Retinoic Acid-inducible Gene I Signaling Research Articles

Characterisation of LGP2 complex multitranscript system in humans: role in the innate immune response and evolution from non-human primates.

Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I, MDA5 and LGP2, recognize viral RNA to mount an antiviral interferon (IFN) response RLRs share three different protein domains: C-terminal domain, DExD/H box RNA helicase domain, and an N-terminal domain with two tandem repeats (CARDs). LGP2 lacks tandem CARD and is not able to induce an IFN response. However, LGP2 positively enhances MDA5 and negatively regulates RIG-I signaling. In this study, we determined the LGP2 alternative transcripts in humans to further comprehend the mechanism of its regulation, their evolutionary origin, and the isoforms functionallity. The results showed new eight alternative transcripts in the samples tested. The presence of these transcripts demonstrated that the main mechanisms for the regulation of LGP2 expression are both by insertion of introns and by the loss of exons. The phylogenetic analysis of the comparison between sequences from exon 1 to exon 3 of humans and those previously described in non-human primates showed three well-differentiated groups (lineages) originating from gorillas, suggesting that the transspecies evolution has been maintained for 10 million years. The corresponding protein models (isoforms) were also established, obtaining four isoforms: one complete and three others lacking the C-terminal domain or this domain and the partial or total He2 Helicase domain, which would compromise the functionality of LGP2. In conclusion, this is the first study that elucidate the large genomic organization and complex transcriptional regulation of human LGP2, its pattern of sequence generation, and a mode of evolutionary inheritance across species.

PARP7i Clinical Candidate RBN-2397 Exerts Antiviral Activity by Modulating Interferon-β Associated Innate Immune Response in Macrophages.

Polyadenosine diphosphate-ribose polymerase 7 (PARP7) acts as a suppressor of the type I interferon (IFN) signaling pathway via suppressing TANK-binding protein 1 (TBK1). Research study indicates that inhibition of PARP7 could potentially regulate tumor immunity. However, the effect of PARP7 inhibition on innate antiviral immunity in macrophages as well as the underlying mechanism have not been demonstrated else well. We report herein that PARP7 inhibitor clinical candidate RBN-2397 could augment type I interferon (IFN-I) production in macrophages by elevating retinoic acid-inducible gene I (RIG-I) and stimulator of interferon genes (STING) signaling pathways. Treatment with RBN-2397 leads to increased pattern recognition ligands-induced interferon-β production in primary bone marrow-derived macrophages (BMDM) and RAW264.7 cells. Additionally, RBN-2397 suppresses viral replication efficiency in macrophages infected by vesicular stomatitis virus (VSV) and amplifies the expression of interferon-stimulated chemokine genes (ISGs). Mechanistically, RBN-2397 promotes TBK1 phosphorylation, consequently leading to the amplified activation of RIG-I and STING signaling pathways. Furthermore, RBN-2397 enhances the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT2 induced by IFN-α/β and the expression of chemokine genes in macrophages in response to IFN stimulation. In vivo experiments demonstrated that RBN-2397 enhances innate antiviral immunity in mice infected with VSV, resulting in increased serum IFN-β levels, reduced viral loads, and alleviated pulmonary inflammatory responses of the VSV-infected mice. In conclusion, our findings highlight the potential of RBN-2397 as a promising antiviral therapeutic agent for enhancing the IFN-relative antiviral immune defense in host.

Intra-tumoral delivery of 5'ppp-dsRNA induces robust anti-tumor response via RIG-I activation and Bcl-2 gene downregulation in murine model of prostate cancer.

