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Dong Q, Luo Y, Pan Y, Liu Y, Qin L, Ouyang K, Chen Y, Wei Z, Qin Y, Huang W. The C-terminal nsP1a protein of porcine astrovirus antagonizes interferon beta production by targets MAVS and IRF3 signaling. Vet Microbiol 2025; 305:110530. [PMID: 40339257 DOI: 10.1016/j.vetmic.2025.110530] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2024] [Revised: 04/19/2025] [Accepted: 04/23/2025] [Indexed: 05/10/2025]
Abstract
Astroviruses (AstV) are positive-sense, single-stranded RNA viruses that can infect humans and a broad spectrum of mammalian and avian species. Recently, Astrovirus strains from various hosts have been implicated in extra-intestinal infections, leading to symptoms such as encephalitis, meningitis, hepatitis, nephropathy and gout. These findings highlight the significant risks associated with its pathogenicity and its potential for interspecies transmission. The astrovirus nsP1a polyprotein is known to produce at least four distinct products. However, the precise demarcation of boundaries for these proteins remains unclear yet. Interferons, along with their antiviral immune responses, have important roles during early stages of viral infection. Viruses typically employ their encoded proteins to evade host-generated immune responses during infections through different mechanisms. In this study, we identified nonstructural proteins of PAstV1 that hinder the type I interferon (IFN) pathway. This was achieved using an IFN-β promoter reporter and by analyzing IFN-β mRNA levels in HEK 293 T cells. Our findings indicate that PAstV1 nsP1a/4 inhibits IFN-β promoter activation mediated via components in the RIG-I-like receptor (RLR) signaling pathway, specifically RIG-I, MDA5, MAVS, and IRF3. Further analyses revealed that PAstV1 nsP1a/4 protein interacts with MAVS and IRF3 to impede the RIG-I/MDA5 signaling and affects their ability to induce IFN. Overall, our results demonstrate that the ectopic expression of PAstV1 nsP1a/4 antagonizes IFN-β production by interacts with MAVS and IRF3. This reveals a novel mechanism by which PAstV nonstructural proteins evade the host's innate antiviral immune response.
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Affiliation(s)
- Qinting Dong
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, China; Guangxi Vocational University of Agriculture, Nanning, China
| | - Yuhang Luo
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, China; Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, China; Guangxi Key Laboratory of Animal Reproduction, Breeding and Disease Control, Nanning, China; Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Disease, Nanning, China
| | - Yan Pan
- Guangxi Vocational University of Agriculture, Nanning, China
| | - Yi Liu
- Guangxi State Farms Yongxin Livestock Husbandry Group Company Limited, China
| | - Liangshan Qin
- Guangxi Vocational University of Agriculture, Nanning, China
| | - Kang Ouyang
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, China; Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, China; Guangxi Key Laboratory of Animal Reproduction, Breeding and Disease Control, Nanning, China; Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Disease, Nanning, China
| | - Ying Chen
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, China; Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, China; Guangxi Key Laboratory of Animal Reproduction, Breeding and Disease Control, Nanning, China; Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Disease, Nanning, China
| | - Zuzhang Wei
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, China; Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, China; Guangxi Key Laboratory of Animal Reproduction, Breeding and Disease Control, Nanning, China; Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Disease, Nanning, China
| | - Yifeng Qin
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, China; Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, China; Guangxi Key Laboratory of Animal Reproduction, Breeding and Disease Control, Nanning, China; Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Disease, Nanning, China.
| | - Weijian Huang
- Laboratory of Animal Infectious Diseases and Molecular Immunology, College of Animal Science and Technology, Guangxi University, Nanning, China; Guangxi Zhuang Autonomous Region Engineering Research Center of Veterinary Biologics, Nanning, China; Guangxi Key Laboratory of Animal Reproduction, Breeding and Disease Control, Nanning, China; Guangxi Colleges and Universities Key Laboratory of Prevention and Control for Animal Disease, Nanning, China.
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Liu F, Ma Z, Lu J, Wu J, Chen J, Li J, Deng L. Decoding RIG-I ubiquitination in fish EPC Cells: Site identification and antiviral implications. FISH & SHELLFISH IMMUNOLOGY 2025; 163:110393. [PMID: 40334957 DOI: 10.1016/j.fsi.2025.110393] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/05/2025] [Revised: 04/25/2025] [Accepted: 05/05/2025] [Indexed: 05/09/2025]
Abstract
Retinoic-acid-inducible gene-I (RIG-I)-like receptors (RLRs) comprise a family of DExD/H-box RNA helicases that are pivotal in antiviral and inflammatory responses. Ubiquitination serves as a crucial regulatory mechanism for both RIG-I activation and the type I interferon (IFN) signaling pathway in mammals. Although RLRs have been found to be evolutionarily conserved in teleost fish, the functional characterization of RIG-I ubiquitination in this vertebrate group remains largely unexplored. Through the integration of computational prediction with experimental validation, six ubiquitination sites (K115, K118, K145, K163, K168, and K171) were identified on RIG-I in Epithelioma papulosum cyprini (EPC) cells. Among these, K163, K168, and K171 are evolutionarily conserved in mammalian RIG-I orthologs. Biochemical analyses confirmed K63-linked ubiquitination at residues K115, K118, and K163. Functional characterization revealed that mutant RIG-I-K163R and RIG-I-K118R significantly downregulated ifn expression along with three interferon-stimulated genes (ISGs: gig1, mx1, and viperin) in EPC RIG-I knockout (EPCrigi-/-) cells, demonstrating the essential role of these ubiquitination sites in RLR-mediated signaling activation. K118-mediated ubiquitination exerts a more pronounced regulatory effect on RIG-I activation compared to K163, as evidenced by enhanced spring viremia of carp virus (SVCV) proliferation in RIG-I-K118R-transfected EPCrigi-/- cells relative to mutant RIG-I-K163R or wild-type controls. Notably, the mutant RIG-I-K115R exhibited enhanced antiviral activity, characterized by increased type I IFN signaling and reduced viral replication in EPC cells. This unexpected outcome may partially result from the mutant's increased self-oligomerization compared to wild-type RIG-I. It is suggested that a tunable regulatory mechanism mediated by multisite ubiquitination of RIG-I may be conserved in teleosts. These findings provide novel insights into the molecular mechanisms governing the RLR signaling pathway in teleosts, highlighting how multisite ubiquitination of RIG-I can lead to divergent antiviral outcomes.
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Affiliation(s)
- Feihong Liu
- College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, 518055, China
| | - Zhennan Ma
- College of Computer Science and Software Engineering, Shenzhen University, Shenzhen, 518060, China
| | - Jieming Lu
- College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, 518055, China
| | - Jing Wu
- College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, 518055, China
| | - Jie Chen
- College of Computer Science and Software Engineering, Shenzhen University, Shenzhen, 518060, China
| | - Jianqiang Li
- College of Computer Science and Software Engineering, Shenzhen University, Shenzhen, 518060, China
| | - Li Deng
- College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, 518055, China.
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Khatun O, Kaur S, Tripathi S. Anti-interferon armamentarium of human coronaviruses. Cell Mol Life Sci 2025; 82:116. [PMID: 40074984 PMCID: PMC11904029 DOI: 10.1007/s00018-025-05605-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2024] [Revised: 12/15/2024] [Accepted: 01/23/2025] [Indexed: 03/14/2025]
Abstract
Cellular innate immune pathways are formidable barriers against viral invasion, creating an environment unfavorable for virus replication. Interferons (IFNs) play a crucial role in driving and regulating these cell-intrinsic innate antiviral mechanisms through the action of interferon-stimulated genes (ISGs). The host IFN response obstructs viral replication at every stage, prompting viruses to evolve various strategies to counteract or evade this response. Understanding the interplay between viral proteins and cell-intrinsic IFN-mediated immune mechanisms is essential for developing antiviral and anti-inflammatory strategies. Human coronaviruses (HCoVs), including SARS-CoV-2, MERS-CoV, SARS-CoV, and seasonal coronaviruses, encode a range of proteins that, through shared and distinct mechanisms, inhibit IFN-mediated innate immune responses. Compounding the issue, a dysregulated early IFN response can lead to a hyper-inflammatory immune reaction later in the infection, resulting in severe disease. This review provides a brief overview of HCoV replication and a detailed account of its interaction with host cellular innate immune pathways regulated by IFN.
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Affiliation(s)
- Oyahida Khatun
- Emerging Viral Pathogens Laboratory, Centre for Infectious Disease Research, Indian Institute of Science, Bengaluru, India
- Microbiology & Cell Biology Department, Biological Sciences Division, Indian Institute of Science, Bengaluru, India
| | - Sumandeep Kaur
- Emerging Viral Pathogens Laboratory, Centre for Infectious Disease Research, Indian Institute of Science, Bengaluru, India
- Microbiology & Cell Biology Department, Biological Sciences Division, Indian Institute of Science, Bengaluru, India
| | - Shashank Tripathi
- Emerging Viral Pathogens Laboratory, Centre for Infectious Disease Research, Indian Institute of Science, Bengaluru, India.
- Microbiology & Cell Biology Department, Biological Sciences Division, Indian Institute of Science, Bengaluru, India.
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4
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Landman SL, Ressing ME, Gram AM, Tjokrodirijo RTN, van Veelen PA, Neefjes J, Hoeben RC, van der Veen AG, Berlin I. Epstein-Barr virus nuclear antigen EBNA3A modulates IRF3-dependent IFNβ expression. J Biol Chem 2024; 300:107645. [PMID: 39127175 PMCID: PMC11403517 DOI: 10.1016/j.jbc.2024.107645] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2022] [Revised: 07/22/2024] [Accepted: 07/24/2024] [Indexed: 08/12/2024] Open
Abstract
Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, persistently infects over 90% of the human adult population and is associated with several human cancers. To establish life-long infection, EBV tampers with the induction of type I interferon (IFN I)-dependent antiviral immunity in the host. How various EBV genes help orchestrate this crucial strategy is incompletely defined. Here, we reveal a mechanism by which the EBV nuclear antigen 3A (EBNA3A) may inhibit IFNβ induction. Using proximity biotinylation we identify the histone acetyltransferase P300, a member of the IFNβ transcriptional complex, as a binding partner of EBNA3A. We further show that EBNA3A also interacts with the activated IFN-inducing transcription factor interferon regulatory factor 3 that collaborates with P300 in the nucleus. Both events are mediated by the N-terminal domain of EBNA3A. We propose that EBNA3A limits the binding of interferon regulatory factor 3 to the IFNβ promoter, thereby hampering downstream IFN I signaling. Collectively, our findings suggest a new mechanism of immune evasion by EBV, affected by its latency gene EBNA3A.
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Affiliation(s)
- Sanne L Landman
- Department of Cell and Chemical Biology, Leiden University Medical Center (LUMC), Leiden, the Netherlands; Oncode Institute, Leiden University Medical Center (LUMC), Leiden, the Netherlands
| | - Maaike E Ressing
- Department of Cell and Chemical Biology, Leiden University Medical Center (LUMC), Leiden, the Netherlands
| | - Anna M Gram
- Department of Cell and Chemical Biology, Leiden University Medical Center (LUMC), Leiden, the Netherlands
| | | | | | - Jacques Neefjes
- Department of Cell and Chemical Biology, Leiden University Medical Center (LUMC), Leiden, the Netherlands; Oncode Institute, Leiden University Medical Center (LUMC), Leiden, the Netherlands
| | - Rob C Hoeben
- Department of Cell and Chemical Biology, Leiden University Medical Center (LUMC), Leiden, the Netherlands
| | | | - Ilana Berlin
- Department of Cell and Chemical Biology, Leiden University Medical Center (LUMC), Leiden, the Netherlands; Oncode Institute, Leiden University Medical Center (LUMC), Leiden, the Netherlands.
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5
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Xu X, Hong Y, Fan H, Guo Z. Nucleic Acid Materials-Mediated Innate Immune Activation for Cancer Immunotherapy. ChemMedChem 2024; 19:e202400111. [PMID: 38622787 DOI: 10.1002/cmdc.202400111] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2024] [Revised: 04/13/2024] [Accepted: 04/15/2024] [Indexed: 04/17/2024]
Abstract
Abnormally localized nucleic acids (NAs) are considered as pathogen associated molecular patterns (PAMPs) in innate immunity. They are recognized by NAs-specific pattern recognition receptors (PRRs), leading to the activation of associated signaling pathways and subsequent production of type I interferons (IFNs) and pro-inflammatory cytokines, which further trigger the adaptive immunity. Notably, NAs-mediated innate immune activation is highly dependent on the conformation changes, especially the aggregation of PRRs. Evidence indicates that the characteristics of NAs including their length, concentration and even spatial structure play essential roles in inducing the aggregation of PRRs. Therefore, nucleic acid materials (NAMs) with high valency of NAs and high-order structures hold great potential for activating innate and adaptive immunity, making them promising candidates for cancer immunotherapy. In recent years, a variety of NAMs have been developed and have demonstrated significant efficacy in achieving satisfactory anti-tumor immunity in multiple mouse models, exhibiting huge potential for clinical application in cancer treatment. This review aims to discuss the mechanisms of NAMs-mediated innate immune response, and summarize their applications in cancer immunotherapy.
