectable analysis of IFN-β–treated HFF-gLGP2_6 cells revealed a specific IRF3 phosphorylation (Fig. 4D). This time point corresponded with reduction of LGP2 protein level, but neither RIG-I nor MDA5

Esser-Nobis et al. PNAS Latest Articles | 5of11 Downloaded by guest on September 30, 2021 A D of LGP2 FL, LGP2-K30A, or LGP2-K634A/K651A in A549 cells cytosolic RNA cytosolic DNA efficiently blocked the acute activation of IRF3 and IFIT1 in-

Mock 0.5 123 4 5 h poly(I:C) duction 6 h after SeV infection while expression of the helicase RIG-I/MDA5 cGAS IRF3 S386* cGAMP IRF3 S386 constructs (1-546 or HA-tagged 1-475) or the regulatory domain MAVS STING IRF3 (476-678) alone did not impact SeV-induced innate immune ac- TBK1 TBK1 Actin tivation (Fig. 5E). We note that, 24 h after SeV infection, IFIT1 IRF3 IRF3 was induced in all cell lines, but it was unclear if this was due to Mock poly(I:C) 1h B Mock ctDNA E incomplete block of signaling by ectopic LGP2 or continued accumulation of SeV PAMPs to overcome LGP2 inhibition of

2 4 16 2 4 16 h p.t. WCL MIC MAM Mito Cyto WCL MIC MAM Mito Cyto IRF3 S386 LGP2 signaling (SI Appendix,Fig.S5B).Thenegativeregulationof IRF3 Calnexin SeV-induced innate immune activation by wild-type LGP2, Actin CoxIV and the ATP-binding and RNA-binding LGP2 mutants was also confirmed in HFFs expressing each construct (SI Appendix, C Mock ctDNA 4h F Mock poly(I:C) 2h Fig. S5C). We next examined the subcellular distribution of LGP2 con- WCL MIC MAM Mito Cyto WCL MIC MAM Mito Cyto WCL MIC MAM Mito Cyto WCL MIC MAM Mito Cyto structs in A549 cells to determine the association of LGP2 reg- RIG-I LGP2 LGP2 Calnexin ulation of innate immune signaling with specific translocation CoxIV CoxIV dynamics of each. We examined the enrichment of LGP2 protein in Mito fractions after dsRNA treatment of A549 cells (SI Ap- Fig. 4. Temporal correlation of LGP2 translocation and IRF3 activation ki- pendix, Fig. S5 D–J). We found that wild-type LGP2 (LGP2 FL), netics. (A) Pathways leading to IRF3 activation upon stimulation with cytosolic RNA [poly(I:C)] or cytosolic ctDNA. (B) IRF3 activation in A549-RML cells HA-tagged LGP2 (LGP2 FL_HA), LGP2-K30A, and LGP2- upon ctDNA transfection (1 μg/mL). Cells were harvested at the indicated K634A/K651A each accumulated in the Mito fraction after time points and analyzed by Western blot. (C) Subcellular fractionation of dsRNA treatment of cells (summarized in Fig. 5G, data shown in A549-RML cells after 4 h of ctDNA transfection (1 μg/mL). Fractions were SI Appendix, Fig. S5 D, G, H, and J). Conversely, the helicase analyzed by Western blot for presence of LGP2 and RIG-I (n = 2). (D) Early mutants 1-546 or HA-tagged 1-475 (1-475_HA) and the regu- kinetics of poly(I:C)-induced IRF3 activation. A549-RML cells were lysed at the latory domain 476-678 did not change in their distribution, indicated time points after poly(I:C) transfection, and IRF3 phosphorylation remaining chiefly in the Cyto and MIC fractions (Fig. 5G and SI at Serine residues S386 and S396 was analyzed by Western blot. One rep- Appendix, Fig. S5 E, F, and I). We further assessed the in- resentative out of two independent experiments is depicted. (E and F) Kinetics teraction of the LGP2 mutant proteins with MAVS in MIC of LGP2 to mitochondria translocation analyzed by subcellular fractionation of A549-RML cells after 1 h (E)or2h(F) of poly(I:C) transfection. The fractions from mock-treated samples (Fig. 5 F and G and SI four independent fractionations performed for this set of experiments Appendix, Fig. S5K). In line with the observed translocation dy- comprise two fractionations per time point. *, An asterisk indicates longer namics and their inhibitory capacity, LGP2 and both point mu- exposure. tants (ATP-binding mutant, RNA-binding mutant) interacted with MAVS in MIC fractions while 1-475_HA and the regulatory domain (476-678) did not (Fig. 5 F and G and SI Appendix, Fig. expression were affected (Fig. 5B). Comparison of the response S5K). Interestingly, we found evidence for an interaction of the to acute SeV infection of control cells harboring nontargeting helicase construct 1-546 with MAVS in MIC fractions, impli- guide RNA (HFF-gNT) and HFF-gLGP2_6 cells demonstrated cating the region of amino acids 476 to 546 as a potential LGP2- that loss of LGP2 expression confers enhanced kinetics of IRF3 MAVS interaction site. We note however, that possible mis- activation, as marked by enhanced virus-induced IRF3 S386 folding of the 1-475 or 476-678 constructs might impact their phosphorylation in the HFF-gLGP2_6 cells (Fig. 5C). Moreover, ability to bind MAVS. Altogether, these data support the notion −/− we found that mouse embryonic fibroblasts (MEFS) from LGP2 that LGP2 acts as a negative regulator of innate immune sig- mice had significantly enhanced Mx1 mRNA expression kinetics in naling by binding to MAVS at the ER, MAM, or other mem- response to SeV infection compared to wild-type MEFs (SI Ap- branes that are part of the microsome fraction, thus preventing pendix, Fig. S5A). Conversely, HFFs with ectopic LGP2 expression IRF3 activation. exhibited a suppressed response to SeV infection, exhibiting reduced IRF3 activation, with slower activation kinetics and mark- IRF3 Activation Does Not Take Place on Mitochondria. The presence edly reduced IFIT1 concomitant with increased viral protein of LGP2 at membranes contained in the microsome fraction, abundance (Fig. 5C). Together these data affirm LGP2 as a where it interacts with MAVS in resting cells, suggests that IRF3 negative regulator of RIG-I-MAVS innate immune signaling and activation is regulated through LGP2 at microsomal sites of in- suggest that the major effect of LGP2 regulatory actions on RLR nate immune signaling, including ER, and likely MAM, and signaling is exerted after IFN-mediated induction of LGP2 ex- peroxisomes. To better understand where IRF3 activation takes pression (8–11). We note that SeV is known to primarily stimulate place, we assessed SeV-induced innate immune signaling kinetics innate immune activation via RIG-I, such that these analyses do and component activation in A549-RML cells (Fig. 6A). IRF3 not assess the impact of LGP2 on MDA5-driven IRF3 activation and TBK1 activation was detectable early at 2.5 and 5 h post- (13–16). infection (h.p.i.), and phosphorylation/activation of each was strongly increased through 20 h. A reduction in MAVS protein LGP2-Mediated Feedback Inhibition of RLR Signaling Requires Binding level, marking MAVS activation (48), was observed from 10 h to MAVS. LGP2 has RNA-binding activity and is an ATPase (2). after infection and associated with strong IRF3 activation. At To determine the requirement for each activity in regulation of 40 h after infection, innate immune signaling was reduced/down- RLR signaling and mitochondrial translocation, we generated regulated as determined by lower abundance of phospho-IRF3 A549 cells stably expressing full-length LGP2 (LGP2 FL) or (Fig. 6A). LGP2 deletion mutants containing only the helicase domain (1- We also examined the subcellular localization of activated 546 or HA-tagged 1-475) or the C-terminal regulatory domain IRF3 following early (6 h), mid (10 h, and late (20 h) time points (476-678), or full-length constructs harboring a Walker A = of SeV infection (Fig. 6 B–D). At all time points phosphorylated/ ATP-binding point mutant (K30A), or a double point mutation activated IRF3 (IRF3 S386P) was absent from Mito but present that abrogates RNA binding (K634A/K651A) (Fig. 5D) (10, 16, in MIC, MAM, and Cyto fractions (Fig. 6 B–D). This distribution 45–47). In comparison to empty vector (EV) control cells, expression was also confirmed for IRF3 S396P (SI Appendix, Fig. S6A).

