Receptor Retinoic Acid-inducible Gene I Research Articles

Cleavage of 14-3-3ε by the enteroviral 3C protease dampens RIG-I-mediated antiviral signaling.

Viruses have evolved diverse strategies to evade the host innate immune response and promote infection. The retinoic acid-inducible gene I (RIG-I)-like receptors RIG-I and MDA5 are antiviral factors that sense viral RNA and trigger downstream signal via mitochondrial antiviral-signaling protein (MAVS) to activate type I interferon expression. 14-3-3ε is a key component of the RIG-I translocon complex that interacts with MAVS at the mitochondrial membrane; however, the exact role of 14-3-3ε in this pathway is not well understood. In this study, we demonstrate that 14-3-3ε is a direct substrate of both the poliovirus and coxsackievirus B3 (CVB3) 3C proteases (3Cpro) and that it is cleaved at Q236↓G237, resulting in the generation of N- and C-terminal fragments of 27.0 and 2.1 kDa, respectively. While the exogenous expression of wild-type 14-3-3ε enhances IFNB mRNA production during poly(I:C) stimulation, expression of the truncated N-terminal fragment does not. The N-terminal 14-3-3ε fragment does not interact with RIG-I in co-immunoprecipitation assays, nor can it facilitate RIG-I translocation to the mitochondria. Probing the intrinsically disordered C-terminal region identifies key residues responsible for the interaction between 14-3-3ε and RIG-I. Finally, overexpression of the N-terminal fragment promotes CVB3 infection in mammalian cells. The strategic enterovirus 3Cpro-mediated cleavage of 14-3-3ε antagonizes RIG-I signaling by disrupting critical interactions within the RIG-I translocon complex, thus contributing to evasion of the host antiviral response. IMPORTANCE Host antiviral factors work to sense virus infection through various mechanisms, including a complex signaling pathway known as the retinoic acid-inducible gene I (RIG-I)-like receptor pathway. This pathway drives the production of antiviral molecules known as interferons, which are necessary to establish an antiviral state in the cellular environment. Key to this antiviral signaling pathway is the small chaperone protein 14-3-3ε, which facilitates the delivery of a viral sensor protein, RIG-I, to the mitochondria. In this study, we show that the enteroviral 3C protease cleaves 14-3-3ε during infection, rendering it incapable of facilitating this antiviral response. We also find that the resulting N-terminal cleavage fragment dampens RIG-I signaling and promotes virus infection. Our findings reveal a novel viral strategy that restricts the antiviral host response and provides insights into the mechanisms underlying 14-3-3ε function in RIG-I antiviral signaling.

Role of CIV NS1 Protein in Innate Immunity and Viral Replication.

The innate immune pathway serves as the first line of defense against viral infections and plays a crucial role in the host's immune response in clearing viruses. Prior research has indicated that the influenza A virus has developed various strategies to avoid host immune responses. Nevertheless, the role of the NS1 protein of the canine influenza virus (CIV) in the innate immune pathway remains unclear. In this study, eukaryotic plasmids of NS1, NP, PA, PB1, and PB2 were constructed, and it was found that these proteins interact with melanoma differentiation-associated gene 5 (MDA5) and antagonize the activation of IFN-β promoters by MDA5. We selected the NS1 protein for further study and found that NS1 does not affect the interaction between the viral ribonucleoprotein (RNP) subunit and MDA5, but that it downregulates the expression of the laboratory of genetics and physiology 2 (LGP2) and retinoic acid-inducible gene-I (RIG-I) receptors in the RIG-I pathway. Additionally, NS1 was found to inhibit the expression of several antiviral proteins and cytokines, including MX dynamin like GTPase 1 (MX1), 2'-5'oligoadenylate synthetase (OAS), Signal Transducers and Activators of Transcription (STAT1), tripartite motif 25 (TRIM25), interleukin-2 (IL-2), IFN, IL-8, and IL-1β. To further investigate the role of NS1, a recombinant H3N2 virus strain (rH3N2) and an NS1-null virus (rH3N2ΔNS1) were rescued using reverse-genetic technology. The rH3N2ΔNS1 virus exhibited lower viral titers compared to rH3N2, but had a stronger activation effect on the receptors LGP2 and RIG-I. Furthermore, when compared to rH3N2, rH3N2ΔNS1 exhibited a more pronounced activation of antiviral proteins such as MX1, OAS, STAT1, and TRIM25, as well as antiviral cytokines such as IL-6, IFN-β, and IL-1β. These findings suggest a new mechanism by which NS1, a nonstructural protein of CIV, facilitates innate immune signaling and provides new avenues for the development of antiviral strategies.

