RIG-I-like Receptors Signaling Research Articles

H-Ras Exerts Opposing Effects on Type I Interferon Responses Depending on Its Activation Status.

Using shRNA high-throughput screening, we identified H-Ras as a regulator of antiviral activity, whose depletion could enhance Sindbis virus replication. Further analyses indicated that depletion of H-Ras results in a robust increase in vesicular stomatitis virus infection and a decrease in Sendai virus (SeV)-induced retinoic acid-inducible gene-I-like receptor (RLR) signaling. Interestingly, however, ectopic expression of wild-type H-Ras results in a biphasic mode of RLR signaling regulation: while low-level expression of H-Ras enhances SeV-induced RLR signaling, high-level expression of H-Ras significantly inhibits this signaling. The inhibitory effects correlate with the activation status of H-Ras. As a result, oncogenic H-Ras, H-RasV12, strongly inhibits SeV-induced IFN-β promoter activity and type I interferon signaling. Conversely, the positive effects exerted by H-Ras on RLR signaling are independent of its signaling activity, as a constitutively inactive form of H-Ras, H-RasN17, also positively regulates RLR signaling. Mechanistically, we demonstrate that depletion of H-Ras reduces the formation of MAVS–TNF receptor-associated factor 3 signaling complexes. These results reveal that the H-Ras protein plays a role in promoting MAVS signalosome assembly in the mitochondria, whereas oncogenic H-Ras exerts a negative effect on type I IFN responses.

CXCL10 is produced in hepatitis A virus-infected cells in an IRF3-dependent but IFN-independent manner

Acute hepatitis A caused by hepatitis A virus (HAV) infection is accompanied by severe liver injury in adult patients, and the liver injury is associated with the production of chemokines. Herein, we investigated the mechanism of how HAV infection induces the production of CXCR3 and CCR5 chemokines, such as CXCL10, CCL4 and CCL5. The production of CXCL10, CCL4 and CCL5 was markedly increased by HAV (HM-175/18f) infection in the culture of primary human hepatocytes and HepG2 cells. In particular, CXCL10 was produced in HAV-infected cells, not in neighboring uninfected cells. Moreover, these chemokines were significantly increased in the sera of acute hepatitis A patients. The production of IFN-λs was also robustly induced by HAV infection, and the blocking of secreted IFN-λs partially abrogated the production of CCL4 and CCL5 in HAV-infected cells. However, CXCL10 production was not decreased by the blocking of IFN-λs. Instead, CXCL10 production was reduced by silencing the expression of RIG-I-like receptor (RLR) signal molecules, such as mitochondrial antiviral signaling protein and interferon regulatory factor 3, in HAV-infected cells. In conclusion, HAV infection strongly induces the production of helper 1 T cell-associated chemokines, particularly CXCL10 via RLR signaling, even without secreted IFNs.

Pattern recognition receptors in grass carp Ctenopharyngodon idella: I. Organization and expression analysis of TLRs and RLRs

Pattern recognition receptors (PRRs) play indispensable roles in the immune responses against invading pathogens. In the present study, we systematically identified and characterized Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) as well as their adaptors in grass carp (Ctenopharyngodon idella). A comprehensive analysis of BLAST and other bioinformatics methods showed that C. idella TLR family consist of 21 members and their adaptors contain four members. Phylogenetic analyses confirmed the existence of six TLR subfamilies (TLR1, 3, 4, 5, 7 and 11 subfamily) in C. idella and revealed their homologous relationships with other species. Most C. idella TLRs possess three typical structural features of TLR protein family: LRR, TM and TIR domains. Meanwhile, RLR family consist of three conserved members (RIG-I, MDA5 and LGP2) as well as two adaptors (IPS-1 and STING) in C. idella. mRNA expression analyses of TLRs, RLRs and their adaptors indicated that most members are sustainably expressed in multiple tissues before and after grass carp reovirus (GCRV) or Aeromonas hydrophila infection, while TLR9, TLR20a/b, TLR25, TIRAP, SARM1 and STING are transiently expressed in specific tissues. TLRs are transmembrane receptors with few introns, while RLRs are cytoplasmic receptors with plenty of introns. TLRs and RLRs interact with adaptors to perform their functions via various signaling pathways. In conclusion, this study systematically explores the characteristics of TLRs and RLRs in C. idella and provides evidence for the response patterns after viral and/or bacterial infection in vivo. These results contribute to studying the regulation mechanisms of TLR and RLR signaling pathways, and deeply understanding fish immune responses against pathogen infection.