6of11 | www.pnas.org/cgi/doi/10.1073/pnas.1921861117 Esser-Nobis et al. Downloaded by guest on September 30, 2021 A untreated B HFF C HFF IFN-b 1 gNT gLGP2_6 LGP2 Rpl13a 0 -1 gNT gLGP2_4 gLGP2_5 gLGP2_6 Mock 3h 6h 24h -2 Mock 3h 6h 24h - + - + - + - + IFN-b Mock 3h 6h 24h SeV -3 RIG-I IRF3 S386* -4 MDA5 IRF3 S386 -5 LGP2 IRF3

RLR expression rel to RLR expression rel to IFIT1 IFIT1 HFF HFF HFF Vero Vero Vero A549 A549 A549 Actin

MRC5 MRC5 MRC5 MAVS

PH5CH8 PH5CH8 PH5CH8 SeV LGP2 RIG-I MDA5 Actin LGP2 FL 1-678 Helicase CTD D E A549 LGP2 1-546 Helicase LGP2 1-475 Helicase LGP2 476-678 CTD K30A 1-546 Helicase CTD EV FL LGP2 1-475-HA 476-678 K30A K634A/K651A (Walker A-mut) kD - +- +- +- +- +- +- + SeV 6h K634A/K651A Helicase CTD IRF3 S386 (RNA-bind-mut) TBK1 S172 IFIT1 MAVS IP from MIC frx F IRF3 Input Eluate TBK1 75

Input Eluate LGP2(N) 50

50 kD LGP2(C) IgG ctrl K634A/K651A IgG ctrl LGP2 FL 476-678 K30A LGP2 FL K30A K634A/K651A 476-678 75 LGP2 25 ◄ LGP2 25 MAVS 75 Calnexin HA 50 Input Eluate Actin IMMUNOLOGY AND INFLAMMATION

kD IgG ctrl LGP2 FL 1-546 476-678 K30A K634A/K651A IgG ctrl LGP2 FL 1-546 476-678 K30A K634A/K651A 75 LGP2

50 LGP2 FL 1-546 1-476 476-678 K30A K634A/K651A MAVS interaction + + - - + + LGP2* ◄ Translocation to mito + - - - + + Inhibition IRF3 activation + - - - + + 25 LGP2 MAVS Calnexin

Fig. 5. LGP2 is a negative feedback regulator of RIG-I signaling. (A) SYBR green qPCR analysis of RLR expression levels in various cell lines. The indicated cell lines were left untreated or treated with 500 IU/mL IFN-β for 8 h. cDNA was subjected to qPCR analysis using primer pairs specific for LGP2, RIG-I, or MDA5. Data were normalized to Rpl13a housekeeping gene expression and are derived from two (MRC5, Vero) or three (A549, PH5CH8, HFF hTert) independent experiments measured in triplicate. Presented are mean and SD on a log10 scale. (B) Western blot analysis of CRISPR/Cas9-mediated LGP2 knockout in HFF hTert cells. HFF hTert cells were transduced with different guide RNAs (nontargeting [gNT] or targeting LGP2 [gLGP2_4, gLGP2_5, gLGP2_6]) and treated with 500 IU/mL IFN-β for 24 h to monitor LGP2, RIG-I, and MDA5 protein expression. (C) Western blot analysis of innate immune signaling in HFF hTert cells with LGP2 KO (gLGP2_6), LGP2 expression (LGP2), or nontargeting control cells (gNT). Cells were mock infected or infected with SeV (40 hemagglutinating unit [HAU]/mL) and harvested at the indicated time points. (D) Illustration of LGP2 deletion and point mutants. Stars indicate position of point mutations. (E) A549 cells were transduced with lentiviral particles carrying empty vector (EV) or different LGP2 constructs. Cells were selected for stable expression with Blasticidin. Cells were infected with 40 HAU/mL SeV for 6 h and subjected to Western blot analysis. LGP2(C), antibody directed against the LGP2 C-terminal region; LGP2(N), antibody directed against the LGP2 N-terminal region. Depicted is one representative out of two independent experiments (n = 2). (F) MAVS protein was immunoprecipitated from MIC fractions of subcellular fractionation experiments performed with A549 cells expressing the indicated LGP2 mutants (SI Appendix, Fig. S5). Inputs and eluates were analyzed using antibodies directed against LGP2, MAVS, and Calnexin. A polyclonal IgG antibody incubated with the MIC fraction served as control for unspecific binding. The presented data are each derived from one representative out of two independent experiments (n = 2). Arrowheads indicate signals derived from IgG heavy chain or light chain. (G) Graphic summary of the observations made with wild-type LGP2 and LGP2 mutants. *, An asterisk indicates longer exposure. −, indicates no interaction with MAVS, no translocation to mito, regular IRF3 activation; +, indicates interaction with MAVS, translocation to mito, inhibition of IRF3 activation induced by SeV infection.