NAD+ precursor supplementation prevents mtRNA/RIG-I-dependent inflammation during kidney injury.

Our understanding of how global changes in cellular metabolism contribute to human kidney disease remains incompletely understood. Here we show that nicotinamide adenine dinucleotide (NAD+) deficiency drives mitochondrial dysfunction causing inflammation and kidney disease development. Using unbiased global metabolomics in healthy and diseased human kidneys, we identify NAD+ deficiency as a disease signature. Furthermore using models of cisplatin- or ischaemia-reperfusion induced kidney injury in male mice we observed NAD+ depletion Supplemental nicotinamide riboside or nicotinamide mononucleotide restores NAD+ levels and improved kidney function. We find that cisplatin exposure causes cytosolic leakage of mitochondrial RNA (mtRNA) and activation of the cytosolic pattern recognition receptor retinoic acid-inducible gene I (RIG-I), both of which can be ameliorated by restoring NAD+. Male mice with RIG-I knock-out (KO) are protected from cisplatin-induced kidney disease. In summary, we demonstrate that the cytosolic release of mtRNA and RIG-I activation is an NAD+-sensitive mechanism contributing to kidney disease.

RIG-I immunotherapy overcomes radioresistance in p53-positive malignant melanoma.

Radiotherapy induces DNA damage, resulting in cell-cycle arrest and activation of cell-intrinsic death pathways. However, the radioresistance of some tumour entities such as malignant melanoma limits its clinical application. The innate immune sensing receptor retinoic acid-inducible gene I (RIG-I) is ubiquitously expressed and upon activation triggers an immunogenic form of cell death in a variety of tumour cell types including melanoma. To date, the potential of RIG-I ligands to overcome radioresistance of tumour cells has not been investigated. Here, we demonstrate that RIG-I activation enhanced the extent and immunogenicity of irradiation-induced tumour cell death in human and murine melanoma cells in vitro and improved survival in the murine B16 melanoma model invivo. Transcriptome analysis pointed to a central role for p53, which was confirmed using p53-/- B16 cells. In vivo, the additional effect of RIG-I in combination with irradiation on tumour growth was absent in mice carrying p53-/- B16 tumours, while the antitumoural response to RIG-I stimulation alone was maintained. Our results identify p53 as a pivotal checkpoint that is triggered by RIG-I resulting in enhanced irradiation-induced tumour cell death. Thus, the combined administration of RIG-I ligands and radiotherapy is a promising approach to treating radioresistant tumours with a functional p53 pathway, such as melanoma.

Transient RNA structures cause aberrant influenza virus replication and innate immune activation.

During infection, the influenza A virus RNA polymerase produces both full-length and aberrant RNA molecules, such as defective viral genomes (DVGs) and mini viral RNAs (mvRNAs). Subsequent innate immune activation involves the binding of host pathogen receptor retinoic acid–inducible gene I (RIG-I) to viral RNAs. However, it is not clear what factors determine which influenza A virus RNAs are RIG-I agonists. Here, we provide evidence that RNA structures, called template loops (t-loops), stall the viral RNA polymerase and contribute to innate immune activation by mvRNAs during influenza A virus infection. Impairment of replication by t-loops depends on the formation of an RNA duplex near the template entry and exit channels of the RNA polymerase, and this effect is enhanced by mutation of the template exit path from the RNA polymerase active site. Overall, these findings are suggestive of a mechanism involving polymerase stalling that links aberrant viral replication to the activation of the innate immune response.

The intrinsically disordered CARDs-Helicase linker in RIG-I is a molecular gate for RNA proofreading.