Onco-immunotherapy via blocking checkpoint-inhibitors has revolutionized the treatment-landscape of several malignancies, though not in the metastatic castration-resistant prostate cancer (PCa) owing to immunosuppressive and poorly immunogenic "cold" tumor microenvironment (TME). Turning up the heat of such cold TME via triggering innate immunity is now of increasing interest to restore immune-surveillance. Retinoic acid-inducible gene- I (RIG-I)-like receptors (RLRs) are cytosolic innate-sensors that can detect exogenous RNAs and induce type-I interferons and other pro-inflammatory signaling. RIG-I activation is suggested to be a valuable addition to the treatment approaches for several cancers. However, the knowledge about RIG-I signaling in PCa remains elusive. The present study evaluated the expression of two important RLRs, RIG-I and melanoma differentiation-associated protein 5 (MDA5) along with their downstream partners, mitochondrial antiviral-signaling protein (MAVS) and ERA G-protein-like 1 (ERAL1) during PCa progression in the transgenic adenocarcinoma of mouse prostate (TRAMP) model. The early stage of PCa revealed a significant increment in the expression of RLRs, but not MAVS. However, the advanced stage showed downregulated RLR signaling. Further, the therapeutic implication of 5'ppp-dsRNA, a synthetic RIG-I agonist and Bcl2 gene silencer has been investigated in vitro and in vivo. Intra-tumoral delivery of 5'ppp-dsRNA regressed tumor growth via triggering tumor cells apoptosis, immunomodulation, and inducing phagocytic "eat me" signals. These findings highlight that, for the first time, RIG-I activation and Bcl-2 silencing with 5'ppp-dsRNA can serve as a potent tumor-suppressor strategy in PCa and has a significant clinical implication in transforming "cold" TME into immunogenic "hot" TME of PCa.

Evaluation of immune sensor responses to a viral small noncoding RNA.

Gammaherpesviruses are widespread pathogens causing persistent infections linked to the development of numerous types of lymphomas in humans. During latency, most of the viral protein-coding genes are suppressed, facilitating evasion of adaptive immune recognition of protein antigens. In contrast, many noncoding RNA (ncRNA) molecules are expressed in infected cells and can regulate key cellular pathways while simultaneously evading adaptive immune recognition. To counteract this, many cells express internal pattern recognition receptors that can intrinsically sense ongoing infections and initiate cellular defenses. Murine gammaherpesvirus 68 (MHV68) is a valuable model to study in vivo aspects of gammaherpesvirus pathogenesis. The MHV68 ncRNA TMER4 (tRNA-miRNA-encoding RNA 4) promotes lymph node egress of infected B cells: in the absence of TMER4, MHV68-infected B cells accumulate in the lymph node in a manner similar to B cells activated through specific antigen encounter. We hypothesized that TMER4 may alter intrinsic immune activation. In research described here, we aimed to explore the immunomodulatory functions of TMER4 by evaluating its impact on signaling through the critical immune sensors Toll-like receptor 4 (TLR4), TLR3, TLR7, and retinoic acid-inducible gene I (RIG-I). To accomplish this, we developed a system to test noncoding RNAs using commercially available reporter cell lines. We optimized the experimental procedure to ensure ncRNA expression and to quantify immune sensory molecule induction or inhibition by the expressed ncRNA. Expression of TMER4 RNAs from plasmid constructs did not alter TLR or RIG-I signaling. This study provides a clear experimental framework that can be applied to test other small ncRNAs for their impact on various innate immune sensor proteins.

Deep profiling of potential substrate atlas of porcine epidemic diarrhea virus 3C-like protease.

Coronavirus (CoV) 3C-like protease (3CLpro) is essential for viral replication and is involved in immune escape by proteolyzing host proteins. Deep profiling the 3CLpro substrates in the host proteome extends our understanding of viral pathogenesis and facilitates antiviral drug discovery. Here, 3CLpro from porcine epidemic diarrhea virus (PEDV), an enteropathogenic CoV, was used as a model which to identify the potential 3CLpro cleavage motifs in all porcine proteins. We characterized the selectivity of PEDV 3CLpro at sites P5-P4'. We then compiled the 3CLpro substrate preferences into a position-specific scoring matrix and developed a 3CLpro profiling strategy to delineate the protein substrate landscape of CoV 3CLpro. We identified 1,398 potential targets in the porcine proteome containing at least one putative cleavage site and experimentally validated the reliability of the substrate degradome. The PEDV 3CLpro-targeted pathways are involved in mRNA processing, translation, and key effectors of autophagy and the immune system. We also demonstrated that PEDV 3CLpro suppresses the type 1 interferon (IFN-I) cascade via the proteolysis of multiple signaling adaptors in the retinoic acid-inducible gene I (RIG-I) signaling pathway. Our composite method is reproducible and accurate, with an unprecedented depth of coverage for substrate motifs. The 3CLpro substrate degradome establishes a comprehensive substrate atlas that will accelerate the investigation of CoV pathogenicity and the development of anti-CoV drugs.IMPORTANCECoronaviruses (CoVs) are major pathogens that infect humans and animals. The 3C-like protease (3CLpro) encoded by CoV not only cleaves the CoV polyproteins but also degrades host proteins and is considered an attractive target for the development of anti-CoV drugs. However, the comprehensive characterization of an atlas of CoV 3CLpro substrates is a long-standing challenge. Using porcine epidemic diarrhea virus (PEDV) 3CLpro as a model, we developed a method that accurately predicts the substrates of 3CLpro and comprehensively maps the substrate degradome of PEDV 3CLpro. Interestingly, we found that 3CLpro may simultaneously degrade multiple molecules responsible for a specific function. For instance, it cleaves at least four adaptors in the RIG-I signaling pathway to suppress type 1 interferon production. These findings highlight the complexity of the 3CLpro substrate degradome and provide new insights to facilitate the development of anti-CoV drugs.