Interestingly, by using the anti-IRF3 D83B9 antibody (4302; Cell that were mock infected or infected with Mengovirus, an RNA Signaling), which has a higher sensitivity for the detection of base- virus that signals innate immune activation through MDA5 (2), we line variants of total IRF3, nonphosphorylated IRF3, and hypo- observed a similar distribution of activated and total IRF3 (SI phosphorylated IRF3 (30), we revealed a differential distribution of Appendix, Fig. S6B). We note that, in these experiments, although these two IRF3 phospho-species. While hypophosphorylated activated/phosphorylated TBK1 (TBK1 S172) was present in the IRF3 (upper protein band) was mainly present in MIC and Cyto, Mito fraction, no activated IRF3 was present in the Mito fraction nonphosphorylated IRF3 (lower protein band) was enriched in but instead was detected in MAM, microsomes, and cytosol, in- Mito (SI Appendix,Fig.S6A; see IRF3 (CST #4302)). Curiously, dicating that IRF3 activation does not take place on mitochondria nonphosphorylated IRF3 was previously suggested to represent a (Fig. 6 B–D). dormant IRF3 pool while hypophosphorylated IRF3 was sug- Similar to the dsRNA-induced spatiotemporal dynamics of gested to be primed for activation (30). In PH5CH8-RML cells RIG-I and MDA5, SeV infection resulted in acute enrichment of

Esser-Nobis et al. PNAS Latest Articles | 7of11 Downloaded by guest on September 30, 2021 ABA549 RML A549 RML CA549 RML Mock SeV 6h Mock SeV 10h

Mock 2.5 5 10 15 20 40 SeV h.p.i. WCL MIC MAM Mito Cyto WCL MIC MAM Mito Cyto

IRF3 S386 WCL MIC MAM Mito Cyto WCL MIC MAM Mito Cyto IRF3 RIG-I RIG-I IFIT1 ◄ MDA5 ◄ MDA5 TBK1 S172 LGP2 LGP2 IRF3 S386 TBK1 IRF3 S386 IRF3 MAVS IRF3 TBK1 S172 LGP2 TBK1 S172 TBK1 TBK1 VDAC TRAF2 TRAF2 Actin MAVS MAVS MFN2 MFN2 Calnexin Calnexin CoxIV CoxIV α-tubulin α-tubulin D A549 RML E Mock SeV 20h A549 MAVS IP, SeV 5h Input Eluate

WCL MIC MAM Mito Cyto WCL MIC MAM Mito Cyto gNT gLGP2 gNT gLGP2 RIG-I RML RM RML RM MDA5 gNT gLGP2_6 LGP2 MAM MIC - + - + IFN-b/TNF-a MIC MAM MIC MAM MIC MAM IRF3 S386 LGP2 RIG-I* IRF3 IFIT1 RIG-I TBK1 S172 MAVS LGP2* TBK1 Actin LGP2 TRAF2 MAVS MAVS Calnexin MFN2 Calnexin CoxIV α-tubulin F

RNA virus replication Cytosol

RNA PAMP detection by RIG-I/MDA5 Nucleus

IRF3 activation Antiviral gene transcription and IFN-induced LGP2 expression (A) LGP2 protein level ↑ RNA PAMP level ↓ (B)

Microsomes (ER?) Mitochondria LGP2 binds MAVS blocking signaling LGP2 protein level ↑ RNA PAMP level ↔, dsRNA stim

Resolution of innate immune signaling RNA PAMP triggers LGP2 release from MAVS translocation to mitochondria Sequestration?