West Nile virus antagonism of a RIG-I, type I interferon, and STAT5 signaling axis in human dendritic cells

Abstract West Nile virus (WNV) is a neurotropic flavivirus and the leading cause of mosquito-borne encephalitis in the United States. Murine models have found dendritic cells (DCs) are pivotal for viral control and programming of antiviral immunity in a RIG-I like receptor (RLR)- and type I interferon (IFN)-dependent manner. To better understand human WNV infection, we evaluated infection of monocyte derived DCs (moDCs). To define the antiviral landscape within moDCs, we combined mRNA sequencing with weighted gene co-expression analysis. Using molecular signatures for RLR and IFN signaling as filters, we found WNV partially induced expression of genes related to antiviral immunity. Specifically, WNV induced delayed IFN responses and failed to up-regulate inflammatory (IL-1β, IL-6, IL-12, TNF) or T cell co-stimulatory molecules (CD86, CD40, MHC II). Using cis-regulatory sequence analysis, STAT5 was identified as a regulatory node involved in RLR- and IFN-induced DC responses, but was minimally activated during WNV infection. Infected DCs failed to secrete multiple STAT5 signaling cytokines (GM-CSF, IL-4, IL-15) and STAT5 was not phosphorylated during WNV infection. Mechanistically, WNV blocked STAT5 phosphorylation downstream of RIG-I, IFN, and IL-4 signaling. Zika virus (ZIKV), a closely related flavivirus that also fails to activate human DCs, also blocked STAT5 phosphorylation. Our findings suggest STAT5 antagonism is a conserved mechanism used by pathogenic flaviviruses to subvert host immunity. In summary, we employed a systems biology approach to define the antiviral landscape within human DCs, identifying viral antagonism of a RIG-I, IFN, and STAT5 signaling axis that subverts DC immunogenicity during WNV and ZIKV infection.

Transmissible gastroenteritis virus infection induces NF-κB activation through RLR-mediated signaling

Transmissible gastroenteritis virus (TGEV) is a porcine enteric coronavirus which causes lethal severe watery diarrhea in piglets. The pathogenesis of TGEV is strongly associated with inflammation. In this study, we found that TGEV infection activates transcription factors NF-κB, IRF3 and AP-1 in a time- and dose-dependent manner in porcine kidney cells. Treatment with the NF-κB-specific inhibitor BAY11-7082 significantly decreased TGEV-induced proinflammatory cytokine production, but did not affect virus replication. Phosphorylation of NF-κB subunit p65 and proinflammatory cytokine production were greatly decreased after knockdown of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) or its adaptors MAVS and STING, while only slight reduction was observed in cells following silencing of Toll-like receptor adaptors, MyD88 and TRIF. Furthermore, TGEV infection significantly upregulated mRNA expression of RIG-I and MDA5. Taken together, our results indicate that the RLR signaling pathway is involved in TGEV-induced inflammatory responses.

Negative regulators of the RIG-I-like receptor signaling pathway.