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Affiliation(s)
- Xinyu Xu
- State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, 210023, China
| | - Yuxuan Hong
- State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, 210023, China
| | - Huanhuan Fan
- State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, 210023, China
| | - Zijian Guo
- State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, 210023, China
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Xu X, Li S, Yu W, Yao S, Fan H, Guo Z. Activation of RIG-I/MDA5 Signaling and Inhibition of CD47-SIRPα Checkpoint with a Dual siRNA-Assembled Nanoadjuvant for Robust Cancer Immunotherapy. Angew Chem Int Ed Engl 2024; 63:e202318544. [PMID: 38194267 DOI: 10.1002/anie.202318544] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2023] [Revised: 12/30/2023] [Accepted: 01/09/2024] [Indexed: 01/10/2024]
Abstract
Antigen-presenting cells (APCs) play a crucial role in the anti-tumor immunity as they are responsible for capturing, processing, and presenting tumor antigens to T cells. However, their activation is often limited by the absence of adjuvants and the suppressive effects of immune checkpoints, such as CD47-SIRPα. Herein, we present a nanoadjuvant that is self-assembled from long RNA building blocks generated through rolling circle transcription (RCT) reaction and further modified with cationic liposomes. Owing to the high load of densely packed RNA, this nanoadjuvant could robustly activate RIG-I/MDA5 signaling in APCs, leading to the maturation of dendritic cells (DCs) and the polarization of tumor-associated macrophages (TAMs) toward an anti-tumor M1-like phenotype. In addition, with a well-designed template, the generated long RNA from RCT reaction includes two kinds of siRNA targeting both CD47 in tumor cells and SIRPα in APCs. This dual gene silencing results in efficient inhibition of the CD47-SIRPα checkpoint. Collectively, the robust activation of RIG-I/MDA5 signaling and efficient inhibition of CD47-SIRPα checkpoint enhance the phagocytic activity of APCs, which in turn promotes the cross-priming of effector T cells and the activation of anti-tumor immune responses. This study therefore provides a simple and robust RNA nanoadjuvant for cancer immunotherapy.
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Affiliation(s)
- Xinyu Xu
- State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, 210023, China
| | - Shumeng Li
- State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, 210023, China
| | - Wenhao Yu
- State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, 210023, China
| | - Shankun Yao
- State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, 210023, China
| | - Huanhuan Fan
- State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, 210023, China
| | - Zijian Guo
- State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Chemistry and Biomedicine Innovation Center (ChemBIC), Nanjing University, Nanjing, 210023, China
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7
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Magalhães VG, Lukassen S, Drechsler M, Loske J, Burkart SS, Wüst S, Jacobsen EM, Röhmel J, Mall MA, Debatin KM, Eils R, Autenrieth S, Janda A, Lehmann I, Binder M. Immune-epithelial cell cross-talk enhances antiviral responsiveness to SARS-CoV-2 in children. EMBO Rep 2023; 24:e57912. [PMID: 37818799 DOI: 10.15252/embr.202357912] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2023] [Revised: 09/26/2023] [Accepted: 09/28/2023] [Indexed: 10/13/2023] Open
Abstract
The risk of developing severe COVID-19 rises dramatically with age. Schoolchildren are significantly less likely than older people to die from SARS-CoV-2 infection, but the molecular mechanisms underlying this age-dependence are unknown. In primary infections, innate immunity is critical due to the lack of immune memory. Children, in particular, have a significantly stronger interferon response due to a primed state of their airway epithelium. In single-cell transcriptomes of nasal turbinates, we find increased frequencies of immune cells and stronger cytokine-mediated interactions with epithelial cells, resulting in increased epithelial expression of viral sensors (RIG-I, MDA5) via IRF1. In vitro, adolescent peripheral blood mononuclear cells produce more cytokines, priming A549 cells for stronger interferon responses to SARS-CoV-2. Taken together, our findings suggest that increased numbers of immune cells in the airways of children and enhanced cytokine-based interactions with epithelial cells tune the setpoint of the epithelial antiviral system. Our findings shed light on the molecular basis of children's remarkable resistance to COVID-19 and may suggest a novel concept for immunoprophylactic treatments.
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Affiliation(s)
- Vladimir G Magalhães
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Sören Lukassen
- Center for Digital Health, Berlin Institute of Health at the Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Maike Drechsler
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Jennifer Loske
- Molecular Epidemiology Unit, Center for Digital Health, Berlin Institute of Health at the Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Sandy S Burkart
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany
- Faculty of Biosciences, Heidelberg University, Heidelberg, Germany
| | - Sandra Wüst
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Eva-Maria Jacobsen
- Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm University, Ulm, Germany
| | - Jobst Röhmel
- Department of Pediatric Respiratory Medicine, Immunology and Critical Care Medicine, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
| | - Marcus A Mall
- Department of Pediatric Respiratory Medicine, Immunology and Critical Care Medicine, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany
- German Center for Lung Research, Associated Partner, Berlin, Germany
- Berlin Institute of Health at the Charité - Universitätsmedizin Berlin, Berlin, Germany
| | - Klaus-Michael Debatin
- Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm University, Ulm, Germany
| | - Roland Eils
- Center for Digital Health, Berlin Institute of Health at the Charité - Universitätsmedizin Berlin, Berlin, Germany
- German Center for Lung Research, Associated Partner, Berlin, Germany
- Health Data Science Unit, Faculty of Medicine, University of Heidelberg, Heidelberg, Germany
| | - Stella Autenrieth
- Research Group "Dendritic Cells in Infection and Cancer" (F171), German Cancer Research Center (DKFZ), Heidelberg, Germany
- Department of Hematology, Oncology, Clinical Immunology and Rheumatology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany
| | - Aleš Janda
- Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm University, Ulm, Germany
| | - Irina Lehmann
- Molecular Epidemiology Unit, Center for Digital Health, Berlin Institute of Health at the Charité - Universitätsmedizin Berlin, Berlin, Germany
- German Center for Lung Research, Associated Partner, Berlin, Germany
| | - Marco Binder
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany
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de Regt AK, Anand K, Ciupka K, Bender F, Gatterdam K, Putschli B, Fusshöller D, Hilbig D, Kirchhoff A, Hunkler C, Wolter S, Grünewald A, Wallerath C, Schuberth-Wagner C, Ludwig J, Paeschke K, Bartok E, Hagelueken G, Hartmann G, Zillinger T, Geyer M, Schlee M. A conserved isoleucine in the binding pocket of RIG-I controls immune tolerance to mitochondrial RNA. Nucleic Acids Res 2023; 51:11893-11910. [PMID: 37831086 PMCID: PMC10681732 DOI: 10.1093/nar/gkad835] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2023] [Revised: 09/01/2023] [Accepted: 09/21/2023] [Indexed: 10/14/2023] Open
Abstract
RIG-I is a cytosolic receptor of viral RNA essential for the immune response to numerous RNA viruses. Accordingly, RIG-I must sensitively detect viral RNA yet tolerate abundant self-RNA species. The basic binding cleft and an aromatic amino acid of the RIG-I C-terminal domain(CTD) mediate high-affinity recognition of 5'triphosphorylated and 5'base-paired RNA(dsRNA). Here, we found that, while 5'unmodified hydroxyl(OH)-dsRNA demonstrated residual activation potential, 5'-monophosphate(5'p)-termini, present on most cellular RNAs, prevented RIG-I activation. Determination of CTD/dsRNA co-crystal structures and mutant activation studies revealed that the evolutionarily conserved I875 within the CTD sterically inhibits 5'p-dsRNA binding. RIG-I(I875A) was activated by both synthetic 5'p-dsRNA and endogenous long dsRNA within the polyA-rich fraction of total cellular RNA. RIG-I(I875A) specifically interacted with long, polyA-bearing, mitochondrial(mt) RNA, and depletion of mtRNA from total RNA abolished its activation. Altogether, our study demonstrates that avoidance of 5'p-RNA recognition is crucial to prevent mtRNA-triggered RIG-I-mediated autoinflammation.
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Affiliation(s)
- Ann Kristin de Regt
- Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
| | - Kanchan Anand
- Institute of Structural Biology, University Hospital Bonn, Bonn, Germany
| | - Katrin Ciupka
- Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
| | - Felix Bender
- Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
| | - Karl Gatterdam
- Institute of Structural Biology, University Hospital Bonn, Bonn, Germany
| | - Bastian Putschli
- Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
| | - David Fusshöller
- Institute of Structural Biology, University Hospital Bonn, Bonn, Germany
| | - Daniel Hilbig
- Department of Oncology, Hematology and Rheumatology, University Hospital Bonn, Bonn, Germany
| | - Alexander Kirchhoff
- Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
| | - Charlotte Hunkler
- Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
| | - Steven Wolter
- Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
| | - Agathe Grünewald
- Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
| | - Christina Wallerath
- Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
| | | | - Janos Ludwig
- Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
| | - Katrin Paeschke
- Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
- Department of Oncology, Hematology and Rheumatology, University Hospital Bonn, Bonn, Germany
| | - Eva Bartok
- Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
- Institute of Experimental Haematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
- Unit of Experimental Immunology, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium
| | - Gregor Hagelueken
- Institute of Structural Biology, University Hospital Bonn, Bonn, Germany
| | - Gunther Hartmann
- Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
| | - Thomas Zillinger
- Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
- Department of Biomedicine, Aarhus University, Aarhus, Denmark
| | - Matthias Geyer
- Institute of Structural Biology, University Hospital Bonn, Bonn, Germany
| | - Martin Schlee
- Department of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
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Burkart SS, Schweinoch D, Frankish J, Sparn C, Wüst S, Urban C, Merlo M, Magalhães VG, Piras A, Pichlmair A, Willemsen J, Kaderali L, Binder M. High-resolution kinetic characterization of the RIG-I-signaling pathway and the antiviral response. Life Sci Alliance 2023; 6:e202302059. [PMID: 37558422 PMCID: PMC10412806 DOI: 10.26508/lsa.202302059] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2023] [Revised: 07/24/2023] [Accepted: 07/27/2023] [Indexed: 08/11/2023] Open
Abstract
RIG-I recognizes viral dsRNA and activates a cell-autonomous antiviral response. Upon stimulation, it triggers a signaling cascade leading to the production of type I and III IFNs. IFNs are secreted and signal to elicit the expression of IFN-stimulated genes, establishing an antiviral state of the cell. The topology of this pathway has been studied intensively, however, its exact dynamics are less understood. Here, we employed electroporation to synchronously activate RIG-I, enabling us to characterize cell-intrinsic innate immune signaling at a high temporal resolution. Employing IFNAR1/IFNLR-deficient cells, we could differentiate primary RIG-I signaling from secondary signaling downstream of the IFN receptors. Based on these data, we developed a comprehensive mathematical model capable of simulating signaling downstream of dsRNA recognition by RIG-I and the feedback and signal amplification by IFN. We further investigated the impact of viral antagonists on signaling dynamics. Our work provides a comprehensive insight into the signaling events that occur early upon virus infection and opens new avenues to study and disentangle the complexity of the host-virus interface.
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Affiliation(s)
- Sandy S Burkart
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center, Heidelberg, Germany
- Faculty of Biosciences, Heidelberg University, Heidelberg, Germany
| | - Darius Schweinoch
- Institute of Bioinformatics & Center for Functional Genomics of Microbes, University Medicine Greifswald, Greifswald, Germany
| | - Jamie Frankish
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center, Heidelberg, Germany
- Faculty of Biosciences, Heidelberg University, Heidelberg, Germany
| | - Carola Sparn
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center, Heidelberg, Germany
- Faculty of Biosciences, Heidelberg University, Heidelberg, Germany
| | - Sandra Wüst
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center, Heidelberg, Germany
| | - Christian Urban
- Technical University of Munich, School of Medicine, Institute of Virology, Munich, Germany
| | - Marta Merlo
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center, Heidelberg, Germany
- Faculty of Biosciences, Heidelberg University, Heidelberg, Germany
| | - Vladimir G Magalhães
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center, Heidelberg, Germany
| | - Antonio Piras
- Technical University of Munich, School of Medicine, Institute of Virology, Munich, Germany
| | - Andreas Pichlmair
- Technical University of Munich, School of Medicine, Institute of Virology, Munich, Germany
- German Center for Infection Research (DZIF), Munich Partner Site, Munich, Germany
| | - Joschka Willemsen
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center, Heidelberg, Germany
| | - Lars Kaderali
- Institute of Bioinformatics & Center for Functional Genomics of Microbes, University Medicine Greifswald, Greifswald, Germany
| | - Marco Binder
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center, Heidelberg, Germany
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10
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Kong B, Kim Y, Kim EH, Suk JS, Yang Y. mRNA: A promising platform for cancer immunotherapy. Adv Drug Deliv Rev 2023; 199:114993. [PMID: 37414361 PMCID: PMC11797636 DOI: 10.1016/j.addr.2023.114993] [Citation(s) in RCA: 17] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2023] [Revised: 06/22/2023] [Accepted: 07/03/2023] [Indexed: 07/08/2023]
Abstract
Messenger RNA (mRNA) is now in the limelight as a powerful tool for treating various human diseases, especially malignant tumors, thanks to the remarkable clinical outcomes of mRNA vaccines using lipid nanoparticle technology during the COVID-19 pandemic. Recent promising preclinical and clinical results that epitomize the advancement in mRNA and nanoformulation-based delivery technologies have highlighted the tremendous potential of mRNA in cancer immunotherapy. mRNAs can be harnessed for cancer immunotherapy in forms of various therapeutic modalities, including cancer vaccines, adoptive T-cell therapies, therapeutic antibodies, and immunomodulatory proteins. This review provides a comprehensive overview of the current state and prospects of mRNA-based therapeutics, including numerous delivery and therapeutic strategies.
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Affiliation(s)
- Byoungjae Kong
- Center for Nanomedicine, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA
| | - Yelee Kim
- Biomedical Research Division, Korea Institute of Science and Technology (KIST), Seoul, Republic of Korea; Department of Life Sciences, Korea University, Seoul 02841, Republic of Korea
| | - Eun Hye Kim
- Biomedical Research Division, Korea Institute of Science and Technology (KIST), Seoul, Republic of Korea; Department of Life Sciences, Korea University, Seoul 02841, Republic of Korea
| | - Jung Soo Suk
- Center for Nanomedicine, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA; Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
| | - Yoosoo Yang
- Biomedical Research Division, Korea Institute of Science and Technology (KIST), Seoul, Republic of Korea.