Ongoing innate immune signaling

Fig. 6. IRF3 activation does not take place on mitochondria but occurs at the ER-derived membrane network. (A) Innate immune signaling kinetics in A549- RML cells upon SeV infection. A549-RML cells were infected with 40 HAU/mL SeV and harvested for Western blot analysis at the indicated time points. Shown is one representative Western blot derived from two independent experiments (n = 2). (B–D) Subcellular fractionation of A549-RML cells after 6 h (B), 10 h (C), and 20 h (D) of SeV infection. Six independent fractionations were conducted for this experiment, comprising two fractionations per time point. (E, Left) CRISPR/Cas9-mediated LGP2 knockout in A549 cells. A549 cells were transduced with gNT or gLGP2_6 and treated with 500 IU/mL IFN-β + 10 ng/mL TNF-α 48 h prior to Western blot analysis to monitor LGP2 protein expression. (E, Right) MAVS immunoprecipitation from MIC and MAM fractions after subcellular fractionation of A549 gNT RML and A549 gLGP2 RM cells infected with 40 HAU/mL SeV for 5 h. Depicted is one representative out of two independent experiments (n = 2). Asterisks (*) indicate longer exposure. (F) Hypothetical model of RLR-mediated innate immune signaling. Upon RNA virus infection and RNA replication in viral replication compartments that are often generated at the ER, viral RNA PAMPs are detected by RIG-I/MDA5. This leads to interaction with MAVS at ER-derived membranes and IRF3 activation resulting in antiviral gene transcription, inhibition of virus replication, and IFN-induced up- regulation of LGP2 protein expression. In the case of viral PAMP reduction (A), LGP2 binds to MAVS, blocking an interaction of MAVS and RIG-I supporting the resolution of innate immune signaling. In the case of viral persistence or resistance (B), high viral RNA levels trigger LGP2 release from MAVS and its translocation to mitochondria, possibly to sequester LGP2 and allow ongoing innate immune signaling. Arrowheads point to a specific protein band.

both RLRs in MAM and Mito fractions. LGP2 translocation to are known to be central components of the MAVS signalosome mitochondria occurred relatively late at 20 h post-SeV infection (RLRs, MAVS, TRAF2, TBK1, and IRF3) were present in MIC in comparison to dsRNA signaling (Fig. 6 B–D; compare to across the time course. Fig. 2 B–E). Similar translocation dynamics were also observed for We next assessed if LGP2 regulates the MAVS–RIG-I in- TRAF2 and TBK1. In contrast to Mito, all analyzed factors that teraction in response to SeV infection. Therefore, we produced