Upon recognition of specific molecular patterns on microbes, host cells trigger an innate immune response, which culminates in the production of type I interferons, proinflammatory cytokines and chemokines, and restricts pathogen replication and spread within the host. At each stage of this response, there are stimulatory and inhibitory signals that regulate the magnitude, quality, and character of the response. Positive regulation promotes an antiviral state to control and eventually clear infection, whereas negative regulation dampens inflammation and prevents immune-mediated tissue damage. An overexuberant innate response can lead to cell and tissue destruction, and the development of spontaneous autoimmunity. The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), RIG-I and melanoma differentiation-associated gene 5 (MDA5), belong to a family of cytosolic host RNA helicases that recognize distinct nonself RNA signatures and trigger innate immune responses against several RNA viruses by signaling through the essential adaptor protein mitochondrial antiviral signaling (MAVS). The RLR signaling pathway is tightly regulated to maximize antiviral immunity and minimize immune-mediated pathology. This review highlights contemporary findings on negative regulators of the RLR signaling pathway, with specific focus on the proteins and biological processes that directly regulate RIG-I, MDA5 and MAVS signaling function.

Grass Carp Laboratory of Genetics and Physiology 2 Serves As a Negative Regulator in Retinoic Acid-Inducible Gene I- and Melanoma Differentiation-Associated Gene 5-Mediated Antiviral Signaling in Resting State and Early Stage of Grass Carp Reovirus Infection.

Laboratory of genetics and physiology 2 (LGP2) is a key component of RIG-I-like receptors (RLRs). However, the lack of the caspase recruitment domains (CARDs) results in its controversial functional performance as a negative or positive regulator in antiviral responses. Especially, no sufficient evidence uncovers the functional mechanisms of LGP2 in RLR signaling pathways in teleost. Here, negative regulation mechanism of LGP2 in certain situations in retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5)-mediated antiviral responses was identified in Ctenopharyngodon idella kidney cells. LGP2 overexpression inhibits synthesis and phosphorylation of interferon regulatory factor 3/7 (IRF3/7), and mRNA levels and promoter activities of IFNs and NF-κBs in resting state and early phase of grass carp reovirus (GCRV) infection. Knockdown of LGP2 obtains opposite effects. Luciferase report assay indicates that LGP2 works at the upstream of RIG-I and MDA5. LGP2 binds to RIG-I and MDA5 with diverse domain preference and which is independent of GCRV infection. Furthermore, LGP2 restrains K63-linked ubiquitination of RIG-I and MDA5 in various degrees. These differences result in disparate repressive mechanisms of LGP2 to RIG-I- and MDA5-mediated signal activations of IFN-β promoter stimulator 1 and mediator of IRF3 activation. Interestingly, LGP2 also inhibits K48-linked RIG-I and MDA5 ubiquitination to suppress proteins degradation, which guarantees the basal protein levels for subsequently rapid signal activation. All these results reveal a mechanism that LGP2 functions as a suppressor in RLR signaling pathways to maintain cellular homeostasis in resting state and early phase during GCRV infection.

Non-Canonical Role of IKKα in the Regulation of STAT1 Phosphorylation in Antiviral Signaling.

Non-self RNA is recognized by retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), inducing type I interferons (IFNs). Type I IFN promotes the expression of IFN-stimulated genes (ISGs), which requires the activation of signal transducer and activator of transcription-1 (STAT1). We previously reported that dsRNA induced STAT1 phosphorylation via a type I IFN-independent pathway in addition to the well-known type I IFN-dependent pathway. IκB kinase α (IKKα) is involved in antiviral signaling induced by dsRNA; however, its role is incompletely understood. Here, we explored the function of IKKα in RLR-mediated STAT1 phosphorylation. Silencing of IKKα markedly decreased the level of IFN-β and STAT1 phosphorylation inHeH response to dsRNA. However, the inhibition of IKKα did not alter the RLR signaling-mediated dimerization of interferon responsive factor 3 (IRF3) or the nuclear translocation of nuclear factor-κB (NFκB). These results suggest a non-canonical role of IKKα in RLR signaling. Furthermore, phosphorylation of STAT1 was suppressed by IKKα knockdown in cells treated with a specific neutralizing antibody for the type I IFN receptor (IFNAR) and in IFNAR-deficient cells. Collectively, the dual regulation of STAT1 by IKKα in antiviral signaling suggests a role for IKKα in the fine-tuning of antiviral signaling in response to non-self RNA.

