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1
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Ye S, Liang Y, Chang Y, Lai B, Zhong J. Dengue Virus Replicative-Form dsRNA Is Recognized by Both RIG-I and MDA5 to Activate Innate Immunity. J Med Virol 2025; 97:e70194. [PMID: 39873327 DOI: 10.1002/jmv.70194] [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: 12/11/2024] [Revised: 01/12/2025] [Accepted: 01/13/2025] [Indexed: 01/30/2025]
Abstract
RIG-I like receptors (RLRs) are a family of cytosolic RNA sensors that sense RNA virus infection to activate innate immune response. It is generally believed that different RNA viruses are recognized by either RIG-I or MDA5, two important RLR members, depending on the nature of pathogen-associated molecular patterns (PAMPs) that are generated by RNA virus replication. Dengue virus (DENV) is an important RNA virus causing serious human diseases. Despite extensive investigations, the molecular basis of the DENV PAMP recognized by the host RLR has been poorly defined. Here, we demonstrated that the DENV infection-induced interferon response is dependent upon both RIG-I and MDA5, with RIG-I playing a predominant role. Next we purified the DENV PAMP RNA from the DENV-infected cells, and demonstrated that the purified DENV PAMP is viral full-length double-stranded RNA bearing 5'ppp modifications, likely representing the viral replicative-form RNA. Finally, we confirmed the nature of the DENV PAMP by reconstituting the viral replicative-form RNA from in vitro synthesized DENV genomic RNA. In conclusion, our work not only defined the molecular basis of the RLR-PAMP interaction during DENV infection, but also revealed the previously underappreciated recognition of a distinct moiety of the same PAMP by different RLRs in innate immunity against RNA viruses.
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Affiliation(s)
- Sichao Ye
- CAS Key Laboratory of Molecular Virology and Immunology, Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences, Shanghai, China
- Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China
| | - Yisha Liang
- CAS Key Laboratory of Molecular Virology and Immunology, Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences, Shanghai, China
- Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
| | - Yu Chang
- CAS Key Laboratory of Molecular Virology and Immunology, Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences, Shanghai, China
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
| | - Bailiang Lai
- CAS Key Laboratory of Molecular Virology and Immunology, Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences, Shanghai, China
- Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China
| | - Jin Zhong
- CAS Key Laboratory of Molecular Virology and Immunology, Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences, Shanghai, China
- Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
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2
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Jang I, Yum K, Han S, Moon S, Lee JB. A virus-inspired RNA mimicry approach for effective cancer immunotherapy. J Mater Chem B 2025; 13:1619-1629. [PMID: 39834198 DOI: 10.1039/d4tb02301c] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2025]
Abstract
Current cancer treatments, including chemotherapy, surgery, and radiation, often present significant challenges such as severe side effects, drug resistance, and damage to healthy tissues. To address these issues, we introduce a virus-inspired RNA mimicry approach, specifically through the development of uridine-rich nanoparticles (UNPs) synthesized using the rolling circle transcription (RCT) technique. These UNPs are designed to mimic the poly-U tail sequences of viral RNA, effectively engaging RIG-I-like receptors (RLRs) such as MDA5 and LGP2 in cancer cells. Activation of these receptors leads to the upregulation of pro-inflammatory cytokines and the initiation of apoptosis, resulting in targeted cancer cell death. Importantly, this strategy overcomes the limitations of traditional therapies and enhances the effectiveness of existing RIG-I stimulators, such as poly(I:C), which has often exhibited toxicity in clinical settings due to delivery methods. Our in vivo studies further demonstrate the ability of UNPs to significantly reduce tumor growth without adverse effects, highlighting their potential as a novel and effective approach in cancer immunotherapy. This approach offers new therapeutic strategies that leverage the body's innate antiviral mechanisms for cancer treatment.