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11
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Sun H, Chan JFW, Yuan S. Cellular Sensors and Viral Countermeasures: A Molecular Arms Race between Host and SARS-CoV-2. Viruses 2023; 15:352. [PMID: 36851564 PMCID: PMC9962416 DOI: 10.3390/v15020352] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/25/2022] [Revised: 01/23/2023] [Accepted: 01/24/2023] [Indexed: 01/28/2023] Open
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic that has caused disastrous effects on the society and human health globally. SARS-CoV-2 is a sarbecovirus in the Coronaviridae family with a positive-sense single-stranded RNA genome. It mainly replicates in the cytoplasm and viral components including RNAs and proteins can be sensed by pattern recognition receptors including toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptors (NLRs) that regulate the host innate and adaptive immune responses. On the other hand, the SARS-CoV-2 genome encodes multiple proteins that can antagonize the host immune response to facilitate viral replication. In this review, we discuss the current knowledge on host sensors and viral countermeasures against host innate immune response to provide insights on virus-host interactions and novel approaches to modulate host inflammation and antiviral responses.
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Affiliation(s)
- Haoran Sun
- Department of Infectious Disease and Microbiology, The University of Hong Kong-Shenzhen Hospital, Shenzhen 518009, China
| | - Jasper Fuk-Woo Chan
- Department of Infectious Disease and Microbiology, The University of Hong Kong-Shenzhen Hospital, Shenzhen 518009, China
- State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
- Centre for Virology, Vaccinology and Therapeutics, Hong Kong Science and Technology Park, Hong Kong, China
| | - Shuofeng Yuan
- Department of Infectious Disease and Microbiology, The University of Hong Kong-Shenzhen Hospital, Shenzhen 518009, China
- State Key Laboratory of Emerging Infectious Diseases, Carol Yu Centre for Infection, Department of Microbiology, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China
- Centre for Virology, Vaccinology and Therapeutics, Hong Kong Science and Technology Park, Hong Kong, China
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12
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Endogenous Retroviruses as Modulators of Innate Immunity. Pathogens 2023; 12:pathogens12020162. [PMID: 36839434 PMCID: PMC9963469 DOI: 10.3390/pathogens12020162] [Citation(s) in RCA: 27] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2022] [Revised: 01/11/2023] [Accepted: 01/16/2023] [Indexed: 01/21/2023] Open
Abstract
Endogenous retroviruses (ERVs), or LTR retrotransposons, are a class of transposable elements that are highly represented in mammalian genomes. Human ERVs (HERVs) make up roughly 8.3% of the genome and over the course of evolution, HERV elements underwent positive selection and accrued mutations that rendered them non-infectious; thereby, the genome could co-opt them into constructive roles with important biological functions. In the past two decades, with the help of advances in sequencing technology, ERVs are increasingly considered to be important components of the innate immune response. While typically silenced, expression of HERVs can be induced in response to traumatic, toxic, or infection-related stress, leading to a buildup of viral transcripts and under certain circumstances, proteins, including functionally active reverse transcriptase and viral envelopes. The biological activity of HERVs in the context of the innate immune response can be based on the functional effect of four major viral components: (1) HERV LTRs, (2) HERV-derived RNAs, (3) HERV-derived RNA:DNA duplexes and cDNA, and (4) HERV-derived proteins and ribonucleoprotein complexes. In this review, we will discuss the implications of HERVs in all four contexts in relation to innate immunity and their association with various pathological disease states.
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13
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Thoresen DT, Galls D, Götte B, Wang W, Pyle AM. A rapid RIG-I signaling relay mediates efficient antiviral response. Mol Cell 2023; 83:90-104.e4. [PMID: 36521492 PMCID: PMC9825657 DOI: 10.1016/j.molcel.2022.11.018] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2022] [Revised: 08/11/2022] [Accepted: 11/21/2022] [Indexed: 12/15/2022]
Abstract
RIG-I is essential for host defense against viral pathogens, as it triggers the release of type I interferons upon encounter with viral RNA molecules. In this study, we show that RIG-I is rapidly and efficiently activated by small quantities of incoming viral RNA and that it relies exclusively on the constitutively expressed resident pool of RIG-I receptors for a strong antiviral response. Live-cell imaging of RIG-I following stimulation with viral or synthetic dsRNA reveals that RIG-I signaling occurs without mass aggregation at the mitochondrial membrane. By contrast, interferon-induced RIG-I protein becomes embedded in cytosolic aggregates that are functionally unrelated to signaling. These findings suggest that endogenous RIG-I efficiently recognizes viral RNA and rapidly relays an antiviral signal to MAVS via a transient signaling complex and that cellular aggregates of RIG-I have a function that is distinct from signaling.
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Affiliation(s)
- Daniel T Thoresen
- Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA
| | - Drew Galls
- Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA
| | - Benjamin Götte
- Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA
| | - Wenshuai Wang
- Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA
| | - Anna M Pyle
- Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA; Department of Chemistry, Yale University, New Haven, CT, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA.
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14
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Zhao X, Dan C, Gong XY, Li YL, Qu ZL, Sun HY, An LL, Guo WH, Gui JF, Zhang YB. Zebrafish MARCH8 downregulates fish IFN response by targeting MITA and TBK1 for protein degradation. DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 2022; 135:104485. [PMID: 35764162 DOI: 10.1016/j.dci.2022.104485] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/12/2022] [Revised: 06/17/2022] [Accepted: 06/23/2022] [Indexed: 06/15/2023]
Abstract
Recent studies have related the membrane-associated RING-CH-type finger (MARCH) family proteins to host innate immune response. Zebrafish (Danio rerio) MARCH8 is reported to target SVCV glycoprotein for degradation; however, little is known about whether fish MARCH8 is involved in innate interferon (IFN) response. In this study, zebrafish march8 was significantly induced by SVCV infection. Overexpression of MARCH8 diminished fish IFN-mediated antiviral response, thus promoting the replication of SVCV and GCRV in fish cells. Mechanistically, MARCH8 interacts with and degrades MITA and TBK1 proteins to inhibit IFN response. Moreover, MARCH8 has an E3 ligase activity and enhances MITA and TBK1 polyubiquitination. Our findings reveal a mechanism whereby zebrafish MARCH8 downregulates fish IFN response and facilitates viral replication by targeting MITA and TBK1 for protein degradation.
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Affiliation(s)
- Xiang Zhao
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China; University of Chinese Academy of Sciences, Beijing, 10049, China
| | - Cheng Dan
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China; University of Chinese Academy of Sciences, Beijing, 10049, China
| | - Xiu-Ying Gong
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China; University of Chinese Academy of Sciences, Beijing, 10049, China
| | - Yi-Lin Li
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China; University of Chinese Academy of Sciences, Beijing, 10049, China
| | - Zi-Ling Qu
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China; University of Chinese Academy of Sciences, Beijing, 10049, China
| | - Hao-Yu Sun
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China; University of Chinese Academy of Sciences, Beijing, 10049, China
| | - Li-Li An
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China; University of Chinese Academy of Sciences, Beijing, 10049, China
| | - Wen-Hao Guo
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China; University of Chinese Academy of Sciences, Beijing, 10049, China
| | - Jian-Fang Gui
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China; University of Chinese Academy of Sciences, Beijing, 10049, China; The Innovation Academy of Seed Design, Chinese Academy of Sciences, Wuhan, 430072, China
| | - Yi-Bing Zhang
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China; University of Chinese Academy of Sciences, Beijing, 10049, China; The Innovation Academy of Seed Design, Chinese Academy of Sciences, Wuhan, 430072, China.
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15
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Zander DY, Burkart SS, Wüst S, Magalhães VG, Binder M. Cooperative effects of RIG-I-like receptor signaling and IRF1 on DNA damage-induced cell death. Cell Death Dis 2022; 13:364. [PMID: 35436994 PMCID: PMC9016077 DOI: 10.1038/s41419-022-04797-7] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2021] [Revised: 03/17/2022] [Accepted: 03/30/2022] [Indexed: 12/19/2022]
Abstract
Properly responding to DNA damage is vital for eukaryotic cells, including the induction of DNA repair, growth arrest and, as a last resort to prevent neoplastic transformation, cell death. Besides being crucial for ensuring homeostasis, the same pathways and mechanisms are at the basis of chemoradiotherapy in cancer treatment, which involves therapeutic induction of DNA damage by chemical or physical (radiological) measures. Apart from typical DNA damage response mediators, the relevance of cell-intrinsic antiviral signaling pathways in response to DNA breaks has recently emerged. Originally known for combatting viruses via expression of antiviral factors including interferons (IFNs) and establishing of an antiviral state, RIG-I-like receptors (RLRs) were found to be critical for adequate induction of cell death upon the introduction of DNA double-strand breaks. We here show that presence of IRF3 is crucial in this process, most likely through direct activation of pro-apoptotic factors rather than transcriptional induction of canonical downstream components, such as IFNs. Investigating genes reported to be involved in both DNA damage response and antiviral signaling, we demonstrate that IRF1 is an obligatory factor for DNA damage-induced cell death. Interestingly, its regulation does not require activation of RLR signaling, but rather sensing of DNA double-strand breaks by ATM and ATR. Hence, even though independently regulated, both RLR signaling and IRF1 are essential for full-fledged induction/execution of DNA damage-mediated cell death programs. Our results not only support more broadly developing IRF1 as a biomarker predictive for the effectiveness of chemoradiotherapy, but also suggest investigating a combined pharmacological stimulation of RLR and IRF1 signaling as a potential adjuvant regimen in tumor therapy.
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Affiliation(s)
- David Y Zander
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany
- Department of Infectious Diseases, Molecular Virology, Center for Integrative Infectious Disease Research, Heidelberg University, Heidelberg, Germany
| | - Sandy S Burkart
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany
- Faculty of Biosciences, Heidelberg University, Heidelberg, Germany
| | - Sandra Wüst
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Vladimir G Magalhães
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Marco Binder
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany.
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16
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Kwon S, Kwon M, Im S, Lee K, Lee H. mRNA vaccines: the most recent clinical applications of synthetic mRNA. Arch Pharm Res 2022; 45:245-262. [PMID: 35426547 PMCID: PMC9012156 DOI: 10.1007/s12272-022-01381-7] [Citation(s) in RCA: 29] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2021] [Accepted: 04/05/2022] [Indexed: 12/24/2022]
Abstract
Synthetic mRNA has been considered as an emerging biotherapeutic agent for the past decades. Recently, the SARS-CoV-2 pandemic has led to the first clinical use of synthetic mRNA. mRNA vaccines showed far surpassing influences on the public as compared to other vaccine platforms such as viral vector vaccines and recombinant protein vaccines. It allowed rapid development and production of vaccines that have never been achieved in history. Synthetic mRNA, called in vitro transcribed (IVT) mRNA, is the key component of mRNA vaccines. It has several advantages over conventional gene-expressing systems such as plasmid DNA and viral vectors. It can translate proteins in the cytoplasm by structurally resembling natural mRNA and exhibit various protein expression patterns depending on how it is engineered. Another advantage is that synthetic mRNA enables fast, scalable, and cost-effective production. Therefore, starting with the mRNA vaccine, synthetic mRNA is now in the spotlight as a promising new drug development agent. In this review, we will summarize the latest IVT mRNA technology such as new mRNA structures or large-scale production. In addition, the nature of the innate immunogenicity of IVT mRNA will be discussed along with its roles in the development of vaccines. Finally, the principles of the mRNA vaccine and the future direction of synthetic mRNA will be provided.
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Affiliation(s)
- Suji Kwon
- College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, 03760, Republic of Korea
| | - Minseon Kwon
- College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, 03760, Republic of Korea
| | - Seongeun Im
- College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, 03760, Republic of Korea
| | - Kyuri Lee
- College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang National University, Jinju, 52828, Republic of Korea.
| | - Hyukjin Lee
- College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, 03760, Republic of Korea.
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17
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Kang YG, Kim J, Lee K, Choe JY, Dua P, Lee DK. Retinoic Acid-Inducible Gene I-Mediated Innate Immune Stimulation by Chemically Synthesized Long Double-Stranded RNAs Is Structure and Sequence Dependent. Nucleic Acid Ther 2022; 32:321-332. [PMID: 35263174 DOI: 10.1089/nat.2021.0104] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Double-stranded RNAs (dsRNAs) longer than 30 bp have not been considered desirable RNA interference (RNAi) triggering structures in mammalian cells as they nonspecifically activate innate immune response. However, in earlier studies, not only dsRNA length but also 5'-triphosphate moiety produced by in vitro transcription might have affected the stimulation of innate immune system. Herein, using chemically synthesized long dsRNAs without 5'-triphosphate, we elucidated direct relationship between length of dsRNAs and innate immune stimulation. First, we found that blunt-ended, chemically synthesized 38/40-60 bp-long dsRNAs induced retinoic acid-inducible gene I (RIG-I)-mediated innate immune response, which was suppressed by the introduction of the 2-nt 3' overhang structure. Surprisingly, we discovered that RIG-I activation by these long dsRNAs is also sequence dependent, and the sequence composition at dsRNA termini is important for RIG-I activation. In addition, we identified that long dsRNAs over 38 bp could elicit single- or dual-target gene silencing in a Dicer-independent manner. Taken together, our findings may serve as guidelines to develop an immunostimulatory RNAi trigger to exploit host's innate immune system, as well as a specific dual-gene targeting RNAi therapeutics platform without nonspecific innate immune stimulation by RIG-I.