Nanoparticle Retinoic Acid-Inducible Gene I Agonist for Cancer Immunotherapy.

Pharmacological activation of the retinoic acid-inducible gene I (RIG-I) pathway holds promise for increasing tumor immunogenicity and improving the response to immune checkpoint inhibitors (ICIs). However, the potency and clinical efficacy of 5'-triphosphate RNA (3pRNA) agonists of RIG-I are hindered by multiple pharmacological barriers, including poor pharmacokinetics, nuclease degradation, and inefficient delivery to the cytosol where RIG-I is localized. Here, we address these challenges through the design and evaluation of ionizable lipid nanoparticles (LNPs) for the delivery of 3p-modified stem-loop RNAs (SLRs). Packaging of SLRs into LNPs (SLR-LNPs) yielded surface charge-neutral nanoparticles with a size of ∼100 nm that activated RIG-I signaling in vitro and in vivo. SLR-LNPs were safely administered to mice via both intratumoral and intravenous routes, resulting in RIG-I activation in the tumor microenvironment (TME) and the inhibition of tumor growth in mouse models of poorly immunogenic melanoma and breast cancer. Significantly, we found that systemic administration of SLR-LNPs reprogrammed the breast TME to enhance the infiltration of CD8+ and CD4+ T cells with antitumor function, resulting in enhanced response to αPD-1 ICI in an orthotopic EO771 model of triple-negative breast cancer. Therapeutic efficacy was further demonstrated in a metastatic B16.F10 melanoma model, with systemically administered SLR-LNPs significantly reducing lung metastatic burden compared to combined αPD-1 + αCTLA-4 ICI. Collectively, these studies have established SLR-LNPs as a translationally promising immunotherapeutic nanomedicine for potent and selective activation of RIG-I with the potential to enhance response to ICIs and other immunotherapeutic modalities.

Activation of RIG-I signaling in the early stage of Paragonimus proliferus infection causes lung injury via type I immune response in rat.

Paragonimiasis is a common zoonotic parasitic disease. The retinoic acid-inducible gene I (RIG-I) signaling is very important for the host to recognize invading pathogens (especially viruses and bacteria). However, the role of RIG-I signaling in the early stages of P. proliferus infection remains unclear. Therefore, in this study, Sprague-Dawley (SD) rat models with lung damage caused by P. proliferus were established. Experimental methods including Enzyme-linked Immuno Sorbent Assay (ELISA), real-time fluorescent quantitative polymerase chain reaction (PCR), western blotting, and hematoxylin and eosin (HE) staining were used to explore the mechanisms of lung injury caused by P. proliferus. As a result, the expression of the mRNA and proteins of RIG-I signal-related key target molecules, including RIG-I, tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), interferon regulatory Factor 7 (IRF7), IPS-1, and downstream C-X-C chemokine ligand 10 (CXCL10), were significantly up-regulated immediately after infection, peaked at 3 or 7 days, and showed a downward trend on after 14 days. The levels of pro-inflammatory cytokines interleukin-1 (IL-1), interferon (IFN)-α, -β, and -γ, which represent type 1 immune response, gradually increased and reached a peak by 14 days, which was consistent with the changes in the degree of inflammatory damage observed under HE staining of lung tissues. In conclusion, RIG-I signaling is activated in the early stage (before 14 days) of P. proliferus infection, it is inferred that the lung injury of the host may be related to the activation of RIG-I like signaling to induce type I immune response.

