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Abstract Retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor involved in innate immunity, but its role in adaptive immunity, specifically in the context of CD8+ T-cell antitumour immunity, remains unclear. Here, we demonstrate that RIG-I is upregulated in tumour-infiltrating CD8+ T cells, where it functions as an intracellular checkpoint to negatively regulate CD8+ T-cell function and limit antitumour immunity. Mechanistically, the upregulation of RIG-I in CD8+ T cells is induced by activated T cells, and directly inhibits the AKT/glycolysis signalling pathway. In addition, knocking out RIG-I enhances the efficacy of adoptively transferred T cells against solid tumours, and inhibiting RIG-I enhances the response to PD-1 blockade. Overall, our study identifies RIG-I as an intracellular checkpoint and a potential target for alleviating inhibitory constraints on T cells in cancer immunotherapy, either alone or in combination with an immune checkpoint inhibitor.

Following the initial treatment of nasopharyngeal carcinoma (NPC), tumor progression often portends an adverse prognosis for these patients. MicroRNAs (miRNAs) have emerged as critical regulators of tumor immunity, yet their intricate mechanisms in NPC remain elusive. Through comprehensive miRNA sequencing, tumor tissue microarrays and tissue samples analysis, we identified miR-142-3p as a significantly upregulated miRNA that is strongly associated with poor prognosis in recurrent NPC patients. To elucidate the underlying molecular mechanism, we employed RNA sequencing, coupled with cellular and tissue assays, to identify the downstream targets and associated signaling pathways of miR-142-3p. Our findings revealed two potential targets, CFL2 and WASL, which are directly targeted by miR-142-3p. Functionally, overexpressing CFL2 or WASL significantly reversed the malignant phenotypes induced by miR-142-3p both in vitro and in vivo. Furthermore, signaling pathway analysis revealed that miR-142-3p repressed the RIG-I-mediated immune defense response in NPC by inhibiting the nuclear translocation of IRF3, IRF7 and p65. Moreover, we discovered that ADAR1 physically interacted with Dicer and promoted the formation of mature miR-142-3p in a dose-dependent manner. Collectively, ADAR1-mediated miR-142-3p processing promotes tumor progression and suppresses antitumor immunity, indicating that miR-142-3p may serve as a promising prognostic biomarker and therapeutic target for NPC patients.

Abstract Senecavirus A (SVA) is an emerging virus that poses a threat to swine herds worldwide. To date, the role of tripartite motif 5 (TRIM5) in the replication of viruses has not been evaluated. Here, TRIM5 was reported to inhibit SVA replication by promoting the type I interferon (IFN) antiviral response mediated by retinoic acid-inducible gene I (RIG-I). TRIM5 expression was significantly upregulated in SVA-infected cells, and TRIM5 overexpression inhibited viral replication and promoted IFN-α, IFN-β, interleukin-1beta (IL-1β), IL-6, and IL-18 expression. Conversely, interfering with the expression of TRIM5 had the opposite effect. Viral adsorption and entry assays showed that TRIM5 did not affect the adsorption of SVA but inhibited its entry. In addition, TRIM5 promoted the expression of RIG-I and RIG-I-mediated IFNs and proinflammatory cytokines, and this effect was also proven by inhibiting the expression of TRIM5. These findings expand the scope of knowledge on host factors inhibiting the replication of SVA and indicate that targeting TRIM5 may aid in the development of new agents against SVA.

ObjectivePneumonia is a common clinical condition primarily treated with antibiotics and organ support. Exploring the pathogenesis to identify therapeutic targets may aid in the adjunct treatment of pneumonia and improve survival rates.MethodsTranscriptomic data from peripheral blood of 183 pneumonia patients were analyzed using Gene Set Variation Analysis (GSVA) and univariate Cox regression analysis to identify signaling pathways associated with pneumonia mortality. A pneumonia mouse model was established via airway injection of Klebsiella pneumoniae, and pathway-specific blockers were administered via tail vein infusion to assess whether the identified signaling pathways impact the mortality in pneumonia.ResultsThe combination of GSVA and Cox analysis revealed 17 signaling pathways significantly associated with 28-day mortality in pneumonia patients (P < 0.05). Notably, the RIG-I-like receptor signaling pathway exhibited the highest hazard ratio of 2.501 with a 95% confidence interval of [1.223–5.114]. Infusion of RIG012 via the tail vein effectively inhibited the RIG-I-like receptor signaling pathway, significantly ameliorated lung injury in pneumonia mice, reduced pulmonary inflammatory responses, and showed a trend toward improved survival rates.ConclusionRIG012 may represent a novel adjunctive therapeutic agent for pneumonia.

The incessant arms race between viruses and hosts has led to numerous evolutionary innovations that shape life’s evolution. During this process, the interactions between viral receptors and viruses have garnered significant interest since viral receptors are cell surface proteins exploited by viruses to initiate infection. Our study sheds light on the arms race between the MDA5 receptor and 5’ppp-RNA virus in a lower vertebrate fish, Miichthys miiuy. Firstly, the frequent and independent loss events of RIG-I in vertebrates prompted us to search for alternative immune substitutes, with homology-dependent genetic compensation response (HDGCR) being the main pathway. Our further analysis suggested that MDA5 of M. miiuy and Gallus gallus, the homolog of RIG-I, can replace RIG-I in recognizing 5’ppp-RNA virus, which may lead to redundancy of RIG-I and loss from the species genome during evolution. Secondly, as an adversarial strategy, 5’ppp-RNA SCRV can utilize the m6A methylation mechanism to degrade MDA5 and weaken its antiviral immune ability, thus promoting its own replication and immune evasion. In summary, our study provides a snapshot into the interaction and coevolution between vertebrate and virus, offering valuable perspectives on the ecological and evolutionary factors that contribute to the diversity of the immune system.

Abstract Background Current cancer therapies often fall short in addressing the complexities of malignancies, underscoring the urgent need for innovative treatment strategies. RNA interference technology, which specifically suppresses gene expression, offers a promising new approach in the fight against tumors. Recent studies have identified a novel immunostimulatory small-interfering RNA (siRNA) with a unique sequence (sense strand, 5’-C; antisense strand, 3’-GGG) capable of activating the RIG-I/IRF3 signaling pathway. This activation induces the release of type I and III interferons, leading to an effective antiviral immune response. However, this class of immunostimulatory siRNA has not yet been explored in cancer therapy. Methods IsiBCL-2, an innovative immunostimulatory siRNA designed to suppress the levels of B-cell lymphoma 2 (BCL-2), contains a distinctive motif (sense strand, 5’-C; antisense strand, 3’-GGG). Glioblastoma cells were subjected to 100 nM isiBCL-2 treatment in vitro for 48 h. Morphological changes, cell viability (CCK-8 assay), proliferation (colony formation assay), migration/invasion (scratch test and Transwell assay), apoptosis rate, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were evaluated. Western blotting and immunofluorescence analyses were performed to assess RIG-I and MHC-I molecule levels, and ELISA was utilized to measure the levels of cytokines (IFN-β and CXCL10). In vivo heterogeneous tumor models were established, and the anti-tumor effect of isiBCL-2 was confirmed through intratumoral injection. Results IsiBCL-2 exhibited significant inhibitory effects on glioblastoma cell growth and induced apoptosis. BCL-2 mRNA levels were significantly decreased by 67.52%. IsiBCL-2 treatment resulted in an apoptotic rate of approximately 51.96%, accompanied by a 71.76% reduction in MMP and a 41.87% increase in ROS accumulation. Western blotting and immunofluorescence analyses demonstrated increased levels of RIG-I, MAVS, and MHC-I following isiBCL-2 treatment. ELISA tests indicated a significant increase in IFN-β and CXCL10 levels. In vivo studies using nude mice confirmed that isiBCL-2 effectively impeded the growth and progression of glioblastoma tumors. Conclusions This study introduces an innovative method to induce innate signaling by incorporating an immunostimulatory sequence (sense strand, 5’-C; antisense strand, 3’-GGG) into siRNA, resulting in the formation of RNA dimers through Hoogsteen base-pairing. This activation triggers the RIG-I signaling pathway in tumor cells, causing further damage and inducing a potent immune response. This inventive design and application of immunostimulatory siRNA offer a novel perspective on tumor immunotherapy, holding significant implications for the field.