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Affiliation(s)
- Iksoo Jang
- Department of Chemical Engineering, University of Seoul, Republic of Korea
| | - Kyuha Yum
- Department of Chemical Engineering, University of Seoul, Republic of Korea
| | - Sangwoo Han
- Department of Chemical Engineering, University of Seoul, Republic of Korea
| | - Sunghyun Moon
- Department of Chemical Engineering, University of Seoul, Republic of Korea
| | - Jong Bum Lee
- Department of Chemical Engineering, University of Seoul, Republic of Korea
- Center for Innovative Chemical Processes, Institute of Engineering, University of Seoul, 163 Seoulsiripdaero, Dongdaemun-gu, Seoul, 02504, Republic of Korea.
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3
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Brüggemann Y, Klöhn M, Wedemeyer H, Steinmann E. Hepatitis E virus: from innate sensing to adaptive immune responses. Nat Rev Gastroenterol Hepatol 2024; 21:710-725. [PMID: 39039260 DOI: 10.1038/s41575-024-00950-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 05/29/2024] [Indexed: 07/24/2024]
Abstract
Hepatitis E virus (HEV) infections are a major cause of acute viral hepatitis in humans worldwide. In immunocompetent individuals, the majority of HEV infections remain asymptomatic and lead to spontaneous clearance of the virus, and only a minority of individuals with infection (5-16%) experience symptoms of acute viral hepatitis. However, HEV infections can cause up to 30% mortality in pregnant women, become chronic in immunocompromised patients and cause extrahepatic manifestations. A growing body of evidence suggests that the host immune response to infection with different HEV genotypes is a critical determinant of distinct HEV infection outcomes. In this Review, we summarize key components of the innate and adaptive immune responses to HEV, including the underlying immunological mechanisms of HEV associated with acute and chronic liver failure and interactions between T cell and B cell responses. In addition, we discuss the current status of vaccines against HEV and raise outstanding questions regarding the immune responses induced by HEV and treatment of the disease, highlighting areas for future investigation.
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Affiliation(s)
- Yannick Brüggemann
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
| | - Mara Klöhn
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany
| | - Heiner Wedemeyer
- Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
- German Center for Infection Research (DZIF), Partner Sites Hannover-Braunschweig, Hannover, Germany
- Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany
| | - Eike Steinmann
- Department of Molecular and Medical Virology, Ruhr University Bochum, Bochum, Germany.
- German Center for Infection Research (DZIF), External Partner Site, Bochum, Germany.
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4
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Moon JS, Lee W, Cho YH, Kim Y, Kim GW. The Significance of N6-Methyladenosine RNA Methylation in Regulating the Hepatitis B Virus Life Cycle. J Microbiol Biotechnol 2024; 34:233-239. [PMID: 37942519 PMCID: PMC10940779 DOI: 10.4014/jmb.2309.09013] [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: 09/08/2023] [Revised: 10/16/2023] [Accepted: 10/23/2023] [Indexed: 11/10/2023]
Abstract
N6-methyladenosine (m6A) RNA methylation has recently emerged as a significant co-transcriptional modification involved in regulating various RNA functions. It plays a vital function in numerous biological processes. Enzymes referred to as m6A methyltransferases, such as the methyltransferaselike (METTL) 3-METTL14-Wilms tumor 1 (WT1)-associated protein (WTAP) complex, are responsible for adding m6A modifications, while m6A demethylases, including fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5), can remove m6A methylation. The functions of m6A-methylated RNA are regulated through the recognition and interaction of m6A reader proteins. Recent research has shown that m6A methylation takes place at multiple sites within hepatitis B virus (HBV) RNAs, and the location of these modifications can differentially impact the HBV infection. The addition of m6A modifications to HBV RNA can influence its stability and translation, thereby affecting viral replication and pathogenesis. Furthermore, HBV infection can also alter the m6A modification pattern of host RNA, indicating the virus's ability to manipulate host cellular processes, including m6A modification. This manipulation aids in establishing chronic infection, promoting liver disease, and contributing to pathogenesis. A comprehensive understanding of the functional roles of m6A modification during HBV infection is crucial for developing innovative approaches to combat HBV-mediated liver disease. In this review, we explore the functions of m6A modification in HBV replication and its impact on the development of liver disease.