UBXN9 inhibits the RNA exosome function to promote T cell control of liver tumorigenesis.

Liver tumorigenesis encompasses oncogenic activation and self-adaptation of various biological processes in premalignant hepatocytes to circumvent the pressure of cellular stress and host immune control. Ubiquitin regulatory X domain-containing proteins (UBXNs) participate in the regulation of certain signaling pathways. However, whether UBXN proteins function in the development of liver cancer remains unclear. Here, we demonstrated that UBXN9 (Alveolar Soft Part Sarcoma Chromosomal Region Candidate Gene 1 Protein/Alveolar Soft Part Sarcoma Locus) expression was decreased in autochthonous oncogene-induced mouse liver tumors and ~47.7% of human HCCs, and associated with poor prognosis in patients with HCC. UBXN9 attenuated liver tumorigenesis induced by different oncogenic factors and tumor growth of transplanted liver tumor cells in immuno-competent mice. Mechanistically, UBXN9 significantly inhibited the function of the RNA exosome, resulting in increased expression of RLR-stimulatory RNAs and activation of the retinoic acid-inducible gene-I-IFN-Ι signaling in tumor cells, and hence potentiated T cell recruitment and immune control of tumor growth. Abrogation of the CD8 + T cell response or inhibition of tumor cell retinoic acid-inducible gene-I signaling efficiently counteracted the UBXN9-mediated suppression of liver tumor growth. Our results reveal a modality in which UBXN9 promotes the stimulatory RNA-induced retinoic acid-inducible gene-I-interferon signaling that induces anti-tumor T cell response in liver tumorigenesis. Targeted manipulation of the UBXN9-RNA exosome circuit may have the potential to reinstate the immune control of liver tumor growth.

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.

Oncolytic virotherapy with chimeric VSV-NDV synergistically supports RIG-I-dependent checkpoint inhibitor immunotherapy

Unraveling the complexities of the tumor microenvironment (TME) and its correlation with responsiveness to immunotherapy has become a main focus in overcoming resistance to such treatments. Targeting tumor-intrinsic retinoic acid-inducible gene-I (RIG-I), a sensor for viral RNA, was shown to transform the TME from an immunogenically "cold" state to an inflamed, "hot" lesion, which we demonstrated previously to be a crucial mediator of the efficacy of immune checkpoint inhibition with anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4). In this study, we focus on the chimeric oncolytic virus vesicular stomatitis virus (VSV)-Newcastle disease virus (NDV), comprised of genetic components of VSV and NDV, and we investigate its utility to support tumor-intrinsic RIG-I-dependent therapy with anti-CTLA-4. Overall, we demonstrate that treatment with VSV-NDV efficiently delays tumor growth and significantly prolongs survival in a murine model of malignant melanoma, which was further enhanced in combination with anti-CTLA-4. Although the direct oncolytic and pro-inflammatory effects of VSV-NDV therapy were independent of RIG-I activation, the synergism with anti-CTLA-4 therapy and associated activation of tumor-specific Tcells was critically dependent on active RIG-I signaling in tumor cells. This work highlights the therapeutic value of utilizing an immune-stimulatory oncolytic virus to sensitize tumors to immune checkpoint inhibition.

Viral Protein Accumulation of Zika Virus Variants Links with Regulation of Innate Immunity for Differential Control of Viral Replication, Spread, and Response to Interferon.