Abstract Swine acute diarrhea syndrome coronavirus (SADS-CoV), an emerging Alpha-coronavirus, brings huge economic loss in swine industry. Interferons (IFNs) participate in a frontline antiviral defense mechanism triggering the activation of numerous downstream antiviral genes. Here, we demonstrated that TRIM25 overexpression significantly inhibited SADS-CoV replication, whereas TRIM25 deficiency markedly increased viral yield. We found that SADS-CoV N protein suppressed interferon-beta (IFN-β) production induced by Sendai virus (SeV) or poly(I:C). Moreover, we determined that SADS-CoV N protein interacted with RIG-I N-terminal two caspase activation and recruitment domains (2CARDs) and TRIM25 coiled-coil dimerization (CCD) domain. The interaction of SADS-CoV N protein with RIG-I and TRIM25 caused TRIM25 multimerization inhibition, the RIG-I-TRIM25 interaction disruption, and consequent the IRF3 and TBK1 phosphorylation impediment. Overexpression of SADS-CoV N protein facilitated the replication of VSV-GFP by suppressing IFN-β production. Our results demonstrate that SADS-CoV N suppresses the host IFN response, thus highlighting the significant involvement of TRIM25 in regulating antiviral immune defenses.

RNA 5-methylcytosine (m5C) methylation plays a crucial role in hepatocellular carcinoma (HCC). As reported, aberrant m5C methylation is closely associated with the progression, therapeutic efficacy, and prognosis of HCC. The innate immune system functions as the primary defense mechanism in the body against pathogenic infections and tumors since it can activate innate immune pathways through pattern recognition receptors to exert anti-infection and anti-tumor effects. Recently, m5C methylation has been demonstrated to affect the activation of innate immune pathways including TLR, cGAS-STING, and RIG-I pathways by modulating RNA function, unveiling new mechanisms underlying the regulation of innate immune responses by tumor cells. However, research on m5C methylation and its interplay with innate immune pathways is still in its infancy. Therefore, this review details the biological significance of RNA m5C methylation in HCC and discusses its potential regulatory relationship with TLR, cGAS-STING, and RIG-I pathways, thereby providing fresh insights into the role of RNA methylation in the innate immune mechanisms and treatment of HCC.

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 T cells 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.

Hepatitis E virus (HEV) poses a significant global health threat, with an estimated 20 million infections occurring annually. Despite being a self-limiting illness, in most cases, HEV infection can lead to severe outcomes, particularly in pregnant women and individuals with pre-existing liver disease. In the absence of specific antiviral treatments, the exploration of RNAi interference (RNAi) as a targeted strategy provides valuable insights for urgently needed therapeutic interventions against Hepatitis E. We designed small interfering RNAs (siRNAs) against HEV, which target the helicase domain and the open reading frame 3 (ORF3). These target regions will reduce the risk of viral escape through mutations, as they belong to the most conserved regions in the HEV genome. The siRNAs targeting the ORF3 efficiently inhibited viral replication in A549 cells after HEV infection. Importantly, the siRNA was also highly effective at inhibiting HEV in the persistently infected A549 cell line, which provides a suitable model for chronic infection in patients. Furthermore, we showed that a 5′ triphosphate modification on the siRNA sense strand activates the RIG-I receptor, a cytoplasmic pattern recognition receptor that recognizes viral RNA. Upon activation, RIG-I triggers a signaling cascade, effectively suppressing HEV replication. This dual-action strategy, combining the activation of the adaptive immune response and the inherent RNAi pathway, inhibits HEV replication successfully and may lead to the development of new therapies.

Influenza virus possesses an RNA genome of single-stranded, negative-sensed, and segmented configuration. Influenza virus causes an acute respiratory disease, commonly known as the “flu” in humans. In some individuals, flu can lead to pneumonia and acute respiratory distress syndrome. Influenza A virus (IAV) is the most significant because it causes recurring seasonal epidemics, occasional pandemics, and zoonotic outbreaks in human populations, globally. The host innate immune response to IAV infection plays a critical role in sensing, preventing, and clearing the infection as well as in flu disease pathology. Host cells sense IAV infection through multiple receptors and mechanisms, which culminate in the induction of a concerted innate antiviral response and the creation of an antiviral state, which inhibits and clears the infection from host cells. However, IAV antagonizes and escapes many steps of the innate antiviral response by different mechanisms. Herein, we review those host and viral mechanisms. This review covers most aspects of the host innate immune response, i.e., (1) the sensing of incoming virus particles, (2) the activation of downstream innate antiviral signaling pathways, (3) the expression of interferon-stimulated genes, (4) and viral antagonism and escape.

Mitochondria are key orchestrators of antiviral responses that serve as platforms for the assembly and activation of innate immune-signaling complexes. In response to viral infection, mitochondria can be triggered to release immune-stimulatory molecules that can boost interferon production. These same molecules can be released by damaged mitochondria to induce pathogenic, antiviral-like immune responses in the absence of infection. This review explores how members of the tripartite motif-containing (TRIM) protein family, which are recognized for their roles in antiviral defense, regulate mitochondria-based innate immune activation. In antiviral defense, TRIMs are essential components of immune signal transduction pathways and function as directly acting viral restriction factors. TRIMs carry out conceptually similar activities when controlling immune activation related to mitochondria. First, they modulate immune-signaling pathways that can be activated by mitochondrial molecules. Second, they co-ordinate the direct removal of mitochondria and associated immune-activating factors through mitophagy. These insights broaden the scope of TRIM actions in innate immunity and may implicate TRIMs in diseases associated with mitochondria-derived inflammation.

Retinoic acid-inducible gene I (RIG-I) is a cytosolic viral RNA receptor. Upon viral infection, the protein recognizes and then recruits adapter protein mitochondrial antiviral signaling (MAVS) protein, initiating the production of interferons and proinflammatory cytokines to establish an antiviral state. In the present study, we identify zinc finger protein 205 (ZNF205) which associates with RIG-I and promotes the Sendai virus (SeV)-induced antiviral innate immune response. Overexpression of ZNF205 facilitates interferon-beta (IFN-β) introduction, whereas ZNF205 deficiency restricts its introduction. Mechanistically, the C-terminal zinc finger domain of ZNF205 interacts with the N-terminal tandem caspase recruitment domains (CARDs) of RIG-I; this interaction markedly promotes K63 ubiquitin-linked polyubiquitination of RIG-I, which is crucial for RIG-I activation. Thus, our results demonstrate that ZNF205 is a positive regulator of the RIG-I-mediated innate antiviral immune signaling pathway.

Insulin-like growth factor-1 receptor (IGF-1R) has been made an attractive anticancer target due to its overexpression in cancers. However, targeting it has often produced the disappointing results as the role played by cross talk with numerous downstream signalings. Here, we report a disobliging IGF-1R signaling which promotes growth of cancer through triggering the E3 ubiquitin ligase MEX3A-mediated degradation of RIG-I. The active β-arrestin-2 scaffolds this disobliging signaling to talk with MEX3A. In response to ligands, IGF-1Rβ activated the basal βarr2 into its active state by phosphorylating the interdomain domain on Tyr64 and Tyr250, opening the middle loop (Leu130‒Cys141) to the RING domain of MEX3A through the conformational changes of βarr2. The models of βarr2/IGF-1Rβ and βarr2/MEX3A could interpret the mechanism of the activated-IGF-1R in triggering degradation of RIG-I. The assay of the mutants βarr2Y64A and βarr2Y250A further confirmed the role of these two Tyr residues of the interlobe in mediating the talk between IGF-1Rβ and the RING domain of MEX3A. The truncated-βarr2 and the peptide ATQAIRIF, which mimicked the RING domain of MEX3A could prevent the formation of βarr2/IGF-1Rβ and βarr2/MEX3A complexes, thus blocking the IGF-1R-triggered RIG-I degradation. Degradation of RIG-I resulted in the suppression of the IFN-I-associated immune cells in the TME due to the blockade of the RIG-I-MAVS-IFN-I pathway. Poly(I:C) could reverse anti-PD-L1 insensitivity by recovery of RIG-I. In summary, we revealed a disobliging IGF-1R signaling by which IGF-1Rβ promoted cancer growth through triggering the MEX3A-mediated degradation of RIG-I.