The innate immune receptor RIG‐I provides a first line of defense against viral infections. Viral RNAs are recognized by RIG‐I's C‐terminal domain (CTD), but the RNA must engage the helicase domain to release the signaling CARD (Caspase Activation and Recruitment Domain) domains from their autoinhibitory CARD2:Hel2i interactions. Because the helicase itself lacks RNA specificity, mechanisms to proofread RNAs entering the helicase domain must exist. Although such mechanisms would be crucial in preventing aberrant immune responses by non‐specific RNAs, they remain largely uncharacterized to date. This study reveals a previously unknown proofreading mechanism through which RIG‐I ensures that the helicase engages RNAs explicitly recognized by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs‐Helicase Linker (CHL), which connects the CARDs to the helicase subdomain Hel1. CHL uses its negatively charged regions to antagonize incoming RNAs electrostatically. In addition to this RNA gating function, CHL is essential for stabilization of the CARD2:Hel2i interface. Overall, we uncover that the CHL and CARD2:Hel2i interface work together to establish a tunable gating mechanism that allows CTD‐chosen RNAs to bind the helicase domain, while at the same time blocking non‐specific RNAs. These findings also indicate that CHL could represent a novel target for RIG‐I‐based therapeutics.

Immunogenicity of In Vitro-Transcribed RNA.

In vitro-transcribed RNAs are emerging as new biologics for therapeutic innovation, as exemplified by their application recently in SARS-CoV-2 vaccinations. RNAs prepared by in vitro transcription (IVT) allow transient expression of proteins of interest, conferring safety over DNA- or virus-mediated gene delivery systems. However, in vitro-transcribed RNAs should be used with caution because of their immunogenicity, which is in part triggered by double-stranded RNA (dsRNA) byproducts during IVT. Cellular innate immune response to dsRNA byproducts can lead to undesirable consequences, including suppression of protein synthesis and cell death, which in turn can detrimentally impact the efficacy of mRNA therapy. Thus, it is critical to understand the nature of IVT byproducts and the mechanisms by which they trigger innate immune responses.Our lab has been investigating the mechanisms by which the innate immune system discriminates between "self" and "nonself" RNA, with the focus on the cytoplasmic dsRNA receptors retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated 5 (MDA5). We have biochemically and structurally characterized critical events involving RNA discrimination and signal transduction by RIG-I or MDA5. We have used in vitro-transcribed RNAs as tools to investigate RNA specificity of RIG-I and MDA5, which required optimization of the IVT reaction and purification processes to eliminate the effect of IVT byproducts. In this Account, we summarize our current understanding of RIG-I and MDA5 and IVT reactions and propose future directions for improving IVT as a method to generate both research tools and therapeutics. Other critical proteins in cellular innate immune response to dsRNAs are also discussed. We arrange the contents in the following order: (i) innate immunity sensors for nonself RNA, including the RIG-I-like receptors (RLRs) in the cytosol and the toll-like receptors (TLRs) in the endosome, as well as cytoplasmic dsRNA-responding proteins, including protein kinase R (PKR) and 2',5'-oligoadenylate synthetases (OASes), illustrating the feature of protein-RNA binding and its consequences; (ii) the immunogenicity of IVT byproducts, specifically the generation of dsRNA molecules during IVT; and (iii) methods to reduce IVT RNA immunogenicity, including optimizations of RNA polymerases, reagents, and experimental conditions during IVT and subsequent purification.

RIG-I, a novel DAMPs sensor for myoglobin, activates NF-\u03baB/caspase-3 signaling in CS-AKI model