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Affiliation(s)
- Young Gyu Kang
- Department of Chemistry, Sungkyunkwan University, Suwon, Korea.,OliX Pharmaceuticals, Inc., Suwon, Korea
| | - Jaejin Kim
- Department of Chemistry, Sungkyunkwan University, Suwon, Korea
| | - Kyeongmin Lee
- Department of Chemistry, Sungkyunkwan University, Suwon, Korea
| | - Jeong Yong Choe
- Department of Chemistry, Sungkyunkwan University, Suwon, Korea.,OliX Pharmaceuticals, Inc., Suwon, Korea
| | - Pooja Dua
- OliX Pharmaceuticals, Inc., Suwon, Korea
| | - Dong Ki Lee
- Department of Chemistry, Sungkyunkwan University, Suwon, Korea.,OliX Pharmaceuticals, Inc., Suwon, Korea
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18
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Altstetter SM, Quitt O, Pinci F, Hornung V, Lucko AM, Wisskirchen K, Jung S, Protzer U. Hepatitis-D Virus Infection Is Not Impaired by Innate Immunity but Increases Cytotoxic T-Cell Activity. Cells 2021; 10:3253. [PMID: 34831475 PMCID: PMC8619298 DOI: 10.3390/cells10113253] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2021] [Revised: 11/08/2021] [Accepted: 11/18/2021] [Indexed: 11/22/2022] Open
Abstract
Approximately 70 million humans worldwide are affected by chronic hepatitis D, which rapidly leads to liver cirrhosis and hepatocellular carcinoma due to chronic inflammation. The triggers and consequences of this chronic inflammation, induced by co-infection with the hepatitis D virus (HDV) and the hepatitis B virus (HBV), are poorly understood. Using CRISPR technology, we characterized the recognition of HDV mono- and co-infection by intracellular innate immunity and determined its influence on the viral life cycle and effector T-cell responses using different HBV and HDV permissive hepatoma cell lines. We showed that HDV infection is detected by MDA5 and -after a lag phase -induces a profound type I interferon response in the infected cells. The type I interferon response, however, was not able to suppress HDV replication or spread, thus providing a persistent trigger. Using engineered T-cells directed against the envelope proteins commonly used by HBV and HDV, we found that HDV immune recognition enhanced T-cell cytotoxicity. Interestingly, the T-cell effector function was enhanced independently of antigen presentation. These findings help to explain immune mediated tissue damage in chronic hepatitis D patients and indicate that combining innate triggers with T-cell activating therapies might allow for a curative approach.
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Affiliation(s)
- Sebastian Maximilian Altstetter
- Institute of Virology, School of Medicine, Helmholtz Zentrum München/Technical University of Munich, 81675 Munich, Germany; (S.M.A.); (O.Q.); (A.M.L.); (K.W.)
| | - Oliver Quitt
- Institute of Virology, School of Medicine, Helmholtz Zentrum München/Technical University of Munich, 81675 Munich, Germany; (S.M.A.); (O.Q.); (A.M.L.); (K.W.)
| | - Francesca Pinci
- Gene Center and Department of Biochemistry, Ludwig-Maximilians—University Munich, 81377 Munich, Germany; (F.P.); (V.H.)
| | - Veit Hornung
- Gene Center and Department of Biochemistry, Ludwig-Maximilians—University Munich, 81377 Munich, Germany; (F.P.); (V.H.)
| | - Aaron Michael Lucko
- Institute of Virology, School of Medicine, Helmholtz Zentrum München/Technical University of Munich, 81675 Munich, Germany; (S.M.A.); (O.Q.); (A.M.L.); (K.W.)
| | - Karin Wisskirchen
- Institute of Virology, School of Medicine, Helmholtz Zentrum München/Technical University of Munich, 81675 Munich, Germany; (S.M.A.); (O.Q.); (A.M.L.); (K.W.)
| | - Stephanie Jung
- Institute of Virology, School of Medicine, Helmholtz Zentrum München/Technical University of Munich, 81675 Munich, Germany; (S.M.A.); (O.Q.); (A.M.L.); (K.W.)
- Institute of Cardiovascular Immunology, University Hospital Bonn, University of Bonn, 53127 Bonn, Germany
| | - Ulrike Protzer
- Institute of Virology, School of Medicine, Helmholtz Zentrum München/Technical University of Munich, 81675 Munich, Germany; (S.M.A.); (O.Q.); (A.M.L.); (K.W.)
- German Center for Infection Research (DZIF), Munich Partner Site, 81675 Munich, Germany
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19
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Nicolay W, Moeller R, Kahl S, Vondran FWR, Pietschmann T, Kunz S, Gerold G. Characterization of RNA Sensing Pathways in Hepatoma Cell Lines and Primary Human Hepatocytes. Cells 2021; 10:3019. [PMID: 34831243 PMCID: PMC8616302 DOI: 10.3390/cells10113019] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2021] [Revised: 10/30/2021] [Accepted: 11/02/2021] [Indexed: 11/23/2022] Open
Abstract
The liver is targeted by several human pathogenic RNA viruses for viral replication and dissemination; despite this, the extent of innate immune sensing of RNA viruses by human hepatocytes is insufficiently understood to date. In particular, for highly human tropic viruses such as hepatitis C virus, cell culture models are needed to study immune sensing. However, several human hepatoma cell lines have impaired RNA sensing pathways and fail to mimic innate immune responses in the human liver. Here we compare the RNA sensing properties of six human hepatoma cell lines, namely Huh-6, Huh-7, HepG2, HepG2-HFL, Hep3B, and HepaRG, with primary human hepatocytes. We show that primary liver cells sense RNA through retinoic acid-inducible gene I (RIG-I) like receptor (RLR) and Toll-like receptor 3 (TLR3) pathways. Of the tested cell lines, Hep3B cells most closely mimicked the RLR and TLR3 mediated sensing in primary hepatocytes. This was shown by the expression of RLRs and TLR3 as well as the expression and release of bioactive interferon in primary hepatocytes and Hep3B cells. Our work shows that Hep3B cells partially mimic RNA sensing in primary hepatocytes and thus can serve as in vitro model to study innate immunity to RNA viruses in hepatocytes.
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Affiliation(s)
- Wiebke Nicolay
- TWINCORE—Centre for Experimental and Clinical Infection Research, Institute for Experimental Virology, 30625 Hannover, Germany; (W.N.); (R.M.); (S.K.); (T.P.)
| | - Rebecca Moeller
- TWINCORE—Centre for Experimental and Clinical Infection Research, Institute for Experimental Virology, 30625 Hannover, Germany; (W.N.); (R.M.); (S.K.); (T.P.)
- Center for Emerging Infections and Zoonoses (RIZ), Institute of Biochemistry & Research, University of Veterinary Medicine Hannover, 30625 Hannover, Germany
| | - Sina Kahl
- TWINCORE—Centre for Experimental and Clinical Infection Research, Institute for Experimental Virology, 30625 Hannover, Germany; (W.N.); (R.M.); (S.K.); (T.P.)
| | - Florian W. R. Vondran
- Department of General, Visceral and Transplant Surgery, Hannover Medical School, 30625 Hannover, Germany;
- German Centre for Infection Research (DZIF), 30100 Braunschweig, Germany
| | - Thomas Pietschmann
- TWINCORE—Centre for Experimental and Clinical Infection Research, Institute for Experimental Virology, 30625 Hannover, Germany; (W.N.); (R.M.); (S.K.); (T.P.)
| | - Stefan Kunz
- Institute of Microbiology, Lausanne University Hospital, CH-1011 Lausanne, Switzerland;
| | - Gisa Gerold
- TWINCORE—Centre for Experimental and Clinical Infection Research, Institute for Experimental Virology, 30625 Hannover, Germany; (W.N.); (R.M.); (S.K.); (T.P.)
- Center for Emerging Infections and Zoonoses (RIZ), Institute of Biochemistry & Research, University of Veterinary Medicine Hannover, 30625 Hannover, Germany
- Department of Clinical Microbiology, Virology, Umeå University, SE-90185 Umeå, Sweden
- Wallenberg Centre for Molecular Medicine (WCMM), Umeå University, SE-90185 Umeå, Sweden
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20
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Roca Suarez AA, Testoni B, Baumert TF, Lupberger J. Nucleic Acid-Induced Signaling in Chronic Viral Liver Disease. Front Immunol 2021; 11:624034. [PMID: 33613561 PMCID: PMC7892431 DOI: 10.3389/fimmu.2020.624034] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2020] [Accepted: 12/21/2020] [Indexed: 12/12/2022] Open
Abstract
A hallmark for the development and progression of chronic liver diseases is the persistent dysregulation of signaling pathways related to inflammatory responses, which eventually promotes the development of hepatic fibrosis, cirrhosis and hepatocellular carcinoma (HCC). The two major etiological agents associated with these complications in immunocompetent patients are hepatitis B virus (HBV) and hepatitis C virus (HCV), accounting for almost 1.4 million liver disease-associated deaths worldwide. Although both differ significantly from the point of their genomes and viral life cycles, they exert not only individual but also common strategies to divert innate antiviral defenses. Multiple virus-modulated pathways implicated in stress and inflammation illustrate how chronic viral hepatitis persistently tweaks host signaling processes with important consequences for liver pathogenesis. The following review aims to summarize the molecular events implicated in the sensing of viral nucleic acids, the mechanisms employed by HBV and HCV to counter these measures and how the dysregulation of these cellular pathways drives the development of chronic liver disease and the progression toward HCC.
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MESH Headings
- Carcinoma, Hepatocellular/immunology
- Carcinoma, Hepatocellular/mortality
- Carcinoma, Hepatocellular/pathology
- DNA, Viral/immunology
- Hepacivirus/immunology
- Hepatitis B virus/immunology
- Hepatitis B, Chronic/immunology
- Hepatitis B, Chronic/mortality
- Hepatitis B, Chronic/pathology
- Hepatitis C, Chronic/immunology
- Hepatitis C, Chronic/mortality
- Hepatitis C, Chronic/pathology
- Humans
- Liver Neoplasms/immunology
- Liver Neoplasms/mortality
- Liver Neoplasms/pathology
- RNA, Viral/immunology
- Signal Transduction/immunology
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Affiliation(s)
- Armando Andres Roca Suarez
- INSERM, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France
- Université de Strasbourg, Strasbourg, France
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon (CRCL), Lyon, France
- University of Lyon, Université Claude-Bernard (UCBL), Lyon, France
| | - Barbara Testoni
- INSERM U1052, CNRS UMR-5286, Cancer Research Center of Lyon (CRCL), Lyon, France
- University of Lyon, Université Claude-Bernard (UCBL), Lyon, France
| | - Thomas F. Baumert
- INSERM, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France
- Université de Strasbourg, Strasbourg, France
- Institut Hospitalo-Universitaire, Pôle Hépato-digestif, Nouvel Hôpital Civil, Strasbourg, France
- Institut Universitaire de France (IUF), Paris, France
| | - Joachim Lupberger
- INSERM, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France
- Université de Strasbourg, Strasbourg, France
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21
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Levy ES, Chang R, Zamecnik CR, Dhariwala MO, Fong L, Desai TA. Multi-Immune Agonist Nanoparticle Therapy Stimulates Type I Interferons to Activate Antigen-Presenting Cells and Induce Antigen-Specific Antitumor Immunity. Mol Pharm 2021; 18:1014-1025. [PMID: 33541072 DOI: 10.1021/acs.molpharmaceut.0c00984] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Cancer immunity is mediated by a delicate orchestration between the innate and adaptive immune system both systemically and within the tumor microenvironment. Although several adaptive immunity molecular targets have been proven clinically efficacious, stand-alone innate immunity targeting agents have not been successful in the clinic. Here, we report a nanoparticle optimized for systemic administration that combines immune agonists for TLR9, STING, and RIG-I with a melanoma-specific peptide to induce antitumor immunity. These immune agonistic nanoparticles (iaNPs) significantly enhance the activation of antigen-presenting cells to orchestrate the development and response of melanoma-sensitized T-cells. iaNP treatment not only suppressed tumor growth in an orthotopic solid tumor model, but also significantly reduced tumor burden in a metastatic animal model. This combination biomaterial-based approach to coordinate innate and adaptive anticancer immunity provides further insights into the benefits of stimulating multiple activation pathways to promote tumor regression, while also offering an important platform to effectively and safely deliver combination immunotherapies for cancer.
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Affiliation(s)
- Elizabeth S Levy
- Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California 94158, United States
| | - Ryan Chang
- Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California 94158, United States.,Department of Medicine, University of California San Francisco, San Francisco, California 94143, United States
| | - Colin R Zamecnik
- Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California 94158, United States
| | - Miqdad O Dhariwala
- Department of Dermatology, University of California San Francisco, San Francisco, California 94143, United Stats
| | - Lawrence Fong
- Division of Hematology/Oncology, Department of Medicine, University of California San Francisco, San Francisco, California 94143, United States.,Parker Institute for Cancer Immunotherapy, University of California, San Francisco, San Francisco, California 94143, United States
| | - Tejal A Desai
- Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, California 94158, United States
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22
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Huijser E, Versnel MA. Making Sense of Intracellular Nucleic Acid Sensing in Type I Interferon Activation in Sjögren's Syndrome. J Clin Med 2021; 10:532. [PMID: 33540529 PMCID: PMC7867173 DOI: 10.3390/jcm10030532] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2020] [Revised: 01/26/2021] [Accepted: 01/29/2021] [Indexed: 12/13/2022] Open
Abstract
Primary Sjögren's syndrome (pSS) is a systemic autoimmune rheumatic disease characterized by dryness of the eyes and mucous membranes, which can be accompanied by various extraglandular autoimmune manifestations. The majority of patients exhibit persistent systemic activation of the type I interferon (IFN) system, a feature that is shared with other systemic autoimmune diseases. Type I IFNs are integral to anti-viral immunity and are produced in response to stimulation of pattern recognition receptors, among which nucleic acid (NA) receptors. Dysregulated detection of endogenous NAs has been widely implicated in the pathogenesis of systemic autoimmune diseases. Stimulation of endosomal Toll-like receptors by NA-containing immune complexes are considered to contribute to the systemic type I IFN activation. Accumulating evidence suggest additional roles for cytosolic NA-sensing pathways in the pathogenesis of systemic autoimmune rheumatic diseases. In this review, we will provide an overview of the functions and signaling of intracellular RNA- and DNA-sensing receptors and summarize the evidence for a potential role of these receptors in the pathogenesis of pSS and the sustained systemic type I IFN activation.