RIG-I and MDA5 are members of RIG-I-like receptors (RLRs) that detect viral RNA within the cytoplasm and subsequently initiate antiviral immune responses. Necroptosis is a form of programmed cell death (PCD) executed by mixed lineage kinase domain-like (MLKL), which, upon phosphorylation by receptor-interacting protein kinase 3 (RIPK3), causes necrotic cell death. To date, no link between RLRs and necroptosis has been observed during bacterial infection. Edwardsiella tarda is a zoonotic bacterial pathogen that can thrive in host macrophages. In a previous study, we identified RIG-I and MDA5 as two hub factors of RAW264.7 cells responsive to E. tarda infection. The present study aimed to determine the specific form of cell death triggered by E. tarda and explore the association between RIG-I/MDA5 and PCD in the context of bacterial infection. Our results showed that E. tarda infection induced RIPK3-MLKL-mediated necroptosis, rather than pyroptosis or apoptosis, in RAW264.7 cells. Meanwhile, E. tarda promoted RIG-I/MDA5 production and activated the RIG-I/MDA5 pathways that led to IRF3 phosphorylation, IFN-β secretion, and interferon-stimulated gene (ISG) and cytokine expression. Both RIG-I and MDA5 were essential for E. tarda-triggered necroptosis and required for effective inhibition of intracellular bacterial replication. Furthermore, the regulatory effect of RIG-I/MDA5 on necroptosis was not affected by type I IFN or TNF-α signaling blockage. Together these results revealed that necroptosis could be triggered by intracellular bacterial infection through the RIG-I/MDA5 pathways, and that there existed intricate interplays between PCD and RLRs induced by bacterial pathogen.

ABSTRACT Nervous necrosis virus (NNV), a highly contagious fish virus, has caused huge economic losses to the global aquaculture industry. A previous study showed that protein A (ProA) encoded by orange-spotted grouper NNV triggers type I interferon (IFN) production in fish cells, but the activation and modulation of correlative signal pathways remain unclear. Here, we proved that ProA induces fish cell-specific IFN promoter activation in a dose-dependent manner. In channel catfish ovary (CCO, an NNV-permissive cell), ProA evoked expression and secretion of functional IFN with anti-DNA virus activity, suggesting a good model for IFN signaling research. A contrastive study of signaling in CCO and fathead minnow (FHM, an NNV-nonpermissive cell) using RNAi knockdown or dominant negative mutant overexpression verified that ProA-mediated IFN activation went through retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) pathway (RIG-I/MDA5-MAVS-TRAF3-TBK1-IRF3) while NOD1-RIPK2-NFκB and TLR3-TRIF branches were unnecessary. As RNA receptors upregulated by ProA in FHM, RIG-I and MDA5 promoted ProA-mediated IFN activation to form a positive feedback loop, while LGP2, NOD1, and PKR inhibited this activation as negative modulators. In ProA-expressed, NNV-infected CCO, the transcription of RIG-I, MDA5, MAVS, TBK1, IRF3, and IFN was downregulated, but the expression of LGP2, TRAF3, and NOD1 remained unchanged, suggesting unknown IFN suppression mechanism by NNV infection. IFN inhibition by overexpressing mutated RIG-I greatly enhanced NNV replication in FHM, implying that RIG-I might be the main target for both ProA-mediated activation and NNV infection-induced inhibition. This study provides overviews and foundations for understanding the interaction between betanodavirus-encoded protein and fish innate immune signaling. IMPORTANCE As a major pathogen, nervous necrosis virus (NNV) infects more than 120 fish species worldwide and is virulent to larvae and juvenile fish, hampering the development of the fish fry industry. Understanding virus-host interaction and underlying mechanisms is an important but largely unknown issue in fish virus studies. Here, using channel catfish ovary and fathead minnow cells as models for the study of innate immunity signaling, we found that NNV-encoded ProA activated interferon signaling via the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) pathway which was still suppressed by the infection of wild-type NNV. This finding has important implications for the comprehension of NNV protein function and the immune response from different cells. First, RIG-I is the key node for anti-NNV innate immunity. Second, the response intensity of RLR signaling determines the degree of NNV proliferation. This study expands our knowledge regarding the overview of signal pathways affected by NNV-encoded protein and also highlights potential directions for the control of aquatic viruses.

ABSTRACT Host innate immunity is an important defense line against virus infection and is precisely regulated by various factors. Studying the mechanism of virus-host interaction is essential to developing novel antivirals. Being one of the key host natural antiviral responses, host small non-coding RNAs (miRNAs) possess promising antiviral potential worthy of study. Herein, we found that miR-26a exerted broad-spectrum antiviral effect against multiple viruses, e.g., Hepatitis E virus, Vesicular Stomatitis Virus, and Sendai Virus. Mechanistically, miR-26a specifically targets 3′UTR of mRNA to inhibit USP15 expression. USP15 interacted directly with RIG-I to deubiquitinate K63-linked RIG-I, thus negatively regulating type I interferon (IFN) signaling. Consequently, miR-26a, by downregulating USP15, promotes K63 ubiquitination of RIG-I to enhance type I IFN responses, resulting in an active antiviral state against virus infection. Intriguingly, the activation of type I IFN responses could suppress miR-26a expression, serving as an intrinsic negative feedback loop to avoid dysregulated signal activation. Hence, the broad-spectrum antiviral effects of miR-26a and its mode-of-action enriched the interaction networks between miRNAs and innate immunity, providing insights for the development of broad-spectrum antivirals against viral infection. IMPORTANCE miR-26a serves as a potent positive regulator of type I interferon (IFN) responses. By inhibiting USP15 expression, miR-26a promotes RIG-I K63-ubiquitination to enhance type I IFN responses, resulting in an active antiviral state against viruses. Being an intricate regulatory network, the activation of type I IFN responses could in turn suppress miR-26a expression to avoid the disordered activation that might result in the so-called “type I interferonopathy.” The knowledge gained would be essential for the development of novel antiviral strategies against viral infection.

Abstract Background Multiple host factors are involved in modulating type I interferon expression induced by viruses; however, the mechanism is not fully elucidated. Influenza A virus infection causes severe respiratory symptoms and triggers a series of signaling cascades and host innate immune responses, including interferon production. The co-IP/MS technology was used to screen several antiviral factors in the early stage. Among these factors, ariadne-1 homolog (ARIH1) caught our attention. Methods Western blot assay was performed to detect the level of proteins and software ImageJ was used to analyze the band intensities. Polymerase activity assay was conducted to evaluate the polymerase activity of influenza A virus. Tissue culture infective dose (TCID50) assay was performed to measure influenza A virus titers, and quantitative RT-PCR assay was applied to test the mRNA level of IFN-β, ISG56, and CXCL10. Luciferase reporter assay was used to confirm the target of ARIH1 in RIG-I signaling. Immunoprecipitation assay was performed to detect the interaction and the ubiquitination of the proteins. All data were analyzed by biostatistical methods and presented as means ± standard deviation from three independent experiments. Statistical significance was determined using two-tailed student’s t test. A P value of less than 0.05 was considered statistically significant, and a P value of less than 0.01 was considered highly significant (ns, P ≥ 0.05; *, P

OM-85 is a bacterial lysate from common respiratory tract pathogens, with an excellent safety profile, widely used to prevent recurrent respiratory tract infections. Several studies have been reporting the immunomodulating properties and antiviral roles of OM-85. The COVID-19 pandemic, originating in 2019, has presented a significant global public health crisis. While effective vaccines have been developed, vaccination rates vary considerably, and numerous concerning viral variants continue to emerge. The challenge persists in creating early interventions to halt the progression of the disease to its severe stages. To examine the therapeutic effect of OM-85 after SARS-CoV-2 infection and compared to recombinant human (rhINF-β) we collected nasopharyngeal cells from COVID-19 patients. The cells were treated ex-vivo with OM-85 or hrINF-β and the response was analyzed after 24h for gene expression by real-time PCR. We found that OM-85 decreased the SARS-CoV-2 N1 gene expression and increased RIG-I (DDX58) in these cells. The expression of ACE2 was undetected in these samples. These data support the antiviral effect of OM-85 against SARS-CoV-2.