8of11 | www.pnas.org/cgi/doi/10.1073/pnas.1921861117 Esser-Nobis et al. Downloaded by guest on September 30, 2021 A549-gLGP2_6 cells expressing ectopic RIG-I and MDA5 (gLGP2- studies by Quicke et al. and Parisien et al., who both reported an RM cells) and A549-gNT cells expressing RML (A549-gNT-RML inhibitory effect of the 1-546 LGP2 helicase region construct (45, cells) and probed for RIG-I protein after MAVS immunoprecipi- 46). Underlying reasons for this discrepancy might be the dif- tation from MIC and MAM fractions after SeV infection. Indeed, ferent experimental setups: While we generated A549 cells stably the virus-induced RIG-I–MAVS interaction was highly increased in expressing the 1-546 construct and studied the impact on en- the absence of LGP2 (Fig. 6E and SI Appendix,Fig.S6C). Thus dogenous IRF3 activation and IFIT1 protein expression by West- LGP2 regulates the RIG-I–MAVS interaction. ern blot analysis, the other groups employed transient transfection of LGP2 1-546 into HEK293 cells and measured IFN-β promoter- Discussion driven luciferase expression from an ectopic plasmid construct. Our observations support a model where the low level of LGP2 When comparing the effects of LGP2 KO and ectopic LGP2 ex- present in resting cells basally occupies interaction with MAVS pression on SeV-induced IRF3 activation in our cell models, we on microsomal membranes, such as ER, and including peroxi- found that increased expression of LGP2 in our ectopic expression somes, and MAM. Upon RNA virus infection, viral replication cell models had a much stronger effect on innate immune signal- takes place in close proximity to membranes that are mostly ing. Thus, we propose that LGP2 exerts its main regulatory func- derived from the ER (21) so that viral PAMPs, including dsRNA tion following its increased expression by IFN. In this respect, and other viral RNAs, accumulate at these membrane sites. LGP2 functions as a negative feedback regulator contributing to Once activated by PAMP binding, RIG-I (and possibly MDA5) resolution of RIG-I (and possibly MDA5) signaling after virus in- mediate a stable interaction with MAVS at microsomal mem- fection. The impact of baseline LGP2 on the initiation of RLR- branes that could displace LGP2 for sequestration at mitochon- MAVS signaling is likely to differ between different cell lines and dria via ER-to-mitochondria translocation, thus initiating innate in vivo cell types, considering the variability of basal LGP2 ex- immune signaling and the activation of IRF3 at microsomal pression level demonstrated in our study. membrane sites independent of the OMM. This process facilitates We found that LGP2 translocation from microsomal MAVS innate immune activation marked by the expression of IRF3- to the OMM occurs coincident with high burden of viral PAMP target genes, IFN production, and ISG expression that includes and IRF3 activation. Therefore, it is plausible that LGP2 release increased RLR expression. Upon IFN-induced suppression of vi- from MAVS and translocation to mitochondria facilitates on- ral PAMP production and increase of LGP2 protein expression, a going innate immune signaling. We note that LGP2 has been negative feedback loop is initiated in which LGP2 binds to mi- shown to serve as a positive cofactor of MDA5 signaling to fa- crosomal MAVS and disrupts or prevents the RIG-I–MAVS in- cilitate the dsRNA interaction of MDA5 that builds signaling- teraction, resulting in resolution of innate immune signaling. competent MDA5 filaments (13). Although we did not examine IMMUNOLOGY AND INFLAMMATION These actions of LGP2 are summarized in Fig. 6F and are sup- the specific MDA5 signaling regulation by LGP2 in the present ported by the following observations. 1) Only full-length LGP2 study, our model allows for this cofactor action of LGP2 as constructs that bind to MAVS inhibit IRF3 activation, and the LGP2 could execute distinct functions at different subcellular RIG-I–MAVS interaction is stronger in the absence of LGP2, compartments. We found that RNA binding or ATPase activity suggesting that the LGP2–MAVS interaction hinders RIG-I– of LGP2 are not required for MAVS interaction, mitochondrial MAVS binding and thereby inhibits IRF3 activation. 2) The LGP2 translocation, or the negative regulation of SeV-induced IRF3 1-546 construct binds to MAVS but does not block IRF3 activa- activation. Therefore, direct binding of RNA by LGP2 is not the tion, pointing to a possible steric hindrance of RIG-I–MAVS in- inducer of LGP2-to-mitochondria translocation. However, LGP2 teraction by full-length LGP2. 3) We confirmed the LGP2–MAVS release from MAVS might be induced by competitive MAVS interaction, concluding that LGP2 indeed binds to MAVS and binding between RIG-I and LGP2 or be regulated by other that this interaction facilitates disruption of RIG-I signaling, thus RNA-binding proteins. MAVS itself was not required for LGP2 implicating MAVS in LGP2 regulation of RIG-I signaling. Fur- membrane localization or dynamics, suggesting a role for other ther, we propose that, in case of viral persistence and high PAMP factors in directing LGP2 membrane association and re- level, as mimicked by dsRNA treatment, LGP2 is released from distribution. In the context of RLR mitochondrial translocation, MAVS (Fig. 6F, pathway B) and is translocated to mitochondria, the 14-3-3 chaperone proteins have been shown to play a role in possibly to sequester LGP2, allowing ongoing innate immune directing RIG-I and MDA5 to membranes for innate immune signaling. signaling (18, 19). Thus, 14-3-3 proteins might similarly facilitate Previous studies of RLR-MAVS signaling were based on LGP2 translocation to the OMM in response to RLR signaling. confocal microscopy or analysis of crude mitochondrial fractions, It is well established that, at baseline, LGP2 acts as a positive neither of which provide the resolution to distinguish between regulator of MDA5 signaling (13–17, 25) although, at increasing mitochondria and associated organelles. In contrast, here, we concentrations, LGP2 seems to exert MDA5 negative regulation employ a higher resolution biochemical analysis of subcellular (15, 25). In our model, LGP2 binding to MAVS blocks the RIG- fractions spanning a broad range of cellular compartments and I–MAVS interaction while viral PAMP RNA induces LGP2 allowing us to examine highly enriched mitochondrial fractions translocation to the OMM after its levels accumulate following devoid of other organellar membranes (18, 39, 40). This ap- IFN signaling. This process may sequester LGP2 to allow innate proach, in combination with TEM analyses and immunoprecip- immune signaling, or, as noted above, it may also allow MDA5 itation, revealed that, in resting cells, LGP2 is bound to MAVS, filament formation. We also note that LGP2 has not only been possibly involving a region spanning amino acid residues 476 to implicated in regulation of RLR signaling regulation but has 546. An interaction of LGP2 and MAVS was already reported by been assigned other functions, including regulation of DNA- Komuro et al. (8), who demonstrated coimmunoprecipitation of virus and bacteria induced immune responses (49), promoting the two proteins in whole cell lysates of MAVS and LGP2 CD8 T cell expansion and survival (23), and mediating resistance cotransfected cells, thus identifying the C-terminal region and of tumor cells to ionizing radiation (50). It is possible that the transmembrane domain of MAVS as LGP2 interaction domains. intracellular dynamics of LGP2 localization could impact these Interestingly, the authors found that LGP2 blocked an interac- processes where its distribution among different organellar mem- tion of MAVS with IKKe. Our data suggest that, during SeV branes would serve to regulate these cellular functions. infection, LGP2 similarly blocks the interaction of MAVS and In summary, we performed a high resolution analysis on the RIG-I, most likely by steric hindrance through full-length LGP2 spatiotemporal distribution of RLRs, including LGP2, over a broad since an LGP2 deletion mutant (1-546) bound to MAVS but did range of fractions and spanning various subcellular compartments. not block IRF3 activation. This observation is in contrast to We present data that argue against the dogma of mitochondria