MicroRNA-145 regulates the RLR signaling pathway in miiuy croaker after poly(I:C) stimulation via targeting MDA5

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that participate in diverse biological processes via degrading the target mRNAs or repressing translation. In this study, the regulation of miRNA to the RLR (RIG-I-like receptor) signaling pathway by degrading the target mRNAs was researched in miiuy croaker. MDA5, a microRNA-145-5p (miR-145-5p) putative target gene, was predicted by bioinformatics, and the target sites from the 3'untranslated region of MDA5 transcripts were confirmed using luciferase reporter assays. Pre-miR-145 was more effective in inhibiting MDA5 than miR-145-5p mimic, and the effect was dose- and time-dependent. The expression patterns of miR-145-5p and MDA5 were analyzed in liver and kidney from miiuy croaker. Results implied that miR-145-5p may function via degrading the MDA5 mRNAs, thereby regulating the RLR signaling pathway. Studies on miR-145-5p will enrich knowledge of its functions in immune response regulation in fish, as well as offer a basis for regulatory networks that are composed of numerous miRNAs.

Identification of sea perch (Lateolabrax japonicus) ribonucleoprotein PTB-Binding 1 involved in antiviral immune response against RGNNV

RIG-I-like receptors (RLRs) can recognize viral RNA and initiate innate antiviral response. In earlier studies, we demonstrated that RLRs were implicated in the antiviral immunity against RGNNV in the seawater fish sea perch (Lateolabrax japonicus). However, potential regulators of RLRs-mediated signaling pathways involved in RGNNV infection remain unclear. In this study, a novel ribonucleoprotein PTB-binding 1 (Raver1) of sea perch (LjRAVER1) was identified for the first time. The cDNA of LjRAVER1 was 4066 bp in length and encoded a deduced polypeptide of 733 amino acids. Phylogenetic analysis revealed a closer affinity of LjRAVER1 with Larimichthys Crocea Raver1. LjRAVER1 mRNA was constitutively expressed in all 10 sampled tissues, and rapidly and significantly increased in vivo upon RGNNV infection. Time course analysis showed that LjRAVER1 transcripts were significantly increased both in vivo and in vitro after RGNNV infection. Viral infection and poly I:C treatment caused translocation of LjRAVER1 from the nucleus to the cytoplasm. Ectopic expression of LjRAVER1 increased the transcription level of several RLR signaling pathway related genes inducible by poly I:C treatment in vitro. Moreover, the viral gene transcription and virus production of RGNNV were significantly decreased in LjRAVER1 overexpressing cells. Luciferase reporter assays demonstrated that overexpression of LjRAVER1 significantly increased the promoter activity of zebrafish IFN1. Taken together, these findings indicated that LjRAVER1 might be an important component of RLR signaling pathway and involved in RLR pathway-mediated IFN response in sea perch.

Absence of autophagy promotes apoptosis by modulating the ROS-dependent RLR signaling pathway in classical swine fever virus-infected cells

ABSTRACTA growing number of studies have demonstrated that both macroautophagy/autophagy and apoptosis are important inner mechanisms of cell to maintain homeostasis and participate in the host response to pathogens. We have previously reported that a functional autophagy pathway is trigged by infection of classical swine fever virus (CSFV) and is required for viral replication and release in host cells. However, the interplay of autophagy and apoptosis in CSFV-infected cells has not been clarified. In the present study, we demonstrated that autophagy induction with rapamycin facilitates cellular proliferation after CSFV infection, and that autophagy inhibition by knockdown of essential autophagic proteins BECN1/Beclin 1 or MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) promotes apoptosis via fully activating both intrinsic and extrinsic mechanisms in CSFV-infected cells. We also found that RIG-I-like receptor (RLR) signaling was amplified in autophagy-deficient cells during CSFV infection, which was closely linked to the activation of the intrinsic apoptosis pathway. Moreover, we discovered that virus infection of autophagy-impaired cells results in an increase in copy number of mitochondrial DNA and in the production of reactive oxygen species (ROS), which plays a significant role in enhanced RLR signaling and the activated extrinsic apoptosis pathway in cultured cells. Collectively, these data indicate that CSFV-induced autophagy delays apoptosis by downregulating ROS-dependent RLR signaling and thus contributes to virus persistent infection in host cells.