BackgroundAcute kidney injury (AKI) is the main life-threatening complication of crush syndrome (CS), and myoglobin is accepted as the main pathogenic factor. The pattern recognition receptor retinoicacid-inducible gene I (RIG-I) has been reported to exert anti-viral effects function in the innate immune response. However, it is not clear whether RIG-I plays a role in CS-AKI. The present research was carried out to explore the role of RIG-I in CS-AKI.MethodsSprague-Dawley rats were randomly divided into two groups: the sham and CS groups (n = 12). After administration of anesthesia, the double hind limbs of rats in the CS group were put under a pressure of 3 kg for 16 h to mimic crush conditions. The rats in both groups were denied access to food and water. Rats were sacrificed at 12 h or 36 h after pressure was relieved. The successful establishment of the CS-AKI model was confirmed by serum biochemical analysis and renal histological examination. In addition, RNA sequencing was performed on rat kidney tissue to identify molecular pathways involved in CS-AKI. Furthermore, NRK-52E cells were treated with 200 μmol/L ferrous myoglobin to mimic CS-AKI at the cellular level. The cells and cell supernatant samples were collected at 6 h or 24 h. Small interfering RNAs (siRNA) was used to knock down RIG-I expression. The relative expression levels of molecules involved in the RIG-I pathway in rat kidney or cells samples were measured by quantitative Real-time PCR (qPCR), Western blotting analysis, and immunohistochemistry (IHC) staining. Tumor necrosis factor-α (TNF-α) was detected by ELISA. Co-Immunoprecipitation (Co-IP) assays were used to detect the interaction between RIG-I and myoglobin.ResultsRNA sequencing of CS-AKI rat kidney tissue revealed that the different expression of RIG-I signaling pathway. qPCR, Western blotting, and IHC assays showed that RIG-I, nuclear factor kappa-B (NF-κB) P65, p-P65, and the apoptotic marker caspase-3 and cleaved caspase-3 were up-regulated in the CS group (P < 0.05). However, the levels of interferon regulatory factor 3 (IRF3), p-IRF3 and the antiviral factor interferon-beta (IFN-β) showed no significant changes between the sham and CS groups. Co-IP assays showed the interaction between RIG-I and myoglobin in the kidneys of the CS group. Depletion of RIG-I could alleviate the myoglobin induced expression of apoptosis-associated molecules via the NF-κB/caspase-3 axis.ConclusionRIG-I is a novel damage-associated molecular patterns (DAMPs) sensor for myoglobin and participates in the NF-κB/caspase-3 signaling pathway in CS-AKI. In the development of CS-AKI, specific intervention in the RIG-I pathway might be a potential therapeutic strategy for CS-AKI.

Combined Use of Immunoreactivities of RIG-I with Efp/TRIM25 for Predicting Prognosis of Patients With Estrogen Receptor-positive Breast Cancer

BackgroundWe previously identified estrogen-responsive finger protein (Efp) as an estrogen-induced gene, and showed that the positive immunoreactivity of Efp is a poor prognostic factor for patients with breast cancer. We also demonstrated that Efp has distinctive roles in innate immunity by activating pattern recognition receptor retinoic acid-inducible gene I (RIG-I). The clinical value of RIG-I protein expression in breast cancer had not been evaluated in relationship with patients’ prognosis. Patients and MethodsTissue samples of estrogen receptor-positive invasive breast cancer were obtained from 145 female patients with breast cancer who underwent surgical treatment. Immunoreactivities of RIG-I and Efp were analyzed with the antibodies generated for the present study. ResultsPositive immunoreactivity of RIG-I was correlated with lower disease-free survival (P = .032) and was an independent poor prognostic factor (P = .043). RIG-I immunoreactivity was positively correlated with that of Efp (P = .0004). Patients with positive immunoreactivities of both RIG-I and Efp proteins were associated with a lower disease-free survival rate (P = .005). ConclusionsPositive immunoreactivity of RIG-I has clinical significance as a poor prognostic factor in patients with estrogen receptor-positive breast cancer. A positive correlation of RIG-I and Efp immunoreactivities was observed, and the combination of their immunoreactivities can be used to predict patients’ prognosis.

Hepatitis E Virus ORF2 Inhibits RIG-I Mediated Interferon Response.

Understanding the dynamics of host innate immune responses against a pathogen marks the first step toward developing intervention strategies against the pathogen. The cytosolic pattern recognition receptor retinoic acid-inducible gene I (RIG-I) has been shown to be the major innate immune sensor for hepatitis E virus (HEV). Here, we show that HEV capsid protein (ORF2), a 660 amino acid long protein, interferes with the RIG-I signaling. Interestingly, only the full length ORF2 protein but not the 112-608 ORF2 protein inhibited RIG-I dependent interferon response. Both synthetic agonist and virus induced RIG-I activation was modulated by ORF2. Interference of interferon response was confirmed by reporter assays involving different interferon inducible promoters, qRT PCR, ELISA, and immunofluorescence microscopy. Neither glycosylation nor dimerization of the ORF2 protein had any effect on the observed inhibition. Further analyses revealed that the ORF2 protein antagonized Toll-like receptor (TLR) pathways as well. ORF2 inhibited signaling by RIG-I and TLR adapters, IPS-1, MyD88, and TRIF but was unable to inhibit activation by ectopically expressed IRF3 suggesting that it may be acting at a site upstream of IRF3 and downstream of adapter proteins. Our data uncover a new mechanism by which HEV may interfere with the host antiviral signaling.