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Affiliation(s)
- Jae-Su Moon
- Division of Infectious Diseases, Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA
| | - Wooseong Lee
- Center for Convergent Research of Emerging virus Infection, Korea Research Institute of Chemical Technology (KRICT), Daejeon 34114, Republic of Korea
| | - Yong-Hee Cho
- Data Convergence Drug Research Center, Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology (KRICT), Daejeon 34114, Republic of Korea
- Department of Medical Chemistry and Pharmacology, University of Science and Technology (UST), Daejeon 34113, Republic of Korea
| | - Yonghyo Kim
- Data Convergence Drug Research Center, Therapeutics and Biotechnology Division, Korea Research Institute of Chemical Technology (KRICT), Daejeon 34114, Republic of Korea
| | - Geon-Woo Kim
- Department of Microbiology and Molecular Biology, Chungnam National University, Daejeon 34134, Republic of Korea
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5
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Sun H, Zhang Y, Wang G, Yang W, Xu Y. mRNA-Based Therapeutics in Cancer Treatment. Pharmaceutics 2023; 15:pharmaceutics15020622. [PMID: 36839944 PMCID: PMC9964383 DOI: 10.3390/pharmaceutics15020622] [Citation(s) in RCA: 15] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2023] [Revised: 01/28/2023] [Accepted: 01/28/2023] [Indexed: 02/15/2023] Open
Abstract
Over the past two decades, significant technological innovations have led to messenger RNA (mRNA) becoming a promising option for developing prophylactic and therapeutic vaccines, protein replacement therapies, and genome engineering. The success of the two COVID-19 mRNA vaccines has sparked new enthusiasm for other medical applications, particularly in cancer treatment. In vitro-transcribed (IVT) mRNAs are structurally designed to resemble naturally occurring mature mRNA. Delivery of IVT mRNA via delivery platforms such as lipid nanoparticles allows host cells to produce many copies of encoded proteins, which can serve as antigens to stimulate immune responses or as additional beneficial proteins for supplements. mRNA-based cancer therapeutics include mRNA cancer vaccines, mRNA encoding cytokines, chimeric antigen receptors, tumor suppressors, and other combination therapies. To better understand the current development and research status of mRNA therapies for cancer treatment, this review focused on the molecular design, delivery systems, and clinical indications of mRNA therapies in cancer.
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Affiliation(s)
- Han Sun
- Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Yu Zhang
- Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Ge Wang
- Department of Oral Maxillofacial & Head and Neck Oncology, National Center of Stomatology, National Clinical Research Center for Oral Disease, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
| | - Wen Yang
- Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
| | - Yingjie Xu
- Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
- Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
- Correspondence:
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6
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Jain A, Mittal S, Tripathi LP, Nussinov R, Ahmad S. Host-pathogen protein-nucleic acid interactions: A comprehensive review. Comput Struct Biotechnol J 2022; 20:4415-4436. [PMID: 36051878 PMCID: PMC9420432 DOI: 10.1016/j.csbj.2022.08.001] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2022] [Revised: 08/01/2022] [Accepted: 08/01/2022] [Indexed: 12/02/2022] Open
Abstract
Recognition of pathogen-derived nucleic acids by host cells is an effective host strategy to detect pathogenic invasion and trigger immune responses. In the context of pathogen-specific pharmacology, there is a growing interest in mapping the interactions between pathogen-derived nucleic acids and host proteins. Insight into the principles of the structural and immunological mechanisms underlying such interactions and their roles in host defense is necessary to guide therapeutic intervention. Here, we discuss the newest advances in studies of molecular interactions involving pathogen nucleic acids and host factors, including their drug design, molecular structure and specific patterns. We observed that two groups of nucleic acid recognizing molecules, Toll-like receptors (TLRs) and the cytoplasmic retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) form the backbone of host responses to pathogen nucleic acids, with additional support provided by absent in melanoma 2 (AIM2) and DNA-dependent activator of Interferons (IFNs)-regulatory factors (DAI) like cytosolic activity. We review the structural, immunological, and other biological aspects of these representative groups of molecules, especially in terms of their target specificity and affinity and challenges in leveraging host-pathogen protein-nucleic acid interactions (HP-PNI) in drug discovery.