Accessory Factors of Cytoplasmic Viral RNA Sensors Required for Antiviral Innate Immune Response

Type I interferon (IFN) induces many antiviral factors in host cells. RIG-I-like receptors (RLRs) are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs) and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and, thus, cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway.

A systems biology approach unravels the host response to West Nile virus infection in human dendritic cells

Abstract West Nile virus (WNV) is a neurotropic Flavivirus and the leading cause of mosquito-borne encephalitis in the United States. Previous studies in mice have found that dendritic cells (DCs) are pivotal for viral control and programming of antiviral immunity in a RIG-I like receptor (RLR) and type I interferon (IFN) dependent manner. To gain a better understanding of human WNV infection, we evaluated infection of human monocyte DCs (moDCs). We found that moDCs support productive WNV infection and that stimulation of RLR and type I IFN signaling potently restricts viral replication. Most intriguingly, we observed significant variability in viral infection of moDCs generated from different donors, ranging from 6–56% of cells infected by 48 hours post infection. To define the antiviral landscape during WNV infection, we performed mRNA sequencing and found that, in contrast to RLR and type I IFN signaling, WNV infection partially triggers expression of antiviral defense response related genes. Despite strong induction of IFNβ during WNV infection, we observed minimal expression of cytokines that promote antiviral T cell immunity (IL-4, IL-12, IL-7, IL-15) and costimulatory molecules (CD80, CD86, CD40) at the transcriptional and protein level. Using weighted gene co-expression network analysis combined with cis-regulatory sequence analysis, we identified STAT5 as an important regulatory node downstream of RLR signaling. Most intriguingly, we found that STAT5 was not induced during WNV infection, suggesting that WNV dampens DC activation by preventing STAT5 activation. Further studies are underway to better understand how RLR signaling promotes STAT5 activation and to define the viral factors that regulate STAT5 activation.

The role of the RIG-I like receptors in mediating CD8+ T cell immunity during virus infection

Abstract West Nile virus (WNV) is a mosquito-borne Flavivirus that is a major cause of virus-induced encephalitis in the US. Upon virus infection, pattern recognition receptors (PRRs) recognize viral PAMPs to trigger potent antiviral defense programs. In particular, the RIG-I-like receptors (RLRs) are a family of cytosolic PRRs that comprise of RIG-I, MDA5 and LGP2 and are expressed in nearly every host cell, including antigen-presenting cells. We have previously demonstrated that RLRs as well as their central adaptor protein mitochondria antiviral signaling (MAVS) are critical for mediating immune protection against WNV infection. Recently, we discovered that the RLRs function in a non-canonical manner within CD8+ T cells to promote T cell expansion, survival and effector function during virus infection. Specifically, we found that the RLR LGP2 plays an essential role in promoting virus-specific CD8+ T cell survival and effector functions. To better understand this novel RLR pathway in T cells, we evaluated the cell-intrinsic roles of RIG-I and MAVS, two known interacting partners of LGP2. Following in vitro CD8+T cell stimulation, we found that RIG-I−/−, but not MAVS−/−, CD8+ T cells displayed higher proliferation rates than wild type (WT) cells. Adoptive transfer studies into RAG−/− mice revealed that WNV-specific CD8+ T cells from RIG-I−/− or MAVS−/− mice were present in lower percentages than WT CD8+ T cells during virus infection. To define the non-canonical RLR signaling, we are undertaking a genetics approach to screen proteins associated with the RLR interactome in CD8+ T cells. Altogether, our data indicate that RLRs function in a noncanonical manner within CD8+ T cells to regulate antigen-specific responses during virus infection.