Abstract RIG-I-like receptors (RLRs) are intracellular pattern recognition receptors that detect viral or bacterial infection and induce host innate immune responses. The RLRs family comprises retinoic acid-inducible gene 1 (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology 2 (LGP2) that have distinctive features. These receptors not only recognize RNA intermediates from viruses and bacteria, but also interact with endogenous RNA such as the mislocalized mitochondrial RNA, the aberrantly reactivated repetitive or transposable elements in the human genome. Evasion of RLRs-mediated immune response may lead to sustained infection, defective host immunity and carcinogenesis. Therapeutic targeting RLRs may not only provoke anti-infection effects, but also induce anticancer immunity or sensitize “immune-cold” tumors to immune checkpoint blockade. In this review, we summarize the current knowledge of RLRs signaling and discuss the rationale for therapeutic targeting RLRs in cancer. We describe how RLRs can be activated by synthetic RNA, oncolytic viruses, viral mimicry and radio-chemotherapy, and how the RNA agonists of RLRs can be systemically delivered in vivo. The integration of RLRs agonism with RNA interference or CAR-T cells provides new dimensions that complement cancer immunotherapy. Moreover, we update the progress of recent clinical trials for cancer therapy involving RLRs activation and immune modulation. Further studies of the mechanisms underlying RLRs signaling will shed new light on the development of cancer therapeutics. Manipulation of RLRs signaling represents an opportunity for clinically relevant cancer therapy. Addressing the challenges in this field will help develop future generations of cancer immunotherapy.

Abstract Background Oncolytic viral therapy is a promising method for tumor treatment. Currently, several oncolytic viruses (OVs) have been used as tumor therapy at different phases of research and clinical trials. OVs not only directly lyse tumor cells due to viral replication but also initiate host antitumor immune responses. Previous studies have primarily focused on how OVs activate adaptive immune responses in immune cells. However, the role of innate immune responses in tumors induced by OVs remains unclear. Methods To determine the innate immune responses induced by vesicular stomatitis virus (VSV), the mutant VSVΔM51 strain was used for the infection and quantitative polymerase chain reaction (qPCR) was employed to measure the transcriptional levels of antiviral genes. The knockdown efficiency of RIG‐I was examined by qPCR. Viral titers were measured by plaque assays. Tumor models were established by intradermally implanting RIG‐I‐knockdown and control LLC cells into the flank of wild type C57BL/6J mice. When the tumors reached approximately 50mm3, they were infected with VSVΔM51 via intratumoral injections to examine its therapeutic effect. Results Infection with VSVΔM51 triggered remarkable innate immune responses in several tumor cell lines through the cytoplasmic RIG‐I sensing pathway. Moreover, we found that intratumoral injection of VSVΔM51 effectively reduced tumor growth in murine LCC lung cancer model. Importantly, VSVΔM51‐induced antitumor therapy was more effective in murine LLC tumor model established using Rig‐I‐knockdown cells compared with the tumor model established using control cells. Conclusion RIG‐I‐mediated innate immune signaling in tumor cells plays a negative role in regulating antitumor therapy with VSVΔM51 virus.

Abstract Background Hepatocarcinogenesis is driven by necroinflammation or metabolic disorders, and the underlying mechanisms remain largely elusive. We previously found that retinoic acid-inducible gene-I (RIG-I), a sensor for recognizing RNA virus in innate immune cells, is mainly expressed by parenchymal hepatocytes in the liver. However, its roles in hepatocarcinogenesis are unknown, which is intensively investigated in this study. Methods DEN-induced necroinflammation-driven hepatocarcinogenesis and STAM NASH-hepatocarcinogenesis were carried out in hepatocyte-specific RIG-I knockout mice. The post-translational modification of RIG-I was determined by mass spectrometry, and specific antibodies against methylated lysine sites and the RIG-I lysine mutant mice were constructed to identify the functions of RIG-I methylation. Results We interestingly found that DEN-induced hepatocarcinogenesis was enhanced, while NASH-induced hepatocarcinogenesis was suppressed by hepatocyte-specific RIG-I deficiency. Further, IL-6 decreased RIG-I expression in HCC progenitor cells (HcPCs), which then viciously promoted IL-6 effector signaling and drove HcPCs to fully established HCC. RIG-I expression was increased by HFD, which then enhanced cholesterol synthesis and steatosis, and the in-turn NASH and NASH-induced hepatocarcinogenesis. Mechanistically, RIG-I was constitutively mono-methylated at K18 and K146, and demethylase JMJD4-mediated RIG-I demethylation suppressed IL-6-STAT3 signaling. The constitutive methylated RIG-I associated with AMPKα to inhibit HMGCR phosphorylation, thus promoting HMGCR enzymatic activity and cholesterol synthesis. Clinically, RIG-I was decreased in human hepatic precancerous dysplastic nodules while increased in NAFLD livers, which were in accordance with the data in mouse models. Conclusions Decreased RIG-I in HcPCs promotes necroinflammation-induced hepatocarcinogenesis, while increased constitutive methylated RIG-I enhances steatosis and NASH-induced hepatocarcinogenesis. JMJD4-demethylated RIG-I prevents both necroinflammation and NASH-induced hepatocarcinogenesis, which provides mechanistic insight and potential target for preventing HCC.

RationaleA family of short synthetic, triphosphorylated stem-loop RNAs (SLRs) have been designed to activate the retinoic-acid-inducible gene I (RIG-I) pathway and induce a potent interferon (IFN) response, which may have therapeutic potential. We investigated immune response modulation by SLR10. We addressed whether RIG-I pathway activation with SLR10 leads to protection of nonsmoking (NS) and cigarette smoke (CS)-exposed mice after influenza A virus (IAV) infection.MethodsMice were given 25 µg of SLR10 1 day before IAV infection. We compared the survival rates and host immune responses of NS and CS-exposed mice following challenge with IAV.ResultsSLR10 significantly decreased weight loss and increased survival rates in both NS and CS-exposed mice during IAV infection. SLR10 administration repaired the impaired proinflammatory response in CS-exposed mice without causing more lung injury in NS mice as assessed by physiologic measurements. Although histopathologic study revealed that SLR10 administration was likely to result in higher pathological scores than untreated groups in both NS and CS mice, this change was not enough to increase lung injury evaluated by lung-to-body weight ratio. Both qRT-PCR on lung tissues and multiplex immunoassay on bronchoalveolar lavage fluids (BALFs) showed that most IFNs and proinflammatory cytokines were expressed at lower levels in SLR10-treated NS mice than control-treaded NS mice at day 5 post infection (p.i.). Remarkably, proinflammatory cytokines IL-6, IL-12, and GM-CSF were increased in CS-exposed mice by SLR10 at day 5 p.i. Significantly, SLR10 elevated the ratio of the two chemokines (CXCL9 and CCL17) in BALFs, suggesting macrophages were polarized to classically activated (M1) status. In vitro testing also found that SLR10 not only stimulated human alveolar macrophage polarization to an M1 phenotype, but also reversed cigarette smoke extract (CSE)-induced M2 to M1 polarization.ConclusionsOur data show that SLR10 administration in mice is protective for both NS and CS-exposed IAV-infected mice. Mechanistically, SLR10 treatment promoted M1 macrophage polarization in the lung during influenza infection. The protective effects by SLR10 may be a promising intervention for therapy for infections with viruses, particularly those with CS-enhanced susceptibility to adverse outcomes.

ABSTRACT 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.