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Affiliation(s)
- Anuja Jain
- School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi 110067, India
| | - Shikha Mittal
- School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi 110067, India
- Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, Himachal Pradesh, 173234, India
| | - Lokesh P. Tripathi
- National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka, Japan
- Riken Center for Integrative Medical Sciences, Tsurumi, Yokohama, Kanagawa, Japan
| | - Ruth Nussinov
- Computational Structural Biology Section, Basic Science Program, Frederick National, Laboratory for Cancer Research, Frederick, MD 21702, USA
- Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Israel
| | - Shandar Ahmad
- School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi 110067, India
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7
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Wu Z, Hu T, Chen W, Cheng Y, Wang M, Jia R, Zhu D, Liu M, Zhao X, Yang Q, Wu Y, Zhang S, Huang J, Mao S, Ou X, Gao Q, Sun D, Cheng A, Chen S. The autophagy-related degradation of MDA5 by Tembusu virus nonstructural 2B disrupts IFNβ production. FASEB J 2022; 36:e22417. [PMID: 35713934 DOI: 10.1096/fj.202101916rrr] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2021] [Revised: 05/23/2022] [Accepted: 06/06/2022] [Indexed: 12/24/2022]
Abstract
Duck Tembusu virus (TMUV) is a serious avian pathogen causing a decline in egg production, but the mechanism of the virus that breaks through the innate immune system is poorly understood. Here, we show that TMUV inhibits poly(I:C)-induced interferon (IFN) production. Because poly(I:C) transfection can specifically activate the MDA5 pathway in duck primary cells, we found that infection with TMUV can specifically target MDA5 and lead to its degradation. MDA5 downregulation could be blocked by the autophagy inhibitor 3-methyladenine (3-MA) but not a proteasome inhibitor, strongly implicating MDA5 degradation as an autophagy-related degradation pathway. Pretreatment with 3-MA enhanced the expression of MDA5 and inhibited TMUV replication. To screen TMUV proteins that degraded MDA5, the TMUV replicon and MDA5-Flag were cotransfected into cells, and the western blot analysis showed that nonstructural 2B (NS2B) can degrade MDA5 in a dose-dependent manner. Dual-luciferase assays indicate that NS2B alone inhibits MDA5- or poly(I:C)-mediated IFN production. NS2B binds MDA5 in the presence of 3-MA. The deletion of the amino acids of NS2B from residues 51 to 92 (hydrophilic area) restored the expression of MDA5 and relieved the MDA5-mediated IFNβ production inhibition by NS2B, indicating that the hydrophilic area of NS2B is important for its interaction with host innate immunity.
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Affiliation(s)
- Zhen Wu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Tao Hu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Weiqiong Chen
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Yao Cheng
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Mingshu Wang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Renyong Jia
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Dekang Zhu
- Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Mafeng Liu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Xinxin Zhao
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Qiao Yang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Ying Wu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Shaqiu Zhang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Juan Huang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Sai Mao
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Xumin Ou
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Qun Gao
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Di Sun
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Anchun Cheng
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Shun Chen
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
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8
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Kim D, Han S, Ji Y, Moon S, Nam H, Lee JB. Multimeric RNAs for efficient RNA-based therapeutics and vaccines. J Control Release 2022; 345:770-785. [PMID: 35367477 PMCID: PMC8970614 DOI: 10.1016/j.jconrel.2022.03.052] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2021] [Revised: 03/22/2022] [Accepted: 03/27/2022] [Indexed: 11/17/2022]
Abstract
There has been a growing interest in RNA therapeutics globally, and much progress has been made in this area, which has been further accelerated by the clinical applications of RNA-based vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Following these successful clinical trials, various technologies have been developed to improve the efficacy of RNA-based drugs. Multimerization of RNA therapeutics is one of the most attractive approaches to ensure high stability, high efficacy, and prolonged action of RNA-based drugs. In this review, we offer an overview of the representative approaches for generating repetitive functional RNAs by chemical conjugation, structural self-assembly, enzymatic elongation, and self-amplification. The therapeutic and vaccine applications of engineered multimeric RNAs in various diseases have also been summarized. By outlining the current status of multimeric RNAs, the potential of multimeric RNA as a promising treatment strategy is highlighted.