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Affiliation(s)
| | - Marjan A. Versnel
- Department of Immunology, Erasmus MC, University Medical Center Rotterdam, 3015 GD Rotterdam, The Netherlands;
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23
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Mdkhana B, Saheb Sharif-Askari N, Ramakrishnan RK, Goel S, Hamid Q, Halwani R. Nucleic Acid-Sensing Pathways During SARS-CoV-2 Infection: Expectations versus Reality. J Inflamm Res 2021; 14:199-216. [PMID: 33531826 PMCID: PMC7847386 DOI: 10.2147/jir.s277716] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2020] [Accepted: 10/16/2020] [Indexed: 12/22/2022] Open
Abstract
The coronavirus disease 2019 (COVID-19) pandemic has affected millions of people and crippled economies worldwide. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for this pandemic has triggered avid research on its pathobiology to better understand the pathophysiology of COVID-19. In the absence of approved antiviral therapeutic strategies or vaccine platforms capable of effectively targeting this global threat, the hunt for effective therapeutics has led to many candidates being actively evaluated for their efficacy in controlling or preventing COVID-19. In this review, we gathered current evidence on the innate nucleic acid-sensing pathways expected to be elicited by SARS-CoV-2 and the immune evasion mechanisms they have developed to promote viral replication and infection. Within the nucleic acid-sensing pathways, SARS-CoV-2 infection and evasion mechanisms trigger the activation of NOD-signaling and NLRP3 pathways leading to the production of inflammatory cytokines, IL-1β and IL-6, while muting or blocking cGAS-STING and interferon type I and III pathways, resulting in decreased production of antiviral interferons and delayed innate response. Therefore, blocking the inflammatory arm and boosting the interferon production arm of nucleic acid-sensing pathways could facilitate early control of viral replication and dissemination, prevent disease progression, and cytokine storm development. We also discuss the rationale behind therapeutic modalities targeting these sensing pathways and their implications in the treatment of COVID-19.
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Affiliation(s)
- Bushra Mdkhana
- Sharjah Institute of Medical Research, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates
| | - Narjes Saheb Sharif-Askari
- Sharjah Institute of Medical Research, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates
| | - Rakhee K Ramakrishnan
- Sharjah Institute of Medical Research, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates
| | - Swati Goel
- Sharjah Institute of Medical Research, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates
| | - Qutayba Hamid
- Sharjah Institute of Medical Research, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates.,Department of Clinical Sciences, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates.,Meakins-Christie Laboratories, Research Institute of the McGill University Health Center, Montreal, Quebec, Canada
| | - Rabih Halwani
- Sharjah Institute of Medical Research, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates.,Department of Clinical Sciences, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates
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24
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Fukushima Y, Uchida S, Imai H, Nakatomi H, Kataoka K, Saito N, Itaka K. Treatment of ischemic neuronal death by introducing brain-derived neurotrophic factor mRNA using polyplex nanomicelle. Biomaterials 2021; 270:120681. [PMID: 33517206 DOI: 10.1016/j.biomaterials.2021.120681] [Citation(s) in RCA: 27] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2020] [Revised: 01/12/2021] [Accepted: 01/16/2021] [Indexed: 12/19/2022]
Abstract
Ischemic neuronal death causes serious lifelong neurological deficits; however, there is no proven effective treatment that can prevent neuronal death after the ischemia. We investigated the feasibility of mRNA therapeutics for preventing the neuronal death in a rat model of transient global ischemia (TGI). By intraventricular administration of mRNA encoding brain-derived neurotrophic factor (BDNF) using a polymer-based carrier, polyplex nanomicelle, the mRNA significantly increased the survival rate of hippocampal neurons after TGI, with a rapid rise of BDNF in the hippocampus. Interestingly, mRNA administration on Day 2 after TGI provided significantly better survival rate than the administration immediately after TGI. Eventually, dosing twice on Day 2 and 5 exerted long-term therapeutic effects, which were confirmed by a Y-maze behavioral test demonstrating improved spatial memory compared with untreated rats on Day 20. Immunohistochemical analysis showed that astrocytes were chief targets of the BDNF mRNA-loaded nanomicelles, suggesting that the augmented BDNF secretion from astrocytes creates a supportive microenvironment for the neurons to tolerate changes caused by ischemic stresses, and terminate the process of progressive neuronal death after the ischemic attack. Overall, the unique mechanism of action of mRNA therapeutics provide a promising approach for preventing ischemic neuronal death.
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Affiliation(s)
- Yuta Fukushima
- Department of Biofunction Research, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 1010062, Tokyo, Japan; Department of Neurosurgery, Graduate School of Medicine, The University of Tokyo, 1138655, Tokyo, Japan; Innovation Center of NanoMedicine (iCONM), Kawasaki Institute of Industrial Promotion, 2100821, Kawasaki, Kanagawa, Japan
| | - Satoshi Uchida
- Innovation Center of NanoMedicine (iCONM), Kawasaki Institute of Industrial Promotion, 2100821, Kawasaki, Kanagawa, Japan; Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 1138656, Tokyo, Japan
| | - Hideaki Imai
- Department of Neurosurgery, Graduate School of Medicine, The University of Tokyo, 1138655, Tokyo, Japan
| | - Hirofumi Nakatomi
- Department of Neurosurgery, Graduate School of Medicine, The University of Tokyo, 1138655, Tokyo, Japan
| | - Kazunori Kataoka
- Innovation Center of NanoMedicine (iCONM), Kawasaki Institute of Industrial Promotion, 2100821, Kawasaki, Kanagawa, Japan; Institute for Future Initiatives, The University of Tokyo, 1130033, Tokyo, Japan
| | - Nobuhito Saito
- Department of Neurosurgery, Graduate School of Medicine, The University of Tokyo, 1138655, Tokyo, Japan
| | - Keiji Itaka
- Department of Biofunction Research, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), 1010062, Tokyo, Japan; Innovation Center of NanoMedicine (iCONM), Kawasaki Institute of Industrial Promotion, 2100821, Kawasaki, Kanagawa, Japan.
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25
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Long RKM, Moriarty KP, Cardoen B, Gao G, Vogl AW, Jean F, Hamarneh G, Nabi IR. Super resolution microscopy and deep learning identify Zika virus reorganization of the endoplasmic reticulum. Sci Rep 2020; 10:20937. [PMID: 33262363 PMCID: PMC7708840 DOI: 10.1038/s41598-020-77170-3] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2020] [Accepted: 10/28/2020] [Indexed: 01/08/2023] Open
Abstract
The endoplasmic reticulum (ER) is a complex subcellular organelle composed of diverse structures such as tubules, sheets and tubular matrices. Flaviviruses such as Zika virus (ZIKV) induce reorganization of ER membranes to facilitate viral replication. Here, using 3D super resolution microscopy, ZIKV infection is shown to induce the formation of dense tubular matrices associated with viral replication in the central ER. Viral non-structural proteins NS4B and NS2B associate with replication complexes within the ZIKV-induced tubular matrix and exhibit distinct ER distributions outside this central ER region. Deep neural networks trained to distinguish ZIKV-infected versus mock-infected cells successfully identified ZIKV-induced central ER tubular matrices as a determinant of viral infection. Super resolution microscopy and deep learning are therefore able to identify and localize morphological features of the ER and allow for better understanding of how ER morphology changes due to viral infection.
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Affiliation(s)
- Rory K M Long
- Life Sciences Institute, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.,Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.,Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada
| | - Kathleen P Moriarty
- School of Computing Science, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada
| | - Ben Cardoen
- School of Computing Science, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada
| | - Guang Gao
- Life Sciences Institute, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.,Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada
| | - A Wayne Vogl
- Life Sciences Institute, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.,Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada
| | - François Jean
- Life Sciences Institute, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada. .,Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
| | - Ghassan Hamarneh
- School of Computing Science, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.
| | - Ivan R Nabi
- Life Sciences Institute, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada. .,Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada. .,School of Biomedical Engineering, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
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26
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Jung S, von Thülen T, Yang I, Laukemper V, Rupf B, Janga H, Panagiotidis GD, Schoen A, Nicolai M, Schulte LN, Obermann HL, Weber F, Kaufmann A, Bauer S. A ribosomal RNA fragment with 2',3'-cyclic phosphate and GTP-binding activity acts as RIG-I ligand. Nucleic Acids Res 2020; 48:10397-10412. [PMID: 32946572 PMCID: PMC7544222 DOI: 10.1093/nar/gkaa739] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2019] [Revised: 08/19/2020] [Accepted: 08/26/2020] [Indexed: 12/13/2022] Open
Abstract
The RNA helicase RIG-I plays a key role in sensing pathogen-derived RNA. Double-stranded RNA structures bearing 5'-tri- or diphosphates are commonly referred to as activating RIG-I ligands. However, endogenous RNA fragments generated during viral infection via RNase L also activate RIG-I. Of note, RNase-digested RNA fragments bear a 5'-hydroxyl group and a 2',3'-cyclic phosphate. How endogenous RNA fragments activate RIG-I despite the lack of 5'-phosphorylation has not been elucidated. Here we describe an endogenous RIG-I ligand (eRL) that is derived from the internal transcribed spacer 2 region (ITS2) of the 45S ribosomal RNA after partial RNase A digestion in vitro, RNase A protein transfection or RNase L activation. The immunostimulatory property of the eRL is dependent on 2',3'-cyclic phosphate and its sequence is characterized by a G-quadruplex containing sequence motif mediating guanosine-5'-triphosphate (GTP) binding. In summary, RNase generated self-RNA fragments with 2',3'-cyclic phosphate function as nucleotide-5'-triphosphate binding aptamers activating RIG-I.
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Affiliation(s)
- Stephanie Jung
- Institut für Immunologie, Philipps-Universität Marburg, BMFZ, Hans-Meerwein-Straße 2, 35043 Marburg, Germany
| | - Tina von Thülen
- Institut für Immunologie, Philipps-Universität Marburg, BMFZ, Hans-Meerwein-Straße 2, 35043 Marburg, Germany
| | - Ines Yang
- Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Medizinische Hochschule Hannover, Carl Neuberg Straße 1, 30625 Hannover, Germany
| | - Viktoria Laukemper
- Institut für Immunologie, Philipps-Universität Marburg, BMFZ, Hans-Meerwein-Straße 2, 35043 Marburg, Germany
| | - Benjamin Rupf
- Institut für Immunologie, Philipps-Universität Marburg, BMFZ, Hans-Meerwein-Straße 2, 35043 Marburg, Germany
| | - Harshavardhan Janga
- Institut für Lungenforschung/iLung, Philipps-Universität Marburg, BMFZ, Hans-Meerwein-Straße 2, 35043 Marburg, Germany
| | - Georgios-Dimitrios Panagiotidis
- Institut für Virologie, Fachbereich Veterinärmedizin (FB10), Justus-Liebig-Universität Gießen, Schubertstr. 81, 35392 Gießen, Germany
| | - Andreas Schoen
- Institut für Virologie, Fachbereich Veterinärmedizin (FB10), Justus-Liebig-Universität Gießen, Schubertstr. 81, 35392 Gießen, Germany
| | - Marina Nicolai
- Institut für Immunologie, Philipps-Universität Marburg, BMFZ, Hans-Meerwein-Straße 2, 35043 Marburg, Germany
| | - Leon N Schulte
- Institut für Lungenforschung/iLung, Philipps-Universität Marburg, BMFZ, Hans-Meerwein-Straße 2, 35043 Marburg, Germany
- Deutsches Zentrum für Lungenforschung (DZL), 35392 Gießen, Germany
| | - Hannah-Lena Obermann
- Institut für Immunologie, Philipps-Universität Marburg, BMFZ, Hans-Meerwein-Straße 2, 35043 Marburg, Germany
| | - Friedemann Weber
- Institut für Virologie, Fachbereich Veterinärmedizin (FB10), Justus-Liebig-Universität Gießen, Schubertstr. 81, 35392 Gießen, Germany
| | - Andreas Kaufmann
- Institut für Immunologie, Philipps-Universität Marburg, BMFZ, Hans-Meerwein-Straße 2, 35043 Marburg, Germany
| | - Stefan Bauer
- Institut für Immunologie, Philipps-Universität Marburg, BMFZ, Hans-Meerwein-Straße 2, 35043 Marburg, Germany
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27
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Abstract
Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are key sensors of virus infection, mediating the transcriptional induction of type I interferons and other genes that collectively establish an antiviral host response. Recent studies have revealed that both viral and host-derived RNAs can trigger RLR activation; this can lead to an effective antiviral response but also immunopathology if RLR activities are uncontrolled. In this Review, we discuss recent advances in our understanding of the types of RNA sensed by RLRs in the contexts of viral infection, malignancies and autoimmune diseases. We further describe how the activity of RLRs is controlled by host regulatory mechanisms, including RLR-interacting proteins, post-translational modifications and non-coding RNAs. Finally, we discuss key outstanding questions in the RLR field, including how our knowledge of RLR biology could be translated into new therapeutics.
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Affiliation(s)
- Jan Rehwinkel
- Medical Research Council Human Immunology Unit, Medical Research Council Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.
| | - Michaela U Gack
- Department of Microbiology, The University of Chicago, Chicago, IL, USA.
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28
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Bartok E, Hartmann G. Immune Sensing Mechanisms that Discriminate Self from Altered Self and Foreign Nucleic Acids. Immunity 2020; 53:54-77. [PMID: 32668228 PMCID: PMC7359798 DOI: 10.1016/j.immuni.2020.06.014] [Citation(s) in RCA: 117] [Impact Index Per Article: 23.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2020] [Revised: 06/15/2020] [Accepted: 06/16/2020] [Indexed: 12/19/2022]
Abstract
All lifeforms have developed highly sophisticated systems equipped to detect altered self and non-self nucleic acids (NA). In vertebrates, NA-sensing receptors safeguard the integrity of the organism by detecting pathogens, dyshomeostasis and damage, and inducing appropriate responses to eliminate pathogens and reconstitute homeostasis. Effector mechanisms include i) immune signaling, ii) restriction of NA functions such as inhibition of mRNA translation, and iii) cell death pathways. An appropriate effector response is necessary for host defense, but dysregulated NA-sensing can lead to devastating autoimmune and autoinflammatory disease. Their inherent biochemical similarity renders the reliable distinction between self NA under homeostatic conditions and altered or exogenous NA particularly challenging. In this review, we provide an overview of recent progress in our understanding of the closely coordinated and regulated network of innate immune receptors, restriction factors, and nucleases to effectively respond to pathogens and maintain host integrity.