Augmentation of endogenous double-stranded RNA (dsRNA) has become a promising strategy for activating anti-tumor immunity through induction of type I interferon (IFN) in the treatment of ovarian carcinoma. However, the underlying regulatory mechanisms of dsRNA in ovarian carcinoma remain elusive. From The Cancer Genome Atlas (TCGA), we downloaded RNA expression profiles and clinical data of patients with ovarian carcinoma. Using the consensus clustering method, patients can be classified by their expression level of core interferon-stimulated genes (ISGs): IFN signatures high and IFN signatures low. The IFN signatures high group had a good prognosis. Gene set enrichment analysis (GSEA) showed that differentially expressed genes (DEGs) were primarily associated with anti-foreign immune responses. Based on results from protein-protein interaction (PPI) networks and survival analysis, ISG20 was identified as a key gene involved in host anti-tumor immune response. Further, elevated ISG20 expression in ovarian cancer cells led to increased IFN-β production. The elevated interferon improved the immunogenicity of tumor cells and generated chemokines that attract immune cells to infiltrate the area. Upon overexpression of ISG20, endogenous dsRNA accumulated in the cell and stimulated IFN-β production through the Retinoic acid-inducible gene I (RIG-I)-mediated dsRNA sense pathway. The accumulation of dsRNA was associated with the ribonuclease activity of ISG20. This study suggests that targeting ISG20 is a potential immune therapeutic approach to treat ovarian cancer.

Phenuiviridae, a member of the Bunyavirales order, can lead to significant human morbidity and mortality. Various phenuiviruses target specific cellular proteins and have strategies to counteract the effects of type I interferon (IFN). Previous studies have shown that phenuiviruses infection inhibits the synthesis of type I IFNs, and viral nonstructural proteins (NSs) had been further identified as an IFN antagonist. This study found that the NSs proteins of Dabie bandavirus (DBV), Sandfly fever Sicilian virus (SFSV), and Uukuniemi virus (UUKV) can inhibit Sendai virus-induced activation of IFN-β promoter and phosphorylation of IFN regulatory factor 3 (IRF3). Moreover, detailed analysis revealed that the phenuivirus NSs protein could directly interact with retinoic acid inducible gene-I (RIG-I) and degrade it via a proteasome-dependent pathway. In short, this study demonstrate a novel mechanism of phenuiviruses to resist host antiviral immunity, which may help understand these pathogens and suggest novel therapeutic approaches.

Abstract Background Cytosolic RNA sensing can elicit immune responses against viral pathogens. However, antiviral responses must be tightly regulated to avoid the uncontrolled production of type I interferons (IFN) that might have deleterious effects on the host. Upon bacterial infection, the germinal center kinase MST4 can directly phosphorylate the adaptor TRAF6 to limit the inflammatory responses, thereby avoiding the damage caused by excessive immune activation. However, the molecular mechanism of how MST4 regulates virus-mediated type I IFN production remains unknown. Methods The expression levels of IFN-β, IFIT1, and IFIT2 mRNA were determined by RT-PCR. The expression levels of p-IRF3, IRF3, RIG-I, MAVS, and MST4 proteins were determined by Western blot. The effect of secreted level of IFN-β was measured by ELISA. The relationship between MST4 and MAVS was investigated by immunofluorescence staining and coimmunoprecipitation. Results In this study, we reported that MST4 can act as a negative regulator of type I IFN production. Ectopic expression of MST4 suppressed the Poly (I:C) (polyino-sinic-polycytidylic acid)- and Sendai virus (SeV)-triggered production of type I IFN, while the knockdown of MST4 enhanced the production of type I IFN. Mechanistically, upon SeV infection, the MST4 competed with TRAF3 to bind to the 360–540 domain of MAVS, thereby inhibiting the TRAF3/MAVS association. Additionally, MST4 facilitated the interaction between the E3 ubiquitin ligase Smurf1 and MAVS. This promoted the K48-linked ubiquitination of MAVS, thereby accelerating the ubiquitin-mediated proteasome degradation of MAVS. Conclusions Our findings showed that MST4 acted as a crucial negative regulator of RLR-mediated type I IFN production. Video Abstract

Early detection of infection is a central and critical component of our innate immune system. Mammalian cells have developed specialized receptors that detect RNA with unusual structures or of foreign origin – a hallmark of many virus infections. Activation of these receptors induces inflammatory responses and an antiviral state. However, it is increasingly appreciated that these RNA sensors can also be activated in the absence of infection, and that this ‘self-activation’ can be pathogenic and promote disease. Here, we review recent discoveries in sterile activation of the cytosolic innate immune receptors that bind RNA. We focus on new aspects of endogenous ligand recognition uncovered in these studies, and their roles in disease pathogenesis.

Antiviral defense in ecdysozoan invertebrates requires Dicer with a helicase domain capable of ATP hydrolysis. But despite well-conserved ATPase motifs, human Dicer is incapable of ATP hydrolysis, consistent with a muted role in antiviral defense. To investigate this enigma, we used ancestral protein reconstruction to resurrect Dicer’s helicase in animals and trace the evolutionary trajectory of ATP hydrolysis. Biochemical assays indicated ancient Dicer possessed ATPase function, that like extant invertebrate Dicers, is stimulated by dsRNA. Analyses revealed that dsRNA stimulates ATPase activity by increasing ATP affinity, reflected in Michaelis constants. Deuterostome Dicer-1 ancestor, while exhibiting lower dsRNA affinity, retained some ATPase activity; importantly, ATPase activity was undetectable in the vertebrate Dicer-1 ancestor, which had even lower dsRNA affinity. Reverting residues in the ATP hydrolysis pocket was insufficient to rescue hydrolysis, but additional substitutions distant from the pocket rescued vertebrate Dicer-1’s ATPase function. Our work suggests Dicer lost ATPase function in the vertebrate ancestor due to loss of ATP affinity, involving motifs distant from the active site, important for coupling dsRNA binding to the active conformation. By competing with Dicer for viral dsRNA, RIG-I-like receptors important for interferon signaling may have allowed or actively caused loss of ATPase function.

BackgroundMast cells are the major effector cell type for IgE-mediated allergic reactions. Recent studies revealed a role for mast cells in orchestrating the host response to viral infections.ObjectiveWe studied the relationship between FcεRI (high-affinity IgE receptor) and RIG-I-like receptor (RLR)-mediated antiviral signaling pathways.MethodsMast cells (BMMCs) were cultured from bone marrow cells from mice deficient in MAVS or other RLR signaling molecules. MAVS expression was restored by retroviral transduction of MAVS-deficient BMMCs. These cells were stimulated with IgE and antigen and their activation (degranulation and cytokine production/secretion) was quantified. FcεRI-mediated signaling events such as protein phosphorylation and Ca2+ flux were analyzed by western blotting and enzyme assays. WT and mutant mice as well as mast cell-deficient KitW−sh/W−sh mice engrafted with BMMCs were subjected to passive cutaneous anaphylaxis.ResultsUnexpectedly, we found that mast cells devoid of the adaptor molecule MAVS exhibit dramatically increased cytokine production upon FcεRI stimulation, despite near-normal degranulation. Consistent with these observations, MAVS inhibited tyrosine phosphorylation, thus catalytic activity of Syk kinase, the key signaling molecule for FcεRI-mediated mast cell activation. By contrast, mast cells deficient in RIG-I, MDA5 or IRF3, which are antiviral receptor and signaling molecules upstream or downstream of MAVS, exhibited reduced or normal mast cell activation. MAVS-deficient mice showed enhanced late-phase responses in passive cutaneous anaphylaxis.ConclusionThis study demonstrates that the adaptor MAVS in the RLR innate immune pathway uniquely intersects with the adaptive immune FcεRI signaling pathway.

Abstract Background N6-methyladenosine (m6A) RNA modification plays a critical role in various physiological and pathological conditions. However, the role of m6A modification in head and neck squamous cell carcinoma (HNSCC) remains elusive. Methods In this study, the expression of m6A demethylases was detected by HNSCC tissue microarray. m6A-RNA immunoprecipitation (MeRIP) sequencing and RNA sequencing were used to identify downstream targets of ALKBH5. Comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) was used to explore the m6A “readers”. Tumor-infiltrating lymphocytes were analyzed in SCC7-bearing xenografts in C3H mice. Results Here, we demonstrate the downregulation of m6A status and upregulation of two demethylases in HNSCC. Silencing the m6A demethylase alkB homolog 5, RNA demethylase (ALKBH5) suppresses tumor progression in vitro and in vivo. m6A-RNA immunoprecipitation sequencing reveals that ALKBH5 downregulates the m6A modification of DDX58 mRNA. Moreover, RIG-I, encoded by the DDX58 mRNA, reverses the protumorigenic characteristics of ALKBH5. ChIRP-MS demonstrates that HNRNPC binds to the m6A sites of DDX58 mRNA to promote its maturation. ALKBH5 overexpression inhibits RIG-I-mediated IFNα secretion through the IKKε/TBK1/IRF3 pathway. The number of tumor-infiltrating lymphocytes in C3H immunocompetent mice is reduced by ALKBH5 overexpression and restored by IFNα administration. Upregulation of AKLBH5 negatively correlates with RIG-I and IFNα expression in HNSCC patients. Conclusions These findings unveil a novel mechanism of immune microenvironment regulation mediated by m6A modification through the ALKBH5/RIG-I/IFNα axis, providing a rationale for therapeutically targeting epitranscriptomic modulators in HNSCC.