Direct RIG‐I activation in human NK cells induces TRAIL‐dependent cytotoxicity toward autologous melanoma cells

Activation of the innate immune receptor retinoic acid-inducible gene I (RIG-I) by its specific ligand 5'-triphosphate RNA (3pRNA) triggers anti-tumor immunity, which is dependent on natural killer (NK) cell activation and cytokine induction. However, to date, RIG-I expression and the functional consequences of RIG-I activation in NK cells have not been examined. Here, we show for the first time the expression of RIG-I in human NK cells and their activation upon RIG-I ligand (3pRNA) transfection. 3pRNA-activated NK cells killed melanoma cells more efficiently than NK cells activated by type I interferon. Stimulation of RIG-I in NK cells specifically increased the surface expression of membrane-bound TNF-related apoptosis-inducing ligand (TRAIL) on NK cells, while activated NK cell receptors were not affected. RIG-I-induced membrane-bound TRAIL initiated death-receptor-pathway-mediated apoptosis not only in allogeneic but also in autologous human leukocyte antigen (HLA) class I-positive and HLA class I-negative melanoma cells. These results identify the direct activation of RIG-I in NK cells as a novel mechanism for how RIG-I can trigger enhanced NK cell killing of tumor cells, underscoring the potential of RIG-I activation for tumor immunotherapy.

RIG-I Signaling via MAVS Is Dispensable for Survival in Lethal Influenza Infection In Vivo.

Retinoic acid-inducible gene I (RIG-I) is an important regulator of virus-induced antiviral interferons (IFNs) and proinflammatory cytokines. It requires interaction with an adaptor molecule, mitochondrial antiviral-signaling protein (MAVS), to activate downstream signaling pathways. To elucidate the mechanism(s) by which RIG-I-dependent recognition of IAV infection in vivo triggers innate immune responses, we infected mutant mice lacking RIG-I or MAVS with influenza A virus (IAV) and measured their innate immune responses. As has previously been demonstrated with isolated deletion of the virus recognition receptors TLR3, TLR7, and NOD2, RIG-I or MAVS knockout (KO) did not result in higher mortality and did not reduce IAV-induced cytokine responses in mice. Infected RIG-I KO animals displayed similar lung inflammation profiles as did WT mice, in terms of the protein concentration, total cell count, and inflammatory cell composition in the bronchoalveolar lavage fluid. RNA-Seq results demonstrated that all types of mice exhibited equivalent antiviral and inflammatory gene responses following IAV infection. Together, the results indicated that although RIG-I is important in innate cytokine responses in vitro, individual deletion of the genes encoding RIG-I or MAVS did not change survival or innate responses in vivo after IAV infection in mice.

Fish-specific finTRIM FTR36 triggers IFN pathway and mediates inhibition of viral replication

The tripartite motif (TRIM) family involves many cellular processes, including fundamental functions in antiviral immunity. Antiviral activities of TRIMs are reported in a variety of patterns, and one of the most significant channels is related to the activation of the type-I interferon (IFN) pathway. In this study, we described a fintrim (ftr) gene named ftr36, which is mainly expressed in the gills, skin, and intestines. This study shows that ftr36 encodes a protein affording a potent antiviral effect. In vitro, overexpression of FTR36 mediated an upregulated pattern of recognition receptor retinoic acid–inducible gene I (RIG-I), interferon regulatory factor 3/7(IRF3/7), IFN, and IFN-stimulated genes (ISGs) expression. Thereby, FTR36 expression could afford host defense against the spring viremia of carp virus (SVCV) and the giant salamander iridovirus (GSIV). With the deletion of the RING domain or B30.2 domain separately, the antiviral ability of FTR36 was abolished partially and almost lost its ability to activate the IFN-pathway. These findings indicate that both RING and B30.2 domains are indispensable for the antiviral activity of FTR36. Altogether, this study described a finTRIM FTR36, which can activate IFN-pathways and stimulate ISGs to provide host defense against viral infections.