Asian lineage Zika virus (ZIKV) strains emerged globally, causing outbreaks linked with critical clinical disease outcomes unless the virus is effectively restricted by host immunity. We have previously shown that retinoic acid-inducible gene-I (RIG-I) senses ZIKV to trigger innate immunity to direct interferon (IFN) production and antiviral responses that can control ZIKV infection. However, ZIKV proteins have been demonstrated to antagonize IFN. Here, we conducted in vitro analyses to assess how divergent prototypic ZIKV variants differ in virologic properties, innate immune regulation, and infection outcome. We comparatively assessed African lineage ZIKV/Dakar/1984/ArD41519 (ZIKV/Dakar) and Asian lineage ZIKV/Malaysia/1966/P6740 (ZIKV/Malaysia) in a human epithelial cell infection model. De novo viral sequence determination identified amino acid changes within the ZIKV/Dakar genome compared to ZIKV/Malaysia. Viral growth analyses revealed that ZIKV/Malaysia accumulated viral proteins and genome copies earlier and to higher levels than ZIKV/Dakar. Both ZIKV strains activated RIG-I/IFN regulatory factor (IRF3) and NF-κB pathways to induce inflammatory cytokine expression and types I and III IFNs. However, ZIKV/Malaysia, but not ZIKV/Dakar, potently blocked downstream IFN signaling. Remarkably, ZIKV/Dakar protein accumulation and genome replication were rescued in RIG-I knockout (KO) cells late in acute infection, resulting in ZIKV/Dakar-mediated blockade of IFN signaling. We found that RIG-I signaling specifically restricts viral protein accumulation late in acute infection where early accumulation of viral proteins in infected cells confers enhanced ability to limit IFN signaling, promoting viral replication and spread. Our results demonstrate that RIG-I-mediated innate immune signaling imparts restriction of ZIKV protein accumulation, which permits IFN signaling and antiviral actions controlling ZIKV infection. IMPORTANCE ZIKV isolates are classified under African or Asian lineages. Infection with emerging Asian lineage-derived ZIKV strains is associated with increased incidence of neurological symptoms that were not previously reported during infection with African or preemergent Asian lineage viruses. In this study, we utilized in vitro models to compare the virologic properties of and innate immune responses to two prototypic ZIKV strains from distinct lineages: African lineage ZIKV/Dakar and Asian lineage ZIKV/Malaysia. Compared to ZIKV/Dakar, ZIKV/Malaysia accumulates viral proteins earlier, replicates to higher levels, and robustly blocks IFN signaling during acute infection. Early accumulation of ZIKV/Malaysia NS5 protein confers enhanced ability to antagonize IFN signaling, dampening innate immune responses to promote viral spread. Our data identify the kinetics of viral protein accumulation as a major regulator of host innate immunity, influencing host-mediated control of ZIKV replication and spread. Importantly, these findings provide a novel framework for evaluating the virulence of emerging variants.

Replication of Porcine Astrovirus Type 1-Infected PK-15 Cells In Vitro Affected by RIG-I and MDA5 Signaling Pathways.

The interferon (IFN) system is an extremely powerful antiviral response in animal cells. The subsequent effects caused by porcine astrovirus type 1 (PAstV1) IFN activation are important for the host's response to viral infections. Here, we show that this virus, which causes mild diarrhea, growth retardation, and damage of the villi of the small intestinal mucosa in piglets, induces an IFN response upon infection of PK-15 cells. Although IFN-β mRNA was detected within infected cells, this response usually occurs during the middle stages of infection, after genome replication has taken place. Treatment of PAstV1-infected cells with the interferon regulatory factor 3 (IRF3) inhibitor BX795 decreased IFN-β expression, whereas the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) inhibitor BAY11-7082 did not. These findings indicate that PAstV induced the production of IFN-β via IRF3-mediated rather than NF-κB-mediated signaling pathways in PK-15 cells. Moreover, PAstV1 increased the protein expression levels of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) in PK-15 cells. The knockdown of RIG-I and MDA5 decreased the expression levels of IFN-β and the viral loads and increased the infectivity of PAstV1. In conclusion, PAstV1 induced the production of IFN-β via the RIG-I and MDA5 signaling pathways, and the IFN-β produced during PAstV1 infection inhibited viral replication. These results will help provide new evidence that PAstV1-induced IFNs may protect against PAstV replication and pathogenesis. IMPORTANCE Astroviruses (AstVs) are widespread and can infect multiple species. Porcine astroviruses produce mainly gastroenteritis and neurological diseases in pigs. However, astrovirus-host interactions are less well studied, particularly with respect to their antagonism of IFN. Here, we report that PAstV1 acts via IRF3 transcription pathway activation of IFN-β. In addition, the knockdown of RIG-I and MDA5 attenuated the production of IFN-β induced by PAstV1 in PK-15 cells and increased efficient viral replication in vitro. We believe that these findings will help us to better understand the mechanism of how AstVs affect the host IFN response.