The SARS-CoV-2 pandemic has underscored the need for rapidly usable prophylactic and antiviral treatments against emerging viruses. The targeted stimulation of antiviral innate immune receptors can trigger a broad antiviral response that also acts against new, unknown viruses. Here, we used the K18-hACE2 mouse model of COVID-19 to examine whether activation of the antiviral RNA receptor RIG-I protects mice from lethal SARS-CoV-2 infection and reduces disease severity. We found that prophylactic, systemic treatment of mice with the specific RIG-I ligand 3pRNA, but not type I interferon, 1–7 days before viral challenge, improved survival of mice by up to 50%. Survival was also improved with therapeutic 3pRNA treatment starting 1 day after viral challenge. This improved outcome was associated with lower viral load in oropharyngeal swabs and in the lungs and brains of 3pRNA-treated mice. Moreover, 3pRNA-treated mice exhibited reduced lung inflammation and developed a SARS-CoV-2-specific neutralizing antibody response. These results demonstrate that systemic RIG-I activation by therapeutic RNA oligonucleotide agonists is a promising strategy to convey effective, short-term antiviral protection against SARS-CoV-2 infection, and it has great potential as a broad-spectrum approach to constrain the spread of newly emerging viruses until virus-specific therapies and vaccines become available.

Nipah virus (NiV; genus: Henipavirus; family: Paramyxoviridae) naturally infects Old World fruit bats (family Pteropodidae) without causing overt disease. Conversely, NiV infection in humans and other mammals can be lethal. Comparing bat antiviral responses with those of humans may illuminate the mechanisms that facilitate bats’ tolerance. Tripartite motif proteins (TRIMs), a large family of E3-ubiquitin ligases, fine-tune innate antiviral immune responses, and two human TRIMs interact with Henipavirus proteins. We hypothesize that NiV infection induces the expression of an immunosuppressive TRIM in bat, but not human cells, to promote tolerance. Here, we show that TRIM40 is an interferon-stimulated gene (ISG) in pteropodid but not human cells. Knockdown of bat TRIM40 increases gene expression of IFNβ, ISGs, and pro-inflammatory cytokines following poly(I:C) transfection. In Pteropus vampyrus, but not human cells, NiV induces TRIM40 expression within 16 h after infection, and knockdown of TRIM40 correlates with reduced NiV titers as compared to control cells. Bats may have evolved to express TRIM40 in response to viral infections to control immunopathogenesis.

SARS-CoV-2 causes a spectrum of clinical symptoms from respiratory damage to gastrointestinal disorders. Intestinal infection of SARS-CoV-2 triggers immune response. However, the cellular mechanism that how SARS-CoV-2 initiates and induces intestinal immunity is not understood. Here, we exploited SARS-CoV-2-GFP/ΔN trVLP pseudo-virus system and demonstrated that RIG-I and DHX15 are required for sensing SARS-CoV-2 and inducing cellular immune response through MAVS signaling in intestinal epithelial cells (IECs) upon SARS-CoV-2 infection. NLRP6 also engages in the regulation of SARS-CoV-2 immunity by producing IL-18. Furthermore, primary cellular immune response provoked by SARS-CoV-2 in IECs further cascades activation of MAIT cells and produces cytotoxic cytokines including IFN-γ, granzyme B via an IL-18 dependent mechanism. These findings taken together unveil molecular basis of immune recognition in IECs in response to SARS-CoV-2, and provide insights that intestinal immune cross-talk with other immune cells triggers amplified immunity and probably contributes to immunopathogenesis of COVID-19.

Interferons (IFNs), IFN-stimulated genes (ISGs), and inflammatory cytokines mediate innate immune responses, and are essential to establish an antiviral response. Within the innate immune responses, retinoic acid-inducible gene I (RIG-I) is a key sensor of virus infections, mediating the transcriptional induction of IFNs and inflammatory proteins. Nevertheless, since excessive responses could be detrimental to the host, these responses need to be tightly regulated. In this work, we describe, for the first time, how knocking-down or knocking-out the expression of IFN alpha-inducible protein 6 (IFI6) increases IFN, ISG, and pro-inflammatory cytokine expression after the infections with Influenza A Virus (IAV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and Sendai Virus (SeV), or poly(I:C) transfection. We also show how overexpression of IFI6 produces the opposite effect, in vitro and in vivo, indicating that IFI6 negatively modulates the induction of innate immune responses. Knocking-out or knocking-down the expression of IFI6 diminishes the production of infectious IAV and SARS-CoV-2, most likely because of its effect on antiviral responses. Importantly, we report a novel interaction of IFI6 with RIG-I, most likely mediated through binding to RNA, that affects RIG-I activation, providing a molecular mechanism for the effect of IFI6 on negatively regulating innate immunity. Remarkably, these new functions of IFI6 could be targeted to treat diseases associated with an exacerbated induction of innate immune responses and to combat viral infections, such as IAV and SARS-CoV-2.

Sexual transmission of Zika virus (ZIKV) is associated with virus persistence in the testes and shedding in the seminal fluid for months after recovery. We previously demonstrated that ZIKV can establish long-term replication without causing cytotoxicity in human Sertoli cells (SC), responsible for maintaining the immune privileged compartment of seminiferous tubules. Functional gene expression analyses also predicted activation of multiple virus sensing pathways including TLR3, RIG-I, and MDA5. Here, we elucidated which of the RNA virus sensing receptors play a decisive role in restricting ZIKV replication. We show that both poly I:C and IFN-β treatment induced a robust antiviral state and reduced ZIKV replication significantly, suggesting that virus sensing and antiviral signaling are functional in SC. Silencing of TLR3, 7, and 9 did not affect virus replication kinetics; however, both RIG-I and MDA5 played a synergistic role in inducing an anti-ZIKV response. Further, the impact of SC-specific immunosuppressive pathways that collectively regulate SC function, specifically the TGF-β superfamily members, TGF-β, Activin A, and BMP6, on ZIKV replication was investigated. While ZIKV did not modulate the expression of TGF-β and Activin A, BMP6 signaling was suppressed at later stages of infection. Notably, treatment with BMP6 increased IFN-β, p-IRF3, and p-STAT1 levels, and expression of key interferon-stimulated genes including MDA5, suggesting that BMP6 enhances antiviral response in SC. Collectively, this study further delineates the key role of the RIG-I-like receptors in sensing ZIKV in SC, and reveals a novel role of BMP6 in modulating innate immune and antiviral response in the testes.

Abstract Background Although multiple studies have demonstrated a role for exosomes during virus infections, our understanding of the mechanisms by which exosome exchange regulates immune response during viral infections and affects viral pathogenesis is still in its infancy. In particular, very little is known for cytoplasmic single-stranded RNA viruses such as SARS-CoV-2 and Rift Valley fever virus (RVFV). We have used RVFV infection as a model for cytoplasmic single-stranded RNA viruses to address this gap in knowledge. RVFV is a highly pathogenic agent that causes RVF, a zoonotic disease for which no effective therapeutic or approved human vaccine exist. Results We show here that exosomes released from cells infected with RVFV (designated as EXi-RVFV) serve a protective role for the host and provide a mechanistic model for these effects. Our results show that treatment of both naïve immune cells (U937 monocytes) and naïve non-immune cells (HSAECs) with EXi-RVFV induces a strong RIG-I dependent activation of IFN-B. We also demonstrate that this strong anti-viral response leads to activation of autophagy in treated cells and correlates with resistance to subsequent viral infection. Since we have shown that viral RNA genome is associated with EXi-RVFV, RIG-I activation might be mediated by the presence of packaged viral RNA sequences. Conclusions Using RVFV infection as a model for cytoplasmic single-stranded RNA viruses, our results show a novel mechanism of host protection by exosomes released from infected cells (EXi) whereby the EXi activate RIG-I to induce IFN-dependent activation of autophagy in naïve recipient cells including monocytes. Because monocytes serve as reservoirs for RVFV replication, this EXi-RVFV-induced activation of autophagy in monocytes may work to slow down or halt viral dissemination in the infected organism. These findings offer novel mechanistic insights that may aid in future development of effective vaccines or therapeutics, and that may be applicable for a better molecular understanding of how exosome release regulates innate immune response to other cytoplasmic single-stranded RNA viruses.