Amplified RLR signaling activation through an interferon-stimulated gene-endoplasmic reticulum stress-mitochondrial calcium uniporter protein loop.

Type I interferon (IFN-I) is critical for a host against viral and bacterial infections via induction of hundreds of interferon-stimulated genes (ISGs), but the mechanism underlying the regulation of IFN-I remains largely unknown. In this study, we first demonstrate that ISG expression is required for optimal IFN-β levels, an effect that is further enhanced by endoplasmic reticulum (ER) stress. Furthermore, we identify mitochondrial calcium uniporter protein (MCU) as a mitochondrial antiviral signaling protein (MAVS)-interacting protein that is important for ER stress induction and amplified MAVS signaling activation. In addition, by performing an ectopic expression assay to screen a library of 117 human ISGs for effects on IFN-β levels, we found that tumor necrosis factor receptor 1 (TNFR1) significantly increases IFN-β levels independent of ER stress. Altogether, our findings suggest that MCU and TNFR1 are involved in the regulation of RIG-I-like receptors (RLR) signaling.

Measuring Monomer-to-Filament Transition of MAVS as an In Vitro Activity Assay for RIG-I-Like Receptors.

During viral infection, the innate immune RIG-I like receptors (RLRs) recognize viral double stranded RNA (dsRNA) and trigger filament assembly of the adaptor protein Mitochondrial Anti-viral Signaling protein (MAVS). The MAVS filament then activates anti-viral signaling events including the up-regulation of type I interferon expression. In recent years, much insight has been gained into how RLRs recognize dsRNA, but the precise mechanism of how activated RLRs stimulate MAVS filament formation remains less understood. In this chapter, we describe an in vitro reconstitution assay that we have previously developed to study the RLR-catalyzed filament assembly of MAVS. We provide technical guidance for purifying the caspase activation recruitment domain (CARD) of MAVS (MAVS(CARD)) as a functional monomer and also preformed filament seed. We also describe the methods to monitor the monomer-to-filament transition of MAVS(CARD) upon stimulation. This protocol provides a minimalist approach to studying RLR signaling events and can potentially be applied to elucidate signaling mechanisms of other innate immune receptors, such as Toll-like receptors and inflammasomes, that involve higher order assemblies of CARDs or related domains for their downstream signal activation.

TLR and RLR Signaling Are Reprogrammed in Opposite Directions after Detection of Viral Infection

Innate immune recognition of RNA is key for the initiation of immunity in response to viral infection. Although the factors controlling the detection of viral RNA by innate immune receptors in host cells are increasingly well understood, little is known about the dynamic changes in signaling after the initial triggering of these receptors. In this study, we report that preconditioning with the synthetic dsRNA polyinosinic-polycytidylic acid [poly(I:C)], a mimetic of viral RNA, rapidly reprograms murine APCs by simultaneously augmenting sensitivity of endosomal TLRs and inhibiting activation of RIG-I-like receptors (RLRs) in an IFN-β-dependent manner. These changes in receptor sensitivity were also seen in vivo after treatment of mice with poly(I:C). Mechanistically, the increased sensitivity of the TLR pathway was associated with elevated MAPK and NF-κB activity. The RLR response was inhibited downstream of TANK-binding kinase-1, resulting in decreased IFN regulatory factor 3 phosphorylation. Reprogramming of pattern-recognition receptor signaling also occurred after viral infection, because infection of host cells with Sendai virus or their exposure to supernatant from virus-infected cells induced the same changes in TLR and RLR sensitivity as poly(I:C). Thus, innate recognition of viral infection critically modifies responses to pattern-recognition receptor stimulation. These dynamic adaptations to infection may reinforce antiviral immunity and at the same time serve to limit pathological inflammation.