Senecavirus A-induced glycolysis facilitates virus replication by promoting lactate production that attenuates the interaction between MAVS and RIG-I

Senecavirus A (SVA)-induced porcine idiopathic vesicular disease has caused huge economic losses worldwide. Glucose metabolism in the host cell is essential for SVA proliferation; however, the impact of the virus on glucose metabolism in host cells and the subsequent effects are still unknown. Here, glycolysis induced by SVA is shown to facilitate virus replication by promoting lactate production, which then attenuates the interaction between the mitochondrial antiviral-signaling protein (MAVS) and retinoic acid-inducible gene I (RIG-I). SVA induces glycolysis in PK-15 cells, as indicated by significantly increased expression of hexokinase 2 (HK2), 6-phosphofructokinase (PFKM), pyruvate kinase M (PKM), phosphoglycerate kinase 1 (PGK1), hypoxia-inducible factor-1 alpha (HIF-1α), and superoxide dismutase-2 (SOD2) in a dose-and replication-dependent manner, and enhanced lactate production, while reducing ATP generation. Overexpression of PKM, PGK1, HIF-1α, and PDK3 in PK-15 cells and high glucose concentrations promote SVA replication, while glycolytic inhibitors decrease it. Inhibition of RLR signaling allowed better replication of SVA by promoting lactate production to attenuate the interaction between MAVS and RIG-I, and regulatory effect of glycolysis on replication of SVA was mainly via RIG-I signaling. SVA infection in mice increased expression of PKM and PGK1 in tissues and serum yields of lactate. Mice treated with high glucose and administered sodium lactate showed elevated lactate levels and better SVA replication, as well as suppressed induction of RIG-I, interferon beta (IFNβ), IFNα, interferon-stimulated gene 15 (ISG15), and interleukin 6 (IL-6). The inhibitory effect on interferons was lower in mice administered sodium oxamate and low glucose compared to the high glucose, indicating that RLR signaling was inhibited by SVA infection through lactate in vitro and in vivo. These results provide a new perspective on the relationship between metabolism and innate immunity of the host in SVA infection, suggesting that glycolysis or lactate may be new targets against the virus.

The IFN-stimulated gene IFI27 counteracts innate immune responses after viral infections by interfering with RIG-I signaling.

The recognition of viral nucleic acids by host pattern recognition receptors (PRRs) is critical for initiating innate immune responses against viral infections. These innate immune responses are mediated by the induction of interferons (IFNs), IFN-stimulated genes (ISGs) and pro-inflammatory cytokines. However, regulatory mechanisms are critical to avoid excessive or long-lasting innate immune responses that may cause detrimental hyperinflammation. Here, we identified a novel regulatory function of the ISG, IFN alpha inducible protein 27 (IFI27) in counteracting the innate immune responses triggered by cytoplasmic RNA recognition and binding. Our model systems included three unrelated viral infections caused by Influenza A virus (IAV), Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), and Sendai virus (SeV), and transfection with an analog of double-stranded (ds) RNA. Furthermore, we found that IFI27 has a positive effect on IAV and SARS-CoV-2 replication, most likely due to its ability to counteract host-induced antiviral responses, including in vivo. We also show that IFI27 interacts with nucleic acids and PRR retinoic acid-inducible gene I (RIG-I), being the interaction of IFI27 with RIG-I most likely mediated through RNA binding. Interestingly, our results indicate that interaction of IFI27 with RIG-I impairs RIG-I activation, providing a molecular mechanism for the effect of IFI27 on modulating innate immune responses. Our study identifies a molecular mechanism that may explain the effect of IFI27 in counterbalancing innate immune responses to RNA viral infections and preventing excessive innate immune responses. Therefore, this study will have important implications in drug design to control viral infections and viral-induced pathology.