Virus-Like Particles (VLPs) are nanostructures that share conformation and self-assembly properties with viruses, but lack a viral genome and therefore the infectious capacity. In this study, we produced VLPs by co-expression of VSV glycoprotein (VSV-G) and HIV structural proteins (Gag, Pol) that incorporated a strong sequence-optimized 5’ppp-RNA RIG-I agonist, termed M8. Treatment of target cells with VLPs-M8 generated an antiviral state that conferred resistance against multiple viruses. Interestingly, treatment with VLPs-M8 also elicited a therapeutic effect by inhibiting ongoing viral replication in previously infected cells. Finally, the expression of SARS-CoV-2 Spike glycoprotein on the VLP surface retargeted VLPs to ACE2 expressing cells, thus selectively blocking viral infection in permissive cells. These results highlight the potential of VLPs-M8 as a therapeutic and prophylactic vaccine platform. Overall, these observations indicate that the modification of VLP surface glycoproteins and the incorporation of nucleic acids or therapeutic drugs, will permit modulation of particle tropism, direct specific innate and adaptive immune responses in target tissues, and boost immunogenicity while minimizing off-target effects.

Herpesviruses evade host immunity to establish persistent lifelong infection with dormant latent and replicative lytic phases. Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated virus (KSHV) are double-stranded DNA herpesviruses that encode components to activate RNA sensors, (Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5). Yet both viruses can effectively evade the antiviral immune response. The ability of these viruses to disarm RIG-I to evade immunity allowing viral persistency can contribute to the creation of a protected niche that facilitates tumour growth and immune evasion. Alternatively, viral nucleic acids present in the cytosol during the replicative phase of the viral lifecycle can activate pro-inflammatory signaling downstream of RIG-I augmenting tumour promoting inflammation.Understanding how these viral proteins disrupt innate immune pathways could help identify mechanisms to boost immunity, clearing viral infection and enhancing the efficacy of immunotherapy for virally induced cancers. Here we review literature on the strategies EBV and KSHV use to either enhance or inhibit RLR signaling.

Chronic mucosal pathogens have evolved multiple strategies to manipulate the host immune response; consequently, microbes contribute to the development of >2 million cases of cancer/year. Gastric adenocarcinoma is the fourth leading cause of cancer-related death and Helicobacter pylori confers the highest risk for this disease. Gastric innate immune effectors can either eliminate bacteria or mobilize adaptive immune responses including Toll-like receptors (TLRs), and cytosolic DNA sensor/adaptor proteins (e.g., stimulator of interferon genes, STING). The H. pylori strain-specific cag type IV secretion system (T4SS) augments gastric cancer risk and translocates DNA into epithelial cells where it activates the microbial DNA sensor TLR9 and suppresses injury in vivo; however, the ability of H. pylori to suppress additional nucleic acid PRRs within the context of chronic gastric inflammation and injury remains undefined. In this study, in vitro and ex vivo experiments identified a novel mechanism through which H. pylori actively suppresses STING and RIG-I signaling via downregulation of IRF3 activation. In vivo, the use of genetically deficient mice revealed that Th17 inflammatory responses are heightened following H. pylori infection within the context of Sting deficiency in conjunction with increased expression of a known host immune regulator, Trim30a. This novel mechanism of immune suppression by H. pylori is likely a critical component of a finely tuned rheostat that not only regulates the initial innate immune response, but also drives chronic gastric inflammation and injury.

Abstract Background Abdominal aortic aneurysm (AAA), an irreversible cardiovascular disease prevalent in the artery, causes the increase of the aneurysm diameter over time, and is a fatal phenomenon inducing sidewall rupture. Long noncoding RNAs (lncRNAs) serve as promising biomarkers for AAA. In the present study, we sought to define the role of lncRNA growth-arrest-specific transcript 5 (GAS5) in growth of smooth muscle cells (SMC) and progression of AAA. Methods Initially, we established angiotensin II (Ang II)-induced AAA mouse models and Ang II-treated vascular SMC model. RT-qPCR and Western blot analysis were adopted to determine expression of GAS5 and zeste homolog 2 (EZH2). After ectopic expression and depletion experiments in Ang II-treated mice and vascular SMCs, cell apoptosis was detected in SMCs using flow cytometry and in mice using TUNEL staining. The binding of GAS5 and EZH2 was evaluated using RNA binding protein immunoprecipitation (RIP) and Co-IP assays. Results Increased GAS5 and RIG-I but decreased EZH2 were found in aortic tissues of AAA mice. EZH2 overexpression inhibited AAA formation and suppressed SMC apoptosis. Functionally, EZH2 blocked the RIG-I signaling pathway and consequently inhibited SMC apoptosis. GAS5 regulated EZH2 transcription in a negative manner in SMCs. Knockdown of GAS5 attenuated SMC apoptosis, which was reversed by EZH2 inhibition or RIG-I overexpression. Conclusions The current study demonstrated that GAS5 induced SMC apoptosis and subsequent AAA onset by activating EZH2-mediated RIG-I signaling pathway, highlighting GAS5 as a novel biomarker for AAA.

Abstract Background Influenza A virus (IAV) evolves strategies to counteract the host antiviral defense for establishing infection. The influenza A virus (IAV) non-structural protein 1 (NS1) is a key viral factor shown to counteract type I IFN antiviral response mainly through targeting RIG-I signaling. Growing evidence suggests that viral RNA sensors RIG-I, TLR3, and TLR7 function to detect IAV RNA in different cell types to induce type I IFN antiviral response to IAV infection. Yet, it remains unclear if IAV NS1 can exploit a common mechanism to counteract these RNA sensing pathways to type I IFN production at once, then promoting viral propagation in the host. Methods Luciferase reporter assays were conducted to determine the effect of NS1 and its mutants on the RIG-I and TLR3 pathways to the activation of the IFN-β and NF-κB promoters. Coimmunoprecipitation and confocal microscopic analyses were used to the interaction and colocalization between NS1 and TRAF3. Ubiquitination assays were performed to study the effect of NS1 and its mutants on TRAF3 ubiquitination. A recombinant mutant virus carrying NS1 E152A/E153A mutations was generated by reverse genetics for biochemical, ex vivo, and in vivo analyses to explore the importance of NS1 E152/E153 residues in targeting the RNA sensing-TRAF3-type I IFN axis and IAV pathogenicity. Results Here we report that NS1 subverts the RIG-I, TLR3, and TLR7 pathways to type I IFN production through targeting TRAF3 E3 ubiquitin ligase. NS1 harbors a conserved FTEE motif (a.a. 150-153), in which the E152/E153 residues are critical for binding TRAF3 to block TRAF3 ubiquitination and type I IFN production by these RNA sensing pathways. A recombinant mutant virus carrying NS1 E152A/E153A mutations induces higher type I IFN production ex vivo and in vivo, and exhibits the attenuated phenotype in infected mice, indicating the importance of E152/E153 residues in IAV pathogenicity. Conclusions Together our work uncovers a novel mechanism of IAV NS1-mediated immune evasion to promote viral infection through targeting the RNA sensing-TRAF3-type I IFN axis.

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)–messenger RNA (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.