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Affiliation(s)
- Eva Bartok
- Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Venusberg-Campus 1, 53127 Bonn, Germany
| | - Gunther Hartmann
- Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Venusberg-Campus 1, 53127 Bonn, Germany.
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29
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Schweinoch D, Bachmann P, Clausznitzer D, Binder M, Kaderali L. Mechanistic modeling explains the dsRNA length-dependent activation of the RIG-I mediated immune response. J Theor Biol 2020; 500:110336. [PMID: 32446742 DOI: 10.1016/j.jtbi.2020.110336] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2019] [Revised: 05/13/2020] [Accepted: 05/15/2020] [Indexed: 12/25/2022]
Abstract
In cell-intrinsic antiviral immunity, cytoplasmic receptors such as retinoic acid-inducible gene I (RIG-I) detect viral double-stranded RNA (dsRNA) and trigger a signaling cascade activating the interferon (IFN) system. This leads to the transcription of hundreds of interferon-stimulated genes (ISGs) with a wide range of antiviral effects. This recognition of dsRNA not only has to be very specific to discriminate foreign from self but also highly sensitive to detect even very low numbers of pathogenic dsRNA molecules. Previous work indicated an influence of the dsRNA length on the binding behavior of RIG-I and its potential to elicit antiviral signaling. However, the molecular mechanisms behind the binding process are still under debate. We compare two hypothesized RIG-I binding mechanisms by translating them into mathematical models and analyzing their potential to describe published experimental data. The models consider the length of the dsRNA as well as known RIG-I binding motifs and describe RIG-I pathway activation after stimulation with dsRNA. We show that internal RIG-I binding sites in addition to cooperative RIG-I oligomerization are essential to describe the experimentally observed RIG-I binding behavior and immune response activation for different dsRNA lengths and concentrations. The combination of RIG-I binding to internal sites on the dsRNA and cooperative oligomerization compensates for a lack of high-affinity binding motifs and triggers a strong antiviral response for long dsRNAs. Model analysis reveals dsRNA length-dependency as a potential mechanism to discriminate between different types of dsRNAs: It allows for sensitive detection of small numbers of long dsRNAs, a typical by-product of viral replication, while ensuring tolerance against non-harming small dsRNAs.
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Affiliation(s)
- Darius Schweinoch
- University Medicine Greifswald, Institute of Bioinformatics and Center for Functional Genomics of Microbes (C_FunGene), Felix-Hausdorff-Str. 8, 17475 Greifswald, Germany
| | - Pia Bachmann
- University Medicine Greifswald, Institute of Bioinformatics and Center for Functional Genomics of Microbes (C_FunGene), Felix-Hausdorff-Str. 8, 17475 Greifswald, Germany
| | - Diana Clausznitzer
- Technische Universität Dresden, Faculty of Medicine Carl-Gustav Carus, Institute for Medical Informatics and Biometry, Dresden, Germany
| | - Marco Binder
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response", Division Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Lars Kaderali
- University Medicine Greifswald, Institute of Bioinformatics and Center for Functional Genomics of Microbes (C_FunGene), Felix-Hausdorff-Str. 8, 17475 Greifswald, Germany.
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30
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Unfried JP, Fortes P. LncRNAs in HCV Infection and HCV-Related Liver Disease. Int J Mol Sci 2020; 21:ijms21062255. [PMID: 32214045 PMCID: PMC7139329 DOI: 10.3390/ijms21062255] [Citation(s) in RCA: 35] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2020] [Revised: 03/19/2020] [Accepted: 03/20/2020] [Indexed: 12/14/2022] Open
Abstract
Long non-coding RNAs (lncRNAs) are transcripts with poor coding capacity that may interact with proteins, DNA, or other RNAs to perform structural and regulatory functions. The lncRNA transcriptome changes significantly in most diseases, including cancer and viral infections. In this review, we summarize the functional implications of lncRNA-deregulation after infection with hepatitis C virus (HCV). HCV leads to chronic infection in many patients that may progress to liver cirrhosis and hepatocellular carcinoma (HCC). Most lncRNAs deregulated in infected cells that have been described function to potentiate or block the antiviral response and, therefore, they have a great impact on HCV viral replication. In addition, several lncRNAs upregulated by the infection contribute to viral release. Finally, many lncRNAs have been described as deregulated in HCV-related HCC that function to enhance cell survival, proliferation, and tumor progression by different mechanisms. Interestingly, some HCV-related HCC lncRNAs can be detected in bodily fluids, and there is great hope that they could be used as biomarkers to predict cancer initiation, progression, tumor burden, response to treatment, resistance to therapy, or tumor recurrence. Finally, there is high confidence that lncRNAs could also be used to improve the suboptimal long-term outcomes of current HCC treatment options.
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Affiliation(s)
| | - P. Fortes
- Correspondence: ; Tel.: +34-948194700
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31
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Ablasser A, Hur S. Regulation of cGAS- and RLR-mediated immunity to nucleic acids. Nat Immunol 2020; 21:17-29. [PMID: 31819255 DOI: 10.1038/s41590-019-0556-1] [Citation(s) in RCA: 218] [Impact Index Per Article: 43.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2019] [Accepted: 10/29/2019] [Indexed: 12/13/2022]
Abstract
Pathogen-derived nucleic acids are crucial signals for innate immunity. Despite the structural similarity between those and host nucleic acids, mammalian cells have been able to evolve powerful innate immune signaling pathways that originate from the detection of cytosolic nucleic acid species, one of the most prominent being the cGAS-STING pathway for DNA and the RLR-MAVS pathway for RNA, respectively. Recent advances have revealed a plethora of regulatory mechanisms that are crucial for balancing the activity of nucleic acid sensors for the maintenance of overall cellular homeostasis. Elucidation of the various mechanisms that enable cells to maintain control over the activity of cytosolic nucleic acid sensors has provided new insight into the pathology of human diseases and, at the same time, offers a rich and largely unexplored source for new therapeutic targets. This Review addresses the emerging literature on regulation of the sensing of cytosolic DNA and RNA via cGAS and RLRs.
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Affiliation(s)
- Andrea Ablasser
- Global Health Institute, Swiss Federal Institute of Technology, Lausanne, Switzerland.
| | - Sun Hur
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
- Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.
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Cadena C, Hur S. Filament-like Assemblies of Intracellular Nucleic Acid Sensors: Commonalities and Differences. Mol Cell 2019; 76:243-254. [PMID: 31626748 PMCID: PMC6880955 DOI: 10.1016/j.molcel.2019.09.023] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2019] [Revised: 09/13/2019] [Accepted: 09/19/2019] [Indexed: 12/25/2022]
Abstract
Self versus non-self discrimination by innate immune sensors is critical for mounting effective immune responses against pathogens while avoiding harmful auto-inflammatory reactions against the host. Foreign DNA and RNA sensors must discriminate between self versus non-self nucleic acids, despite their shared building blocks and similar physicochemical properties. Recent structural and biochemical studies suggest that multiple steps of filament-like assembly are required for the functions of several nucleic acid sensors. Here, we discuss ligand discrimination and oligomerization of RIG-I-like receptors, AIM2-like receptors, and cGAS. We discuss how filament-like assembly allows for robust and accurate discrimination of self versus non-self nucleic acids and how these assemblies enable sensing of multiple distinct features in foreign nucleic acids, including structure, length, and modifications. We also discuss how individual receptors differ in their assembly and disassembly mechanisms and how these differences contribute to the diversity in nucleic acid specificity and pathogen detection strategies.
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Affiliation(s)
- Cristhian Cadena
- Program in Virology, Division of Medical Sciences, Harvard Medical School, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Program in Cellular and Molecular Medicine, Boston Children's Hospital, MA 02115, USA
| | - Sun Hur
- Program in Virology, Division of Medical Sciences, Harvard Medical School, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Program in Cellular and Molecular Medicine, Boston Children's Hospital, MA 02115, USA.
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Brisse M, Ly H. Comparative Structure and Function Analysis of the RIG-I-Like Receptors: RIG-I and MDA5. Front Immunol 2019; 10:1586. [PMID: 31379819 PMCID: PMC6652118 DOI: 10.3389/fimmu.2019.01586] [Citation(s) in RCA: 247] [Impact Index Per Article: 41.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2019] [Accepted: 06/25/2019] [Indexed: 12/12/2022] Open
Abstract
RIG-I (Retinoic acid-inducible gene I) and MDA5 (Melanoma Differentiation-Associated protein 5), collectively known as the RIG-I-like receptors (RLRs), are key protein sensors of the pathogen-associated molecular patterns (PAMPs) in the form of viral double-stranded RNA (dsRNA) motifs to induce expression of type 1 interferons (IFN1) (IFNα and IFNβ) and other pro-inflammatory cytokines during the early stage of viral infection. While RIG-I and MDA5 share many genetic, structural and functional similarities, there is increasing evidence that they can have significantly different strategies to recognize different pathogens, PAMPs, and in different host species. This review article discusses the similarities and differences between RIG-I and MDA5 from multiple perspectives, including their structures, evolution and functional relationships with other cellular proteins, their differential mechanisms of distinguishing between host and viral dsRNAs and interactions with host and viral protein factors, and their immunogenic signaling. A comprehensive comparative analysis can help inform future studies of RIG-I and MDA5 in order to fully understand their functions in order to optimize potential therapeutic approaches targeting them.
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Affiliation(s)
- Morgan Brisse
- Biochemistry, Molecular Biology, and Biophysics Graduate Program, University of Minnesota, Twin Cities, St. Paul, MN, United States
- Department of Veterinary & Biomedical Sciences, University of Minnesota, Twin Cities, St. Paul, MN, United States
| | - Hinh Ly
- Department of Veterinary & Biomedical Sciences, University of Minnesota, Twin Cities, St. Paul, MN, United States
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Cadena C, Ahmad S, Xavier A, Willemsen J, Park S, Park JW, Oh SW, Fujita T, Hou F, Binder M, Hur S. Ubiquitin-Dependent and -Independent Roles of E3 Ligase RIPLET in Innate Immunity. Cell 2019; 177:1187-1200.e16. [PMID: 31006531 PMCID: PMC6525047 DOI: 10.1016/j.cell.2019.03.017] [Citation(s) in RCA: 146] [Impact Index Per Article: 24.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2018] [Revised: 01/28/2019] [Accepted: 03/07/2019] [Indexed: 01/22/2023]
Abstract
The conventional view posits that E3 ligases function primarily through conjugating ubiquitin (Ub) to their substrate molecules. We report here that RIPLET, an essential E3 ligase in antiviral immunity, promotes the antiviral signaling activity of the viral RNA receptor RIG-I through both Ub-dependent and -independent manners. RIPLET uses its dimeric structure and a bivalent binding mode to preferentially recognize and ubiquitinate RIG-I pre-oligomerized on dsRNA. In addition, RIPLET can cross-bridge RIG-I filaments on longer dsRNAs, inducing aggregate-like RIG-I assemblies. The consequent receptor clustering synergizes with the Ub-dependent mechanism to amplify RIG-I-mediated antiviral signaling in an RNA-length dependent manner. These observations show the unexpected role of an E3 ligase as a co-receptor that directly participates in receptor oligomerization and ligand discrimination. It also highlights a previously unrecognized mechanism by which the innate immune system measures foreign nucleic acid length, a common criterion for self versus non-self nucleic acid discrimination.
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Affiliation(s)
- Cristhian Cadena
- Program in Virology, Division of Medical Sciences, Harvard Medical School, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Program in Cellular and Molecular Medicine, Boston Children's Hospital, MA 02115, USA
| | - Sadeem Ahmad
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Program in Cellular and Molecular Medicine, Boston Children's Hospital, MA 02115, USA
| | - Audrey Xavier
- Program in Cellular and Molecular Medicine, Boston Children's Hospital, MA 02115, USA; Institute of Chemistry and Biochemistry, Free University of Berlin, Germany
| | - Joschka Willemsen
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response" (division F170), German Cancer Research Center, 69120 Heidelberg, Germany
| | - Sehoon Park
- Program in Cellular and Molecular Medicine, Boston Children's Hospital, MA 02115, USA
| | - Ji Woo Park
- Program in Cellular and Molecular Medicine, Boston Children's Hospital, MA 02115, USA; Biology Department, Boston College, Chestnut Hill, MA, USA
| | - Seong-Wook Oh
- Laboratory of Molecular Genetics, Institute for Frontier Life and Medical Sciences, Kyoto University, Japan
| | - Takashi Fujita
- Laboratory of Molecular Genetics, Institute for Frontier Life and Medical Sciences, Kyoto University, Japan
| | - Fajian Hou
- State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, China
| | - Marco Binder
- Research Group "Dynamics of Early Viral Infection and the Innate Antiviral Response" (division F170), German Cancer Research Center, 69120 Heidelberg, Germany
| | - Sun Hur
- Program in Virology, Division of Medical Sciences, Harvard Medical School, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Program in Cellular and Molecular Medicine, Boston Children's Hospital, MA 02115, USA.