Retinoic acid-inducible gene-I-like receptor (RLR)-mediated antiviral innate immune responses in the lower respiratory tract: Roles of TRAF3 and TRAF5

Upon viral infection, the cytoplasmic viral sensor retinoic acid-inducible gene-I (RIG-I) recognizes viral RNA to activate antiviral signaling to induce type I interferon (IFN). RIG-I-like receptors (RLRs) activate antiviral signaling in a tissue-specific manner. The molecular mechanism underlying antiviral signaling in the respiratory system remains unclear. We studied antiviral signaling in the lower respiratory tract (LRT), which is the site of many harmful viral infections. Epithelial cells of the LRT can be roughly divided into two groups: bronchial epithelial cells (BECs) and pulmonary alveolar epithelial cells (AECs). These two cell types exhibit different phenotypes; therefore, we hypothesized that these cells may play different roles in antiviral innate immunity. We found that BECs exhibited higher antiviral activity than AECs. TNF receptor-associated factor 3 (TRAF3) has been shown to be a crucial molecule in RLR signaling. The expression levels of TRAF3 and TRAF5, which have conserved domains that are nearly identical, in the LRT were examined. We found that the bronchus exhibited the highest expression levels of TRAF3 and TRAF5 in the LRT. These findings suggest the importance of the bronchus in antiviral innate immunity in the LRT and indicate that TRAF3 and TRAF5 may contribute to RLR signaling.

ID: 70: A novel function of human Pumilio proteins in antiviral innate immunity

RIG-I-like receptor (RLR) plays a pivotal role in the detection of invading pathogens to initiate type I interferon (IFN) gene transcription. Since aberrant IFN production is harmful, RLR signaling is strictly regulated. However, the regulatory mechanisms are not fully understood. By expression cloning, we identified Pumilio proteins, PUM1 and PUM2, as candidate positive regulators of RIG-I signaling. Overexpression of Pumilio proteins and their knockdown augmented and diminished IFN-β promoter activity induced by Newcastle disease virus (NDV), respectively. Both proteins showed a specific association with LGP2, but not with RIG-I or MDA5. Furthermore, all of these components were recruited to NDV-induced antiviral stress granules. Interestingly, biochemical analyses revealed that Pumilio increased double-stranded (ds) RNA binding affinity of LGP2; however, Pumilio was absent in the dsRNA-LGP2 complex, suggesting that Pumilio facilitates viral RNA recognition by LGP2 through its chaperon-like function. Collectively, our results demonstrate an unknown function of Pumilio in viral recognition by LGP2.

Estrogen receptor alpha inhibits RLR-mediated immune response via ubiquitinating TRAF3

RIG-I-like receptors (RLRs) function as key sentinel receptor for invading viruses. Moderate activation of RLR signaling is critical for efficient viral clearance without harmful immunopathology. Estrogen receptor alpha (ERα) is a member of the nuclear receptor superfamily of ligand-activated transcription factors and is involved in the regulation of innate immune responses. However, the effects of ERα on RLR signaling and the molecular mechanisms are poorly understood. In this study, we identify ERα as a negative regulator of RLR-triggered antiviral immune responses. The expression level of ERα is upregulated following RLR activation in macrophages. In the absence of ligand, VSV infection phosphorylates ERα at serine 167. ERα inhibits VSV-induced IRF3 activation. We further demonstrate that ERα directly interacts with TRAF3 and promotes K48-linked proteasomal degradation of TRAF3. Consistently, ERα inhibits VSV-triggered IFN-β production in macrophages in a ligand independent mechanism. Thus, ERα functions as a negative feedback regulator of RLR-triggered antiviral immune responses. These findings also provide the insights that separate the immune effects of ERα from its ligand-induced hormonal effects.