Foot-and-mouth disease virus (FMDV) is a single-stranded, positive-sense RNA virus containing at least 13 proteins. Many of these proteins show immune modulation capabilities. As a non-structural protein of the FMDV, 2B is involved in the rearrangement of the host cell membranes and the disruption of the host secretory pathway as a viroporin. Previous studies have also shown that FMDV 2B plays a role in the modulation of host type-I interferon (IFN) responses through the inhibition of expression of RIG-I and MDA5, key cytosolic sensors of the type-I IFN signaling. However, the exact molecular mechanism is poorly understood. Here, we demonstrated that FMDV 2B modulates host IFN signal pathway by the degradation of RIG-I and MDA5. FMDV 2B targeted the RIG-I for ubiquitination and proteasomal degradation by recruiting E3 ubiquitin ligase ring finger protein 125 (RNF125) and also targeted MDA5 for apoptosis-induced caspase-3- and caspase-8-dependent degradation. Ultimately, FMDV 2B significantly inhibited RNA virus-induced IFN-β production. Importantly, we identified that the C-terminal amino acids 126-154 of FMDV 2B are essential for 2B-mediated degradation of the RIG-I and MDA5. Collectively, these results provide a clearer understanding of the specific molecular mechanisms used by FMDV 2B to inhibit the IFN responses and a rational approach to virus attenuation for future vaccine development.

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.

The vascular endothelium consists of a highly heterogeneous monolayer of endothelial cells (ECs) which are the primary target for bacterial and viral infections due to EC’s constant and close contact with the bloodstream. Emerging evidence has shown that ECs are a key cell type for innate immunity. Like macrophages, ECs serve as sentinels when sensing invading pathogens or microbial infection caused by viruses and bacteria. It remains elusive how ECs senses danger signals, transduce the signal and fulfil immune functions. Retinoic acid-inducible gene-I (RIG-I, gene name also known as DDX58) is an important member of RIG-I-like receptor (RLR) family that functions as an important pathogen recognition receptor (PRR) to execute immune surveillance and confer host antiviral response. Recent studies have demonstrated that virus infection, dsRNA, dsDNA, interferons, LPS, and 25-hydroxycholesterol (25-HC) can increase RIG-1 expression in ECs and propagate anti-viral response. Of translational significance, RIG-I activation can be inhibited by Panax notoginseng saponins, endogenous PPARγ ligand 15-PGJ2, tryptanthrin and 2-animopurine. Considering the pivotal role of inflammation and innate immunity in regulating endothelial dysfunction and atherosclerosis, here we provided a concise review of the role of RIG-I in endothelial cell function and highlight future direction to elucidate the potential role of RIG-I in regulating cardiovascular diseases as well as virus infectious disease, including COVID-19. Furthered understanding of RIG-I-mediated signaling pathways is important to control disorders associated with altered immunity and inflammation in ECs.

As one of the Mex3 family members, Mex3A is crucial in cell proliferation, migration, and apoptosis in mammals. In this study, a novel gene homologous to mammalian Mex3A (named CiMex3A, MW368974) was cloned and identified in grass carp, which is 1,521 bp in length encoding a putative polypeptide of 506 amino acids. In CIK cells, CiMex3A is upregulated after stimulation with LPS, Z-DNA, and especially with intracellular poly(I:C). CiMex3A overexpression reduces the expressions of IFN1, ISG15, and pro-inflammatory factors IL8 and TNFα; likewise, Mex3A inhibits IRF3 phosphorylation upon treatment with poly(I:C). A screening test to identify potential targets suggested that CiMex3A interacts with RIG-I exclusively. Co-localization analysis showed that Mex3A and RIG-I are simultaneously located in the endoplasmic reticulum, while they rarely appear in the endosome, mitochondria, or lysosome after exposure to poly(I:C). However, RIG-I is mainly located in the early endosome and then transferred to the late endosome following stimulation with poly(I:C). Moreover, we investigated the molecular mechanism underlying CiMex3A-mediated suppression of RIG-I ubiquitination. The results demonstrated that Mex3A truncation mutant (deletion in the RING domain) can still interact physically with RIG-I, but fail to degrade it, suggesting that Mex3A also acts as a RING-type E3 ubiquitin ligase. Taken together, this study showed that grass carp Mex3A can interact with RIG-I in the endoplasmic reticulum following poly(I:C) stimulation, and then Mex3A facilitates the ubiquitination and degradation of RIG-I to inhibit IRF3-mediated innate antiviral immune response.

Dendritic cells (DCs) are important mediators of the induction and regulation of adaptive immune responses following microbial infection and inflammation. Sensing environmental danger signals including viruses, microbial products, or inflammatory stimuli by DCs leads to the rapid transition from a resting state to an activated mature state. DC maturation involves enhanced capturing and processing of antigens for presentation by major histocompatibility complex (MHC) class I and class II, upregulation of chemokines and their receptors, cytokines and costimulatory molecules, and migration to lymphoid tissues where they prime naive T cells. Orchestrating a cellular response to environmental threats requires a high bioenergetic cost that accompanies the metabolic reprogramming of DCs during activation. We previously demonstrated that DCs undergo a striking functional transition after stimulation of the retinoic acid-inducible gene I (RIG-I) pathway with a synthetic 5′ triphosphate containing RNA (termed M8), consisting of the upregulation of interferon (IFN)–stimulated antiviral genes, increased DC phagocytosis, activation of a proinflammatory phenotype, and induction of markers associated with immunogenic cell death. In the present study, we set out to determine the metabolic changes associated with RIG-I stimulation by M8. The rate of glycolysis in primary human DCs was increased in response to RIG-I activation, and glycolytic reprogramming was an essential requirement for DC activation. Pharmacological inhibition of glycolysis in monocyte-derived dendritic cells (MoDCs) impaired type I IFN induction and signaling by disrupting the TBK1-IRF3-STAT1 axis, thereby countering the antiviral activity induced by M8. Functionally, the impaired IFN response resulted in enhanced viral replication of dengue, coronavirus 229E, and Coxsackie B5.

In mammals, the retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) has been demonstrated to play a critical role in activating downstream signaling in response to viral RNA. However, its role in ducks’ antiviral innate immunity is less well understood, and how gene-mediated signaling is regulated is unknown. The regulatory role of the duck laboratory of genetics and physiology 2 (duLGP2) in the duck RIG-I (duRIG-I)-mediated antiviral innate immune signaling system was investigated in this study. In duck embryo fibroblast (DEF) cells, overexpression of duLGP2 dramatically reduced duRIG-I-mediated IFN-promotor activity and cytokine expression. In contrast, the knockdown of duLGP2 led to an opposite effect on the duRIG-I-mediated signaling pathway. We demonstrated that duLGP2 suppressed the duRIG-I activation induced by duck Tembusu virus (DTMUV) infection. Intriguingly, when duRIG-I signaling was triggered, duLGP2 enhanced the production of inflammatory cytokines. We further showed that duLGP2 interacts with duRIG-I, and this interaction was intensified during DTMUV infection. In summary, our data suggest that duLGP2 downregulated duRIG-I mediated innate immunity against the Tembusu virus. The findings of this study will help researchers better understand the antiviral innate immune system’s regulatory networks in ducks.

Viral respiratory infections cause substantial health and economic burden. There is a pressing demand for efficacious vaccination strategies and, therefore, a need for a better understanding of the mechanisms of action of novel potential adjuvants. Here we investigated the effect of a synthetic RIG-I agonist, the dsRNA hairpin 3p10LA9, on the activation of pulmonary myeloid cells. Analysis of early innate immune responses revealed that a single intranasal 3p10LA9 dose induces a transient pulmonary interferon-stimulated gene (ISG) and pro-inflammatory cytokine/chemokine response, which leads to the maturation of three distinct dendritic cell subpopulations in the lungs. While lung resident dendritic cell decrease shortly after 3p10LA9 delivery, their numbers increase in the draining mediastinal lymph node, where they have migrated, maintaining their activated phenotype. At the same time, dsRNA hairpin-induced chemokines attract transiently infiltrating monocytes into the lungs, which causes a short temporary pulmonary inflammation. However, these monocytes are dispensable in controlling influenza infection since in CCR2 deficient mice, lacking these infiltrating cells, the virus load was similar to the wild type mice when infected with the influenza virus at a sublethal dose. In summary, our data suggest that intranasal delivery of dsRNA hairpins, used as a RIG-I targeting adjuvant, represents an attractive strategy to boost type I inteferon-mediated lung dendritic cell maturation, which supports viral reduction in the lungs during infection.