Characterization of signaling pathways regulating the expression of pro-inflammatory long form thymic stromal lymphopoietin upon human metapneumovirus infection

Thymic stromal lymphopoietin (TSLP) is associated with several allergic diseases including asthma. Two isoforms of TSLP exist in humans, a long form (lfTSLP) and a short form (sfTSLP), displaying distinct immunological functions. Recently, TSLP was found to be upregulated in human airway cells upon human metapneumovirus (hMPV) infection, yet it remains unclear if the two isoforms are regulated differently during hMPV infection. Importantly, the molecular mechanisms underlying hMPV-mediated TSLP induction remain undescribed. In this study, we characterized the expression and regulation of TSLP in hMPV-infected human airway cells. We demonstrated that hMPV strongly induced the expression of pro-inflammatory lfTSLP in human airway epithelial cells and lung fibroblasts. Further, knockdown of pattern recognition receptors retinoic acid-inducible gene I (RIG-I) or Toll-like receptor 3 (TLR3), as well as downstream signal transducers, abrogated hMPV-mediated lfTSLP induction. Importantly, silencing of TANK-binding kinase 1 (TBK1) also impaired hMPV-mediated lfTSLP induction, which could be attributed to compromised NF-κB activation. Overall, these results suggest that TBK1 may be instrumental for hMPV-mediated activation of NF-κB downstream RIG-I and TLR3, leading to a specific induction of lfTSLP in hMPV-infected human airway cells.

MCPIP1 attenuates the innate immune response to influenza A virus by suppressing RIG-I expression in lung epithelial cells.

The pattern recognition receptor retinoic acid-inducible gene I (RIG-I) reportedly plays a key role in sensing influenza A virus (IAV) infection and activating type I interferon (IFN) response. MCP-1-induced protein 1 (MCPIP1) can directly degrade cytokine mRNAs, such as IL-6, IL-12, IL-1β, and IL-2, by functioning as an RNase. Here, we initially observed that MCPIP1 exhibited virus supportive functions later in the course of IAV infection in A549 cells, and negatively regulated IAV-induced RIG-I-dependent innate antiviral response. Exogenous overexpression of MCPIP1 suppressed the expression of RIG-I, whereas shRNA-mediated inhibition of endogenous MCPIP1 enhanced RIG-I expression. The results of experiments with actinomycin D and luciferase assay demonstrated that MCPIP1 reduced RIG-I expression through destabilizing its mRNA. Various mutants of functional domains of MCPIP1 further confirmed that the inhibitory effect of MCPIP1 on RIG-I expression required RNase activity but not deubiquitinase activity. Finally, the overexpression of several IAV proteins, which have the ability to inhibit the host IFN response at different levels, induced MCPIP1 expression, especially non-structural protein 1 (NS1). Conclusively, these data demonstrate the MCPIP1 contributes to attenuate IAV-induced host antiviral response by suppressing RIG-I expression.

Abstract B44: Selective stimulation of RIG-I with a novel synthetic RNA induces strong anti-tumor immunity in mouse tumor models

Abstract We describe a novel immunotherapy approach in which a natural viral defense system is harnessed to stimulate anti-tumor immunity. Cancer immunotherapy has revolutionized oncology in recent years, yet many tumors become resistant or do not respond to current treatments such as checkpoint inhibitors. Stimulation of the innate immune system opens a new therapeutic strategy that could be combined effectively with other immunotherapeutic regimens. The ubiquitously expressed cytosolic RNA receptor retinoic acid inducible gene I (RIG-I) recognizes double-stranded RNA bearing a 5'-triphosphate. RIG-I plays a prominent role in antiviral defense. Its activation induces apoptosis preferentially in tumor cells and simultaneously activates the innate immune system via type I interferon (IFN) signaling. We developed an optimized, fully synthetic oligonucleotide, designated RGT100, which is a RIG-I selective ligand. RGT100 activates the RIG-I pathway leading to the induction of cytokines, including IFN-α and IFN-β. The treatment of tumor-bearing mice with RGT100 induced potent anti-tumor activity against large established B16 melanoma and CT26 colon carcinoma tumors. RGT100 also was efficacious in a spontaneous melanoma model. Histological and flow cytometric analysis of the tumors revealed infiltration and activation of immune cells after RGT100 treatment. RGT100 induced a strong and durable type I IFN response in tumors. Treatment of subcutaneous tumors by intratumor injection led to efficacy of both the treated tumors as well as untreated contralateral tumors. Furthermore, systemic delivery of RGT100 was efficacious against both local subcutaneous B16 melanoma as well as lung metastases. Data support both rapid natural killer (NK) cell-mediated and long-term T cell-mediated anti-tumor activities. Depletion of NK cells blocked the immediate anti-tumor activity of RGT100. In summary, Rigontec's RIG-I-selective ligand RGT100 shows strong anti-tumor activity in several clinically relevant mouse tumor models, while bearing an advantageous safety profile. RGT100 is rapidly progressing toward clinical evaluation in cancer patients. This agent will be evaluated both for single-agent activity as well as in combination with checkpoint inhibitors. Citation Format: Jim Barsoum, Marcel Renn, Christine Schuberth, Christopher Jakobs, Anna Schwickart, Martin Schlee, Jasper van den Boorn, Gunther Hartmann. Selective stimulation of RIG-I with a novel synthetic RNA induces strong anti-tumor immunity in mouse tumor models. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B44.