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Affiliation(s)
- Dajeong Kim
- Department of Chemical Engineering, University of Seoul, 163 Seoulsiripdaero, Dongdaemun-gu, Seoul, South Korea
| | - Sangwoo Han
- Department of Chemical Engineering, University of Seoul, 163 Seoulsiripdaero, Dongdaemun-gu, Seoul, South Korea
| | - Yoonbin Ji
- Department of Chemical Engineering, University of Seoul, 163 Seoulsiripdaero, Dongdaemun-gu, Seoul, South Korea
| | - Sunghyun Moon
- Department of Chemical Engineering, University of Seoul, 163 Seoulsiripdaero, Dongdaemun-gu, Seoul, South Korea
| | - Hyangsu Nam
- Department of Chemical Engineering, University of Seoul, 163 Seoulsiripdaero, Dongdaemun-gu, Seoul, South Korea
| | - Jong Bum Lee
- Department of Chemical Engineering, University of Seoul, 163 Seoulsiripdaero, Dongdaemun-gu, Seoul, South Korea.
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9
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Defective Interfering Viral Particle Treatment Reduces Clinical Signs and Protects Hamsters from Lethal Nipah Virus Disease. mBio 2022; 13:e0329421. [PMID: 35297677 PMCID: PMC9040845 DOI: 10.1128/mbio.03294-21] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Defective interfering particles (DIs) contain a considerably smaller genome than the parental virus but retain replication competency. As DIs can directly or indirectly alter propagation kinetics of the parental virus, they offer a novel approach to antiviral therapy, capitalizing on knowledge from natural infection. However, efforts to translate in vitro inhibition to in vivo screening models remain limited. We investigated the efficacy of virus-like particles containing DI genomes (therapeutic infectious particles [TIPs]) in the Syrian hamster model of lethal Nipah virus (NiV) disease. We found that coadministering a high dose of TIPs intraperitoneally with virus challenge improved clinical course and reduced lethality. To mimic natural exposure, we also evaluated lower-dose TIP delivery and virus challenge intranasally, finding equally efficacious reduction in disease severity and overall lethality. Eliminating TIP replicative capacity decreased efficacy, suggesting protection via direct inhibition. These data provide evidence that TIP-mediated treatment can confer protection against disease and lethal outcome in a robust animal NiV model, supporting further development of TIP treatment for NiV and other high-consequence pathogens. IMPORTANCE Here, we demonstrate that treatment with defective interfering particles (DIs), a natural by-product of viral infection, can significantly improve the clinical course and outcome of viral disease. When present with their parental virus, DIs can directly or indirectly alter viral propagation kinetics and exert potent inhibitory properties in cell culture. We evaluated the efficacy of a selection of virus-like particles containing DI genomes (TIPs) delivered intranasally in a lethal hamster model of Nipah virus disease. We demonstrate significantly improved clinical outcomes, including reduction in both lethality and the appearance of clinical signs. This work provides key efficacy data in a robust model of Nipah virus disease to support further development of TIP-mediated treatment against high-consequence viral pathogens.