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35
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Yoshinaga N, Uchida S, Naito M, Osada K, Cabral H, Kataoka K. Induced packaging of mRNA into polyplex micelles by regulated hybridization with a small number of cholesteryl RNA oligonucleotides directed enhanced in vivo transfection. Biomaterials 2019; 197:255-267. [PMID: 30669016 DOI: 10.1016/j.biomaterials.2019.01.023] [Citation(s) in RCA: 37] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2018] [Revised: 12/23/2018] [Accepted: 01/12/2019] [Indexed: 12/22/2022]
Abstract
There has been a progressive interest in the molecular design of polymers and lipids as synthetic carriers for targeting therapeutic mRNA in vivo with the ability to circumvent nuclease attack for treating intractable diseases. Herein, we developed a simple approach to attain one order of magnitude higher nuclease tolerability of mRNA through the formation of polyplex micelles (PMs) by combining ω-cholesteryl (ω-Chol)-poly (ethylene-glycol) (PEG)-polycation block copolymers with mRNA pre-hybridized with cholesterol (Chol)-tethered RNA oligonucleotides (Chol (+)-OligoRNA). Even one or a few short Chol (+)-OligoRNA anchors harboring along the 46-fold longer mRNA strand was sufficient to induce tight mRNA packaging in the PM core, as evidenced by Förster resonance energy transfer (FRET) measurement as well as by a longitudinal relaxation time (T1) measurement using NMR. These results suggest that Chol (+)-OligoRNA on mRNA strand serves as a node to attract ω-Chol moiety of the block copolymers to tighten the mRNA packaging in the PM core. These mRNA loaded PMs showed high tolerability against nuclease attack, and exerted appreciable protein translational activity in cultured cells without any inflammatory responses, achieved by shortening of the length of hybridizing Chol (+)-OligoRNAs to 17 nucleotides. Finally, the Chol (+)-OligoRNA-stabilized PM revealed efficient mRNA introduction into the mouse lungs via intratracheal administration, demonstrating in vivo utility of this formulation.
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Affiliation(s)
- Naoto Yoshinaga
- Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
| | - Satoshi Uchida
- Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan; Innovation Center of NanoMedicine (iCONM), Kawasaki Institute of Industrial Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki, 210-0821, Japan.
| | - Mitsuru Naito
- Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
| | - Kensuke Osada
- National Institute of Radiological Science, 4-9-1 Anagawa, Inage-ku, Chiba-shi, Chiba 263-8555, Japan
| | - Horacio Cabral
- Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
| | - Kazunori Kataoka
- Innovation Center of NanoMedicine (iCONM), Kawasaki Institute of Industrial Promotion, 3-25-14 Tonomachi, Kawasaki-ku, Kawasaki, 210-0821, Japan; Policy Alternatives Research Institute, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
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36
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Fan X, Jin T. Structures of RIG-I-Like Receptors and Insights into Viral RNA Sensing. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1172:157-188. [DOI: 10.1007/978-981-13-9367-9_8] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
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37
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Yang J, Li C, Zhang L, Wang X. Extracellular Vesicles as Carriers of Non-coding RNAs in Liver Diseases. Front Pharmacol 2018; 9:415. [PMID: 29740327 PMCID: PMC5928552 DOI: 10.3389/fphar.2018.00415] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2018] [Accepted: 04/10/2018] [Indexed: 12/12/2022] Open
Abstract
Extracellular vesicles (EVs) are small membranous vesicles secreted from normal, diseased, and transformed cells in vitro and in vivo. EVs have been found to play a critical role in cell-to-cell communication by transferring non-coding RNAs (ncRNAs) including microRNAs (miRNAs), long ncRNAs (lncRNAs) and so on. Emerging evidence shows that transferring biological information through EVs to neighboring cells in intercellular communication not only keep physiological functions, but also participate in the pathogenesis of liver diseases. Liver diseases often promote release of EVs and/or in different cargo sorting into these EVs. Either of these modifications can promote disease pathogenesis. Given this fact, EV-associated ncRNAs, such as miR-192, miR-122 and lncRNA-ROR and so on, can serve as new diagnostic biomarkers and new therapeutic targets for liver disease, because altered EV-associated ncRNAs may reflect the underlying liver disease condition. In this review, we focus on understanding the emerging role of EV-associated ncRNAs in viral hepatitis, liver fibrosis, alcoholic hepatitis (AH), non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) and discuss their utility in biomarker discovery and therapeutics. A better understanding of this multifaceted pattern of communication between different type cells in liver may contribute to developing novel approaches for personalized diagnostics and therapeutics.
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Affiliation(s)
- Junfa Yang
- School of Pharmacy, Anhui Medical University, Hefei, China.,Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui Institute of Innovative Drugs, Hefei, China.,The Key Laboratory of Anti-inflammatory and Immune Medicines, Ministry of Education, Anhui Medical University, Hefei, China
| | - Changyao Li
- School of Pharmacy, Anhui Medical University, Hefei, China.,Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui Institute of Innovative Drugs, Hefei, China.,The Key Laboratory of Anti-inflammatory and Immune Medicines, Ministry of Education, Anhui Medical University, Hefei, China
| | - Lei Zhang
- School of Pharmacy, Anhui Medical University, Hefei, China.,Anhui Province Key Laboratory of Major Autoimmune Diseases, Anhui Institute of Innovative Drugs, Hefei, China.,The Key Laboratory of Anti-inflammatory and Immune Medicines, Ministry of Education, Anhui Medical University, Hefei, China
| | - Xiao Wang
- Department of Radiology, The First Affiliated Hospital of Anhui Medical University, Hefei, China
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38
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Valadkhan S, Fortes P. Regulation of the Interferon Response by lncRNAs in HCV Infection. Front Microbiol 2018; 9:181. [PMID: 29503633 PMCID: PMC5820368 DOI: 10.3389/fmicb.2018.00181] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2017] [Accepted: 01/26/2018] [Indexed: 12/24/2022] Open
Affiliation(s)
- Saba Valadkhan
- Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH, United States
- *Correspondence: Saba Valadkhan, Puri Fortes,
| | - Puri Fortes
- Center for Applied Medical Research, Department of Gene Therapy and Hepatology, Navarra Institute for Health Research (IdiSNA), University of Navarra, Pamplona, Spain
- *Correspondence: Saba Valadkhan, Puri Fortes,
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Barriocanal M, Fortes P. Long Non-coding RNAs in Hepatitis C Virus-Infected Cells. Front Microbiol 2017; 8:1833. [PMID: 29033906 PMCID: PMC5625025 DOI: 10.3389/fmicb.2017.01833] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2017] [Accepted: 09/06/2017] [Indexed: 12/13/2022] Open
Abstract
Hepatitis C virus (HCV) often leads to a chronic infection in the liver that may progress to steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Several viral and cellular factors are required for a productive infection and for the development of liver disease. Some of these are long non-coding RNAs (lncRNAs) deregulated in infected cells. After HCV infection, the sequence and the structure of the viral RNA genome are sensed to activate interferon (IFN) synthesis and signaling pathways. These antiviral pathways regulate transcription of several cellular lncRNAs. Some of these are also deregulated in response to viral replication. Certain viral proteins and/or viral replication can activate transcription factors such as MYC, SP1, NRF2, or HIF1α that modulate the expression of additional cellular lncRNAs. Interestingly, several lncRNAs deregulated in HCV-infected cells described so far play proviral or antiviral functions by acting as positive or negative regulators of the IFN system, while others help in the development of liver cirrhosis and HCC. The study of the structure and mechanism of action of these lncRNAs may aid in the development of novel strategies to treat infectious and immune pathologies and liver diseases such as cirrhosis and HCC.
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Affiliation(s)
| | - Puri Fortes
- Department of Gene Therapy and Hepatology, Navarra Institute for Health Research (IdiSNA), Centro de Investigación Médica Aplicada, University of Navarra, Pamplona, Spain
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40
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Uchida S, Yoshinaga N, Yanagihara K, Yuba E, Kataoka K, Itaka K. Designing immunostimulatory double stranded messenger RNA with maintained translational activity through hybridization with poly A sequences for effective vaccination. Biomaterials 2017; 150:162-170. [PMID: 29031816 DOI: 10.1016/j.biomaterials.2017.09.033] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2017] [Revised: 09/22/2017] [Accepted: 09/25/2017] [Indexed: 12/24/2022]
Abstract
Messenger (m)RNA vaccines require a safe and potent immunostimulatory adjuvant. In this study, we introduced immunostimulatory properties directly into mRNA molecules by hybridizing them with complementary RNA to create highly immunogenic double stranded (ds)RNAs. These dsRNA formulations, comprised entirely of RNA, are expected to be safe and highly efficient due to antigen expression and immunostimulation occurring simultaneously in the same antigen presenting cells. In this strategy, design of dsRNA is important. Indeed, hybridization using full-length antisense (as)RNA drastically reduced translational efficiency. In contrast, by limiting the hybridized portion to the mRNA poly A region, efficient translation and intense immunostimulation was simultaneously obtained. The immune response to the poly U-hybridized mRNAs (mRNA:pU) was mediated through Toll-like receptor (TLR)-3 and retinoic acid-inducible gene (RIG)-I. We also demonstrated that mRNA:pU activation of mouse and human dendritic cells was significantly more effective than activation using single stranded mRNA. In vivo mouse immunization experiments using ovalbumin showed that mRNA:pU significantly enhanced the intensity of specific cellular and humoral immune responses, compared to single stranded mRNA. Our novel mRNA:pU formulation can be delivered using a variety of mRNA carriers depending on the purpose and delivery route, providing a versatile platform for improving mRNA vaccine efficiency.
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Affiliation(s)
- Satoshi Uchida
- Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Bunkyo, Tokyo 113-8656, Japan; Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, Kawasaki, Kanagawa 210-0821, Japan.
| | - Naoto Yoshinaga
- Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, Bunkyo, Tokyo 113-8656, Japan; Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, Kawasaki, Kanagawa 210-0821, Japan
| | - Kayoko Yanagihara
- Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, Kawasaki, Kanagawa 210-0821, Japan; Division of Clinical Biotechnology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan
| | - Eiji Yuba
- Department of Applied Chemistry, Graduate School of Engineering, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
| | - Kazunori Kataoka
- Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, Kawasaki, Kanagawa 210-0821, Japan; Policy Alternatives Research Institute, The University of Tokyo, Bunkyo, Tokyo 113-0033, Japan
| | - Keiji Itaka
- Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, Kawasaki, Kanagawa 210-0821, Japan; Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), Chiyoda, Tokyo 101-0062, Japan.
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Synthetic agonists of NOD-like, RIG-I-like, and C-type lectin receptors for probing the inflammatory immune response. Future Med Chem 2017; 9:1345-1360. [PMID: 28776416 DOI: 10.4155/fmc-2017-0101] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Synthetic agonists of innate immune cells are of interest to immunologists due to their synthesis from well-defined materials, optimized activity, and monodisperse chemical purity. These molecules are used in both prophylactic and therapeutic contexts from vaccines to cancer immunotherapies. In this review we highlight synthetic agonists that activate innate immune cells through three classes of pattern recognition receptors: NOD-like receptors, RIG-I-like receptors, and C-type lectin receptors. We classify these agonists by the receptor they activate and present them from a chemical perspective, focusing on structural components that define agonist activity. We anticipate this review will be useful to the medicinal chemist as a guide to chemical motifs that activate each receptor, ultimately illuminating a chemical space ripe for exploration.
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Phosphorylation-Dependent Feedback Inhibition of RIG-I by DAPK1 Identified by Kinome-wide siRNA Screening. Mol Cell 2017; 65:403-415.e8. [PMID: 28132841 DOI: 10.1016/j.molcel.2016.12.021] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2016] [Revised: 09/28/2016] [Accepted: 12/20/2016] [Indexed: 12/17/2022]
Abstract
Cell-autonomous induction of type I interferon must be stringently regulated. Rapid induction is key to control virus infection, whereas proper limitation of signaling is essential to prevent immunopathology and autoimmune disease. Using unbiased kinome-wide RNAi screening followed by thorough validation, we identified 22 factors that regulate RIG-I/IRF3 signaling activity. We describe a negative-feedback mechanism targeting RIG-I activity, which is mediated by death associated protein kinase 1 (DAPK1). RIG-I signaling triggers DAPK1 kinase activation, and active DAPK1 potently inhibits RIG-I stimulated IRF3 activity and interferon-beta production. DAPK1 phosphorylates RIG-I in vitro at previously reported as well as other sites that limit 5'ppp-dsRNA sensing and virtually abrogate RIG-I activation.
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Abstract
Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) are the most severe coronavirus (CoV)-associated diseases in humans. The causative agents, SARS-CoV and MERS-CoV, are of zoonotic origin but may be transmitted to humans, causing severe and often fatal respiratory disease in their new host. The two coronaviruses are thought to encode an unusually large number of factors that allow them to thrive and replicate in the presence of efficient host defense mechanisms, especially the antiviral interferon system. Here, we review the recent progress in our understanding of the strategies that highly pathogenic coronaviruses employ to escape, dampen, or block the antiviral interferon response in human cells.
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Affiliation(s)
- E Kindler
- University of Bern, Bern, Switzerland; Institute of Virology and Immunology, Bern and Mittelhäusern, Switzerland
| | - V Thiel
- University of Bern, Bern, Switzerland; Institute of Virology and Immunology, Bern and Mittelhäusern, Switzerland
| | - F Weber
- Institute of Virology, Faculty of Veterinary Medicine, Justus Liebig University Giessen, Giessen, Germany.