RIG-I activation induces the release of extracellular vesicles with antitumor activity

ABSTRACTActivation of the innate immune receptor retinoic acid-inducible gene I (RIG-I) by its specific ligand 5′-triphosphate-RNA (3pRNA) triggers antitumor immunity predominantly via NK cell activation and direct apoptosis induction in tumor cells. However, how NK cells are mobilized to attack the tumor cells remains elusive. Here, we show that RIG-I activation induced the secretion of extracellular vesicles (EVs) from melanoma cells, which by themselves revealed antitumor activity in vitro and in vivo. RIG-I-induced EVs from melanoma cells exhibited an increased expression of the NKp30-ligand (BAG6, BAT3) on their surface triggering NK cell-mediated lysis of melanoma cells via activation of the cytotoxicity NK cell-receptor NKp30. Moreover, systemic administration of RIG-I-induced melanoma-EVs showed a potent antitumor activity in a melanoma mouse model in vivo. In conclusion, our data establish a new RIG-I-dependent pathway leading to NK cell-mediated tumor cell killing.

Abstract P3-14-07: Intra-cellular dsRNA receptor RIG-I: A ubiquitous novel target for treatment of chemotherapy drug resistant breast cancer

Abstract INTRODUCTION: Toll-like receptor 3 (TLR3), the cell surface receptor for double stranded RNA (dsRNA), expressed in ∼40% of advanced primary breast cancers (T2/T3/N+) has been shown to be an effective therapeutic target for synthetic dsRNA poly A:U in combination with radiation and adjuvant chemotherapy, producing a significant decrease in risk of metastatic relapse (HR 1.85-2.0; 1.03-3.89; Cancer Res;71:1607). AIM: The present pre-clinical study aimed to explore the therapeutic potential of a ubiquitously expressed intracellular dsRNA receptor, retinoic-acid-inducible gene-I (RIG-I) - a cytoplasmic pathogen recognition receptor directed at pathogen-associated molecular pattern (PAMP) motifs to differentiate viral from cellular dsRNAs. We have shown Ampligen, poly I:C12U (a synthetic dsRNA polymer designed to rapidly degrade in vivo to prevent the toxicity of long dsRNA polymers such as poly I:C) is capable of entering cells in its fragmented dsRNA oligomeric form (&amp;lt;1–2 kb) optimal for activation of intra-cellular RIG-I (J Exp Med 2008;205:1601–1610). METHODOLOGY &amp; RESULTS: Preliminary experiments with Ampligen on several human breast cancer cell lines (MCF-7 &amp; MDA-MB 453) and normal human mammary epithelial &amp; fibroblast cell lines (HMEC &amp; HFC) unexpectedly showed it to consistently cause a significant loss of cell viability (CellTiter-Glo Luminescent Cell Viability Assay) in p53-deficient drug resistant (5-FU/doxorubicin) MDA-MB 453 cell line in contrast to cell growth arrest (Guava cell Cycle Assay) in p53 wild type MCF-7 cancer and the two non-neoplastic cells lines. This selective effect was confirmed using syngeneic clones of MCF-7 breast cancer cells stably transfected with a dominant negative p53 construct or vector alone: p53-function blocked (DD1) vs p53-function active (EV1) MCF-7 cell lines, respectively (J Nucl Med 2006; 47:1525–1530), and shown to be associated with RIG-I specific mRNA induction (RT-PCR) and Type I interferon pathway activation both inhibited by BX795 (selective inhibitor of IRF3 activation and IFN-b production). Decitabine (DNA demethylating drug capable of intra-cellular generation of dsRNA through transcriptional activation of Alu retrotransposons - PNAS 2012; December 10: E89–E98 ) was next tested as an alternative source of intra-cellular dsRNA, and found to produce results similar to Ampligen on DD1 and EV1. Work is in progress to examine dose response and time-course relationships of the effects Ampligen or decitabine added singly or in combination with chemotherapeutic drug (e.g. doxorubicin) on DD1 and triple negative breast cancer cell lines such as MDA-MB 453 to explore potential therapeutically most effective protocols. PROVISIONAL CONCLUSIONS: Intra-cellular dsRNA receptor RIG-I constitutively expressed in all cells offers a more ubiquitous target compared to TLR3 for the treatment of breast cancer. Ampligen and decitabine with their selective growth inhibitory effect on drug resistant p53-deficient breast cancer cell lines, merit testing as novel drugs for treatment of p53-deficient drug resistant breast cancer e.g. triple negative breast cancer frequently (∼70%) associated with drug resistance and altered p53 status. Citation Format: Jasani B, Navabi N, Barrett-Lee P, Thompson A, Chester J, Mason M. Intra-cellular dsRNA receptor RIG-I: A ubiquitous novel target for treatment of chemotherapy drug resistant breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-07.