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10
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Cao X, Cordova AF, Li L. Therapeutic Interventions Targeting Innate Immune Receptors: A Balancing Act. Chem Rev 2021; 122:3414-3458. [PMID: 34870969 DOI: 10.1021/acs.chemrev.1c00716] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
The innate immune system is an organism's first line of defense against an onslaught of internal and external threats. The downstream adaptive immune system has been a popular target for therapeutic intervention, while there is a relative paucity of therapeutics targeting the innate immune system. However, the innate immune system plays a critical role in many human diseases, such as microbial infection, cancer, and autoimmunity, highlighting the need for ongoing therapeutic research. In this review, we discuss the major innate immune pathways and detail the molecular strategies underpinning successful therapeutics targeting each pathway as well as previous and ongoing efforts. We will also discuss any recent discoveries that could inform the development of novel therapeutic strategies. As our understanding of the innate immune system continues to develop, we envision that therapies harnessing the power of the innate immune system will become the mainstay of treatment for a wide variety of human diseases.
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11
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Xing J, Zhang Y, Lin Z, Liu L, Xu Q, Liang J, Yuan Z, Huang C, Liao M, Qi W. 3'UTR SL-IV and DB1 Regions Contribute to Japanese Encephalitis Virus Replication and Pathogenicity. Front Vet Sci 2021; 8:703147. [PMID: 34409089 PMCID: PMC8366024 DOI: 10.3389/fvets.2021.703147] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2021] [Accepted: 07/07/2021] [Indexed: 11/29/2022] Open
Abstract
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus that causes fatal neurological disease in humans, is one of the most important emerging pathogens of public health significance. JEV is maintained in an enzootic cycle and causes reproductive failure in pigs. Notably, the shift in JEV genotypes is not fully protected by existing vaccines, so the development of a candidate vaccine is urgently needed. In this study, we compared pathogenicity between Japanese encephalitis virus SA14 and BJB (isolated from humans in the 1970s) strains. We found that the BJB strain was attenuated in mice and that there was no case fatality rate. The growth rate of BJB was higher than SA14 virus in BHK-21 cells. Based on the sequence alignment of the viral genome between the SA14 and BJB virus strains, some mutations at sites 248, 254, 258, and 307 were observed in the 3′ untranslated region (3′UTR). The 3′UTR of JEV plays a very important role in the viral life cycle. Furthermore, using a reverse genetic system, we conducted and rescued the parental JEV strain SA14 (T248, A254, and A258) and the mutant virus rSA14-3′UTRmut (T248C, A254G, A258G, and 307G). Through an analysis of the RNA secondary structure model of the 3′UTR, we discovered that the mutations of T248C, A254G, and A258G reduced the apiculus ring and increased the lateral ring significantly in the stem-loop structures IV (SL-IV) structure region of 3′UTR. Moreover, the insertion of 307G added a ring to the dumbbell structure 1 (DB1) structure region. Strikingly, these RNA secondary structure changes in 3′UTR of rSA14-3′UTRmut increased viral negative chain RNA production and enhanced the replication ability of the virus in BHK-21 cells. However, in vivo mouse experiments illustrated that the rSA14-3′UTRmut virus significantly decreased the neurovirulence of JEV. These results affirmed that the JEV SL-IV and DB1 regions play an important role in viral proliferation and pathogenicity. Taken together, we complement the study of RNA element function in the 3′UTR region of JEV by providing a new target for the rational design of live attenuated candidate vaccines and the increase of virus production.
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Affiliation(s)
- Jinchao Xing
- Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, China.,National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou, China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, China
| | - Youyue Zhang
- Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, China.,National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou, China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, China
| | - Ziying Lin
- Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, China.,National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou, China
| | - Lele Liu
- Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, China.,National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou, China
| | - Qiang Xu
- Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, China.,National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou, China
| | - Jiaqi Liang
- Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, China.,National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou, China
| | - Zhaoxia Yuan
- College of Animal Sciences and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou, China
| | - Cuiqin Huang
- The Key Laboratory of Fujian Animal Diseases Control, Longyan University, Longyan, China