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44
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Abstract
Immune sensing of foreign nucleic acids among abundant self nucleic acids is a hallmark of virus detection and antiviral defence. Efficient antiviral defence requires a balanced process of sensing foreign nucleic acids and ignoring self nucleic acids. This balance is accomplished by a multilevel, fail-safe system which combines immune sensing of pathogen-specific nucleic acid structures with specific labelling of self nucleic acids and nuclease-mediated degradation. Cellular localization of nucleic acids, nucleic acid secondary structure, nucleic acid sequence and chemical modification all contribute to selective recognition of foreign nucleic acids. Nucleic acid sensing occurs in immune cells and non-immune cells and results in antiviral responses that include the induction of antiviral effector proteins, the secretion of cytokines alarming neighbouring cells, the secretion of chemokines, which attract immune cells, and the induction of cell death. Vertebrate cells cannot completely avoid the occurrence of endogenous self nucleic acid structures with immunostimulatory properties. Therefore, additional mechanisms involving self-nucleic acid modification and nuclease-mediated degradation are necessary to diminish uncontrolled immune activation. Viruses have established sophisticated mechanisms to exploit and adopt endogenous tolerance mechanisms or to avoid the presentation of characteristic molecular features recognized by nucleic acid sensing receptors. The detection of viruses by the immune system is mediated predominantly by the sensing of nucleic acids. Here, the authors review our current understanding of how this complex immune sensory system discriminates self from non-self nucleic acids to reliably detect pathogenic viruses, and discuss the future perspectives and implications for human disease. Innate immunity against pathogens relies on an array of immune receptors to detect molecular patterns that are characteristic of the pathogens, including receptors that are specialized in the detection of foreign nucleic acids. In vertebrates, nucleic acid sensing is the dominant antiviral defence pathway. Stimulation of nucleic acid receptors results in antiviral immune responses with the production of type I interferon (IFN), as well as the expression of IFN-stimulated genes, which encode molecules such as cell-autonomous antiviral effector proteins. This Review summarizes the tremendous progress that has been made in understanding how this sophisticated immune sensory system discriminates self from non-self nucleic acids in order to reliably detect pathogenic viruses.
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Affiliation(s)
- Martin Schlee
- Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Sigmund-Freud-Strasse 25, D-53127 Bonn, Germany
| | - Gunther Hartmann
- Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn, Sigmund-Freud-Strasse 25, D-53127 Bonn, Germany
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45
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Sun X, Xian H, Tian S, Sun T, Qin Y, Zhang S, Cui J. A Hierarchical Mechanism of RIG-I Ubiquitination Provides Sensitivity, Robustness and Synergy in Antiviral Immune Responses. Sci Rep 2016; 6:29263. [PMID: 27387525 PMCID: PMC4937349 DOI: 10.1038/srep29263] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2016] [Accepted: 06/13/2016] [Indexed: 12/12/2022] Open
Abstract
RIG-I is an essential receptor in the initiation of the type I interferon (IFN) signaling pathway upon viral infection. Although K63-linked ubiquitination plays an important role in RIG-I activation, the optimal modulation of conjugated and unanchored ubiquitination of RIG-I as well as its functional implications remains unclear. In this study, we determined that, in contrast to the RIG-I CARD domain, full-length RIG-I must undergo K63-linked ubiquitination at multiple sites to reach full activity. A systems biology approach was designed based on experiments using full-length RIG-I. Model selection for 7 candidate mechanisms of RIG-I ubiquitination inferred a hierarchical architecture of the RIG-I ubiquitination mode, which was then experimentally validated. Compared with other mechanisms, the selected hierarchical mechanism exhibited superior sensitivity and robustness in RIG-I-induced type I IFN activation. Furthermore, our model analysis and experimental data revealed that TRIM4 and TRIM25 exhibited dose-dependent synergism. These results demonstrated that the hierarchical mechanism of multi-site/type ubiquitination of RIG-I provides an efficient, robust and optimal synergistic regulatory module in antiviral immune responses.
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Affiliation(s)
- Xiaoqiang Sun
- Zhong-shan School of Medicine, Sun Yat-sen University, Guangzhou 510089, China
- School of Life Science, Sun Yat-sen University, Guangzhou, 510275, China
- School of Mathematical and Computational Science, Sun Yat-sen University, Guangzhou, 510000, China
| | - Huifang Xian
- School of Life Science, Sun Yat-sen University, Guangzhou, 510275, China
| | - Shuo Tian
- School of Life Science, Sun Yat-sen University, Guangzhou, 510275, China
| | - Tingzhe Sun
- School of Life Sciences, AnQing Normal University, AnQing, 246011, China
| | - Yunfei Qin
- School of Life Sciences, Zhengzhou University, Zhengzhou, Henan, 450001, China
| | - Shoutao Zhang
- School of Life Sciences, Zhengzhou University, Zhengzhou, Henan, 450001, China
| | - Jun Cui
- School of Life Science, Sun Yat-sen University, Guangzhou, 510275, China
- Collaborative Innovation Center of Cancer Medicine, Sun Yat-sen University, Guangzhou, 510060, China
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46
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Ramanathan A, Robb GB, Chan SH. mRNA capping: biological functions and applications. Nucleic Acids Res 2016; 44:7511-26. [PMID: 27317694 PMCID: PMC5027499 DOI: 10.1093/nar/gkw551] [Citation(s) in RCA: 551] [Impact Index Per Article: 61.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2016] [Accepted: 06/03/2016] [Indexed: 12/19/2022] Open
Abstract
The 5′ m7G cap is an evolutionarily conserved modification of eukaryotic mRNA. Decades of research have established that the m7G cap serves as a unique molecular module that recruits cellular proteins and mediates cap-related biological functions such as pre-mRNA processing, nuclear export and cap-dependent protein synthesis. Only recently has the role of the cap 2′O methylation as an identifier of self RNA in the innate immune system against foreign RNA has become clear. The discovery of the cytoplasmic capping machinery suggests a novel level of control network. These new findings underscore the importance of a proper cap structure in the synthesis of functional messenger RNA. In this review, we will summarize the current knowledge of the biological roles of mRNA caps in eukaryotic cells. We will also discuss different means that viruses and their host cells use to cap their RNA and the application of these capping machineries to synthesize functional mRNA. Novel applications of RNA capping enzymes in the discovery of new RNA species and sequencing the microbiome transcriptome will also be discussed. We will end with a summary of novel findings in RNA capping and the questions these findings pose.
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Affiliation(s)
- Anand Ramanathan
- New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA
| | - G Brett Robb
- New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA
| | - Siu-Hong Chan
- New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA
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47
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Sohn J, Hur S. Filament assemblies in foreign nucleic acid sensors. Curr Opin Struct Biol 2016; 37:134-44. [PMID: 26859869 DOI: 10.1016/j.sbi.2016.01.011] [Citation(s) in RCA: 45] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2015] [Revised: 01/20/2016] [Accepted: 01/21/2016] [Indexed: 12/24/2022]
Abstract
Helical filamentous assembly is ubiquitous in biology, but was only recently realized to be broadly employed in the innate immune system of vertebrates. Accumulating evidence suggests that the filamentous assemblies and helical oligomerization play important roles in detection of foreign nucleic acids and activation of the signaling pathways to produce antiviral and inflammatory mediators. In this review, we focus on the helical assemblies observed in the signaling pathways of RIG-I-like receptors (RLRs) and AIM2-like receptors (ALRs). We describe ligand-dependent oligomerization of receptor, receptor-dependent oligomerization of signaling adaptor molecules, and their functional implications and regulations.
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Affiliation(s)
- Jungsan Sohn
- Johns Hopkins University School of Medicine, Baltimore, MD, USA.
| | - Sun Hur
- Harvard Medical School, Boston, MA, USA.
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48
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Ramanathan A, Devarkar SC, Jiang F, Miller MT, Khan AG, Marcotrigiano J, Patel SS. The autoinhibitory CARD2-Hel2i Interface of RIG-I governs RNA selection. Nucleic Acids Res 2016; 44:896-909. [PMID: 26612866 PMCID: PMC4737149 DOI: 10.1093/nar/gkv1299] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2015] [Revised: 10/30/2015] [Accepted: 11/05/2015] [Indexed: 12/24/2022] Open
Abstract
RIG-I (Retinoic Acid Inducible Gene-I) is a cytosolic innate immune receptor that detects atypical features in viral RNAs as foreign to initiate a Type I interferon signaling response. RIG-I is present in an autoinhibited state in the cytoplasm and activated by blunt-ended double-stranded (ds)RNAs carrying a 5' triphosphate (ppp) moiety. These features found in many pathogenic RNAs are absent in cellular RNAs due to post-transcriptional modifications of RNA ends. Although RIG-I is structurally well characterized, the mechanistic basis for RIG-I's remarkable ability to discriminate between cellular and pathogenic RNAs is not completely understood. We show that RIG-I's selectivity for blunt-ended 5'-ppp dsRNAs is ≈3000 times higher than non-blunt ended dsRNAs commonly found in cellular RNAs. Discrimination occurs at multiple stages and signaling RNAs have high affinity and ATPase turnover rate and thus a high katpase/Kd. We show that RIG-I uses its autoinhibitory CARD2-Hel2i (second CARD-helicase insertion domain) interface as a barrier to select against non-blunt ended dsRNAs. Accordingly, deletion of CARDs or point mutations in the CARD2-Hel2i interface decreases the selectivity from ≈3000 to 150 and 750, respectively. We propose that the CARD2-Hel2i interface is a 'gate' that prevents cellular RNAs from generating productive complexes that can signal.
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Affiliation(s)
- Anand Ramanathan
- Robert Wood Johnson Medical School, Department of Biochemistry and Molecular Biology, Rutgers University, Piscataway, NJ 08854, USA
| | - Swapnil C Devarkar
- Robert Wood Johnson Medical School, Department of Biochemistry and Molecular Biology, Rutgers University, Piscataway, NJ 08854, USA
| | - Fuguo Jiang
- Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA
| | - Matthew T Miller
- Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA
| | - Abdul G Khan
- Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA
| | - Joseph Marcotrigiano
- Center for Advanced Biotechnology and Medicine, Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA
| | - Smita S Patel
- Robert Wood Johnson Medical School, Department of Biochemistry and Molecular Biology, Rutgers University, Piscataway, NJ 08854, USA
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49
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Bender S, Reuter A, Eberle F, Einhorn E, Binder M, Bartenschlager R. Activation of Type I and III Interferon Response by Mitochondrial and Peroxisomal MAVS and Inhibition by Hepatitis C Virus. PLoS Pathog 2015; 11:e1005264. [PMID: 26588843 PMCID: PMC4654527 DOI: 10.1371/journal.ppat.1005264] [Citation(s) in RCA: 126] [Impact Index Per Article: 12.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2015] [Accepted: 10/19/2015] [Indexed: 12/22/2022] Open
Abstract
Sensing viruses by pattern recognition receptors (PRR) triggers the innate immune system of the host cell and activates immune signaling cascades such as the RIG-I/IRF3 pathway. Mitochondrial antiviral-signaling protein (MAVS, also known as IPS-1, Cardif, and VISA) is the crucial adaptor protein of this pathway localized on mitochondria, peroxisomes and mitochondria-associated membranes of the endoplasmic reticulum. Activation of MAVS leads to the production of type I and type III interferons (IFN) as well as IFN stimulated genes (ISGs). To refine the role of MAVS subcellular localization for the induction of type I and III IFN responses in hepatocytes and its counteraction by the hepatitis C virus (HCV), we generated various functional and genetic knock-out cell systems that were reconstituted to express mitochondrial (mito) or peroxisomal (pex) MAVS, exclusively. Upon infection with diverse RNA viruses we found that cells exclusively expressing pexMAVS mounted sustained expression of type I and III IFNs to levels comparable to cells exclusively expressing mitoMAVS. To determine whether viral counteraction of MAVS is affected by its subcellular localization we employed infection of cells with HCV, a major causative agent of chronic liver disease with a high propensity to establish persistence. This virus efficiently cleaves MAVS via a viral protease residing in its nonstructural protein 3 (NS3) and this strategy is thought to contribute to the high persistence of this virus. We found that both mito- and pexMAVS were efficiently cleaved by NS3 and this cleavage was required to suppress activation of the IFN response. Taken together, our findings indicate comparable activation of the IFN response by pex- and mitoMAVS in hepatocytes and efficient counteraction of both MAVS species by the HCV NS3 protease. Mammalian cells developed several defense mechanisms against viral infection. One major strategy involves pattern recognition receptors (PRRs) recognizing non-self motifs in viral RNA and triggering the production of type I and III interferon (IFN) that induce an antiviral state. One central signaling molecule in this cascade is MAVS (Mitochondrial Antiviral Signaling protein), residing on mitochondria, mitochondria-associated membranes of the endoplasmic reticulum, and peroxisomes. Here we characterized the role of mitochondrial and peroxisomal MAVS for the activation of the IFN response and their counteraction by the hepatitis C virus (HCV), a major causative agent of chronic liver disease with a high propensity to establish persistence. By using various functional and genetic knock-out cell systems reconstituted to express exclusively mitochondrial or peroxisomal MAVS, we observed comparable activation of type I and III IFN response by either MAVS species. In addition, we found that the HCV protease residing in nonstructural protein 3 (NS3) efficiently cleaves MAVS independent from its subcellular localization. This cleavage is required for suppression of the IFN response and might contribute to HCV persistence. Our results indicate a largely localization-independent activation of the IFN response by MAVS in hepatocytes and its efficient counteraction by the HCV NS3 protease.
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Affiliation(s)
- Silke Bender
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
- Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Antje Reuter
- Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Florian Eberle
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
| | - Evelyne Einhorn
- Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany
- Master BioSciences, Département de Biologie, École Normale Supérieure de Lyon, Université de Lyon, Lyon, France
| | - Marco Binder
- Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
- Division of Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany
- * E-mail:
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50
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Killip MJ, Fodor E, Randall RE. Influenza virus activation of the interferon system. Virus Res 2015; 209:11-22. [PMID: 25678267 PMCID: PMC4638190 DOI: 10.1016/j.virusres.2015.02.003] [Citation(s) in RCA: 140] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2014] [Revised: 01/28/2015] [Accepted: 02/02/2015] [Indexed: 12/24/2022]
Abstract
The host interferon (IFN) response represents one of the first barriers that influenza viruses must surmount in order to establish an infection. Many advances have been made in recent years in understanding the interactions between influenza viruses and the interferon system. In this review, we summarise recent work regarding activation of the type I IFN response by influenza viruses, including attempts to identify the viral RNA responsible for IFN induction, the stage of the virus life cycle at which it is generated and the role of defective viruses in this process.
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Affiliation(s)
- Marian J Killip
- Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK; Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
| | - Ervin Fodor
- Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
| | - Richard E Randall
- Biomedical Sciences Research Complex, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK
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