Esser-Nobis et al. PNAS Latest Articles | 9of11 Downloaded by guest on September 30, 2021 representing the central platform of innate immune signaling but MAM and MIC were pelleted at 100,000 × g for 1 h. Cytosol was concen- instead suggest that innate immune signaling is initiated at the ER trated using an Amicon Ultra-4 filter unit (Millipore Sigma). All membra- including MAM, a subdomain of ER membranes. Our observa- nous fractions were resuspended in lysis buffer (50 mM Tris·HCl, 150 mM tions provide increased details for understanding of the subcellular NaCl, 1% Triton X-100, with freshly added protease and phosphatase in- localization of innate immune signaling components and where hibitors) (Sigma-Aldrich). The protein concentration of each fraction was innate immune signaling is initiated and governed. Such studies measured by Pierce BCA assay (Thermo Fisher Scientific). For Western blot analysis, 2 μg of each fraction were loaded onto a polyacrylamide gel are important to inform strategies in the growing field of RLR- – containing 10% sodium dodecyl sulfate (SDS). Detection of bound horse- based therapeutics (51 55) so that targeting RLRs at relevant radish peroxidase-coupled secondary antibodies was performed using an subcellular compartments can be obtained. Our study demonstrates ECL prime reagent (GE Healthcare) on a ChemiDoc Imager (Bio-Rad). Pol- that LGP2 interaction with MAVS as well as dynamic relocaliza- yvinylidene difluoride membranes were stripped for reprobing with a tion of LGP2 between microsomal membranes and mitochondria harsh stripping buffer containing 2% SDS and 0.8% β-mercaptoethanol. are important features of RLR signaling control. Antibodies are listed in SI Appendix, Table S2.

Materials and Methods Statistical Analysis. Statistical analysis was performed in GraphPad Prism 7.03 Subcellular Fractionation. Subcellular fractionation by Percoll density gradient (GraphPad, La Jolla, CA) and is described in figure legends. centrifugation was performed as described by Bozidis et al. (41). In brief, cells were centrifuged and resuspended in a sucrose homogenization medium. Data Availability. All data, protocols, and reagents are available in the main After breaking up the cells with a Dounce homogenizer, nuclei and un- text or SI Appendix. broken cells were pelleted twice at 600 × g, and the supernatant was sep- arated into crude mitochondria (MAM+mitochondria) and microsomes+cytosol ACKNOWLEDGMENTS. This work was supported by NIH Grants AI04002, × by spinning three times at 10,400 g for 10 min. Crude mitochondria were AI145296, AI118916, and AI127463. We thank B. Schneider at the electron resuspended in buffer Mannitol A, homogenized by douncing, and layered microscopy core facility of the Fred Hutchinson Cancer Research Center; V. onto a 30% Percoll solution. After spinning for 1 h at 95,000 × g,fractionsof Lohmann, M. Binder, and R. Bartenschlager for plasmids; and A. Sekine and MAM and mitochondria were harvested and washed with buffer Mannitol B. K. Voss for mouse embryonic fibroblasts.

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