Mechanisms of length-dependent recognition of viral double-stranded RNA by RIG-I

Retinoic acid-inducible gene I (RIG-I) is the most front-line cytoplasmic viral RNA sensor and induces antiviral immune responses. RIG-I recognizes short double-stranded (dsRNA) (< 500 bp), but not long dsRNA (> 500 bp) to trigger antiviral signaling. Since RIG-I is capable of binding with dsRNA irrespective of size, length-dependent RIG-I signaling remains elusive. Here, we demonstrated that RIG-I bound to long dsRNA with slow kinetics. Remarkably, RIG-I/short dsRNA complex efficiently dissociated in an ATP hydrolysis-dependent manner, whereas RIG-I/long dsRNA was stable and did not dissociate. Our study suggests that the dissociation of RIG-I from RIG-I/dsRNA complex could be a step for efficient antiviral signaling. Dissociated RIG-I exhibited homo-oligomerization, acquiring ability to physically associate with MAVS, and biological activity upon introduction into living cells. We herein discuss common and unique mechanisms of viral dsRNA recognition by RIG-I and MDA5.

Pellino3 ligase negatively regulates influenza B dependent RIG-I signalling through downregulation of TRAF3-mediated induction of the transcription factor IRF3 and IFNβ production.

Viral infection activates the innate immune system, which recognizes viral components by a variety of pattern recognition receptors and initiates signalling cascades leading to the production of pro-inflammatory cytokines. To date, signalling cascades triggered after virus recognition are not fully characterized and are investigated by many research groups. The critical role of the E3 ubiquitin ligase Pellino3 in antibacterial and antiviral response is now widely accepted, but the precise mechanism remains elusive. In this study, we sought to explore Pellino3 role in the retinoic acid-inducible gene I (RIG-I)-dependent signalling pathway. In this work, the molecular mechanisms of the innate immune response, regulated by Pellino3, were investigated in lung epithelial cells during influenza B virus infection. We used wild-type and Pellino3-deficient A549 cells as model cell lines to examine the role of Pellino3 ligase in the type I interferon (IFN) signalling pathway. Our results indicate that Pellino3 is involved in direct ubiquitination and degradation of the TRAF3, suppressing interferon regulatory factor 3 (IRF3) activation and interferon beta (IFNβ) production.

The double-stranded RNA-dependent protein kinase PKR negatively regulates the protein expression of IFN-β induced by RIG-I signaling.

Retinoic acid-inducible gene-I (RIG-I) is a cytoplasmic RNA sensor that plays an important role in innate immune responses to viral RNAs. Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is a eukaryotic initiation factor 2α (eIF2α) kinase that is initially involved in the responses of the translational machinery to dsRNA. PKR is also thought to play an essential role in antiviral innate immunity. However, the coordinated mechanisms of RIG-I and PKR that induce the expression of type I interferons (IFNs), essential cytokines involved in antiviral defense, are not completely understood. In this study, we show that PKR negatively participates in the RIG-I-mediated induction of IFN-β expression. Stress granule (SG) formation is crucial to sequester mRNA to prevent aberrant protein synthesis by various stresses. SG formation in response to dsRNA was triggered by a PKR-mediated antiviral stress response. However, IFN-β mRNA was not sequestered in the SGs of dsRNA-treated cells. dsRNA-induced translational silencing was thought to be PKR dependent. However, our results indicated that some proteins, including IFN-β, were clearly translated despite PKR-mediated translational silencing. This study suggests that RIG-I responds mainly to IFN-β expression in cells to which non-self dsRNA is introduced. In addition, PKR negatively regulates IFN-β protein expression induced by RIG-I signaling. This may explain the essential role of PKR in fine-tuning the expression of IFN-β in RIG-I-mediated antiviral immune responses.

The lncRNAs involved in regulating the RIG-I signaling pathway.

Understanding the targets and interactions of long non-coding RNAs (lncRNAs) related to the retinoic acid-inducible gene-I (RIG-I) signaling pathway is essential for developing interventions, which would enable directing the host inflammatory response regulation toward protective immunity. In the RIG-I signaling pathway, lncRNAs are involved in the important processes of ubiquitination, phosphorylation, and glycolysis, thus promoting the transport of the interferon regulatory factors 3 and 7 (IRF3 and IRF7) and the nuclear factor kappa B (NF-κB) into the nucleus, and activating recruitment of type I interferons (IFN-I) and inflammatory factors to the antiviral action site. In addition, the RIG-I signaling pathway has recently been reported to contain the targets of coronavirus disease-19 (COVID-19)-related lncRNAs. The molecules in the RIG-I signaling pathway are directly regulated by the lncRNA-microRNAs (miRNAs)-messengerRNA(mRNA) axis. Therefore, targeting this axis has become a novel strategy for the diagnosis and treatment of cancer. In this paper, the studies on the regulation of the RIG-I signaling pathway by lncRNAs during viral infections and cancer are comprehensively analyzed. The aim is to provide a solid foundation of information for conducting further detailed studies on lncRNAs and RIG-I in the future and also contribute to clinical drug development.