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

RNAs with 5'-triphosphate (ppp) are detected in the cytoplasm principally by the innate immune receptor Retinoic Acid Inducible Gene-I (RIG-I), whose activation triggers a Type I IFN response. It is thought that self RNAs like mRNAs are not recognized by RIG-I because 5'ppp is capped by the addition of a 7-methyl guanosine (m7G) (Cap-0) and a 2'-O-methyl (2'-OMe) group to the 5'-end nucleotide ribose (Cap-1). Here we provide structural and mechanistic basis for exact roles of capping and 2'-O-methylation in evading RIG-I recognition. Surprisingly, Cap-0 and 5'ppp double-stranded (ds) RNAs bind to RIG-I with nearly identical Kd values and activate RIG-I's ATPase and cellular signaling response to similar extents. On the other hand, Cap-0 and 5'ppp single-stranded RNAs did not bind RIG-I and are signaling inactive. Three crystal structures of RIG-I complexes with dsRNAs bearing 5'OH, 5'ppp, and Cap-0 show that RIG-I can accommodate the m7G cap in a cavity created through conformational changes in the helicase-motif IVa without perturbing the ppp interactions. In contrast, Cap-1 modifications abrogate RIG-I signaling through a mechanism involving the H830 residue, which we show is crucial for discriminating between Cap-0 and Cap-1 RNAs. Furthermore, m7G capping works synergistically with 2'-O-methylation to weaken RNA affinity by 200-fold and lower ATPase activity. Interestingly, a single H830A mutation restores both high-affinity binding and signaling activity with 2'-O-methylated dsRNAs. Our work provides new structural insights into the mechanisms of host and viral immune evasion from RIG-I, explaining the complexity of cap structures over evolution.

Protective Role of LGP2 in Influenza Virus Pathogenesis

Influenza A virus triggers a contagious respiratory disease that can cause considerable morbidity and mortality. Using an in vitro approach, we previously demonstrated that the pattern recognition receptor retinoic acid-inducible gene I (RIG-I) plays a key role in influenza A virus-mediated immune response. However, the importance of RIG-I signaling in vivo has not been thoroughly examined, because of the lack of an appropriate mouse models. To circumvent this issue, we generated a new transgenic mouse overexpressing LGP2 (hereafter, "LGP2 TG mice"), a major regulator of the RIG-I signaling pathway. The time course of several parameters was compared in infected wild-type and LGP2 TG mice. We found that LGP2 TG mice displayed significantly reduced inflammatory mediators and a lower leukocyte infiltration into the bronchoalveolar airspace. More importantly, LGP2 TG mice had a significant survival advantage. Hence, our in vivo study reveals that LGP2 is a major downregulator of the influenza A virus-triggered detrimental inflammatory response.