Human metapneumovirus M2-2 protein inhibits RIG-I signaling by preventing TRIM25-mediated RIG-I ubiquitination.

Retinoic acid-inducible gene I (RIG-I) is a receptor that senses viral RNA and interacts with mitochondrial antiviral signaling (MAVS) protein, leading to the production of type I interferons and inflammatory cytokines to establish an antiviral state. This signaling axis is initiated by the K63-linked RIG-I ubiquitination, mediated by E3 ubiquitin ligases such as TRIM25. However, many viruses, including several members of the family Paramyxoviridae and human respiratory syncytial virus (HRSV), a member of the family Pneumoviridae, escape the immune system by targeting RIG-I/TRIM25 signaling. In this study, we screened human metapneumovirus (HMPV) open reading frames (ORFs) for their ability to block RIG-I signaling reconstituted in HEK293T cells by transfection with TRIM25 and RIG-I CARD (an N-terminal CARD domain that is constitutively active in RIG-I signaling). HMPV M2-2 was the most potent inhibitor of RIG-I/TRIM25-mediated interferon (IFN)-β activation. M2-2 silencing induced the activation of transcription factors (IRF and NF-kB) downstream of RIG-I signaling in A549 cells. Moreover, M2-2 inhibited RIG-I ubiquitination and CARD-dependent interactions with MAVS. Immunoprecipitation revealed that M2-2 forms a stable complex with RIG-I CARD/TRIM25 via direct interaction with the SPRY domain of TRIM25. Similarly, HRSV NS1 also formed a stable complex with RIG-I CARD/TRIM25 and inhibited RIG-I ubiquitination. Notably, the inhibitory actions of HMPV M2-2 and HRSV NS1 are similar to those of V proteins of several members of the Paramyxoviridae family. In this study, we have identified a novel mechanism of immune escape by HMPV, similar to that of Pneumoviridae and Paramyxoviridae family members.

STUB1 activates antiviral response in zebrafish by promoting the expression of RIG-I

Spring viraemia of carp virus (SVCV) is a fierce pathogen causing high mortality in the common carp. At present, the treatment of spring viraemia of carp (SVC) is limited. Innate immunity is the host's first line of defense against microbial pathogens. Retinoic acid-inducible gene I (RIG-I) activation plays an essential role in the antiviral immune response. Virus infection can activate the RIG-I signaling and induce the production of interferon (IFN) and the expression of IFN-stimulated genes (ISGs). STUB1 (STIP1 homology and U-box containing protein 1) is a highly conserved cytoplasmic protein. This protein is known to exist widely in many biological systems and plays an important role in the process of immune regulation, but little is known in fish. To explore the immune function of STUB1 in fish, STUB1 gene was cloned from zebrafish and analyzed in this study. Zebrafish STUB1 showed 77% and 79% amino acid sequence homology with those from human and mouse, respectively. The amino acid sequence of zebrafish STUB1 contains three TPR domains and one U-box domain. Subcellular localization study revealed that STUB1 is located in the cytoplasm. And overexpression of zebrafish STUB1 resulted in the activation of the transcription of IFN1 and ISGs. Functional analysis showed that STUB1 was able to activate RIG-I signaling, and promote the expression of RIG-I, but STUB1 can degrade RIG-I in mammals. The proliferation of SVCV was significantly inhibited after the overexpression of STUB1 and N-terminal TPR domain of STUB1 in EPC cells. And through secondary structure analysis, overexpression of the mutant of STUB1 110 amino acid resulted in weakened antiviral ability. The expression of STUB1 was attenuated by poly(I:C) treatment and SVCV infection. In summary, this study demonstrated for the first time that STUB1 can induce the production of IFN, enhance the expression of ISGs by promoting the expression of RIG-I and inhibiting viral replication in fish. These findings may form the essential basis for the development of antiviral targets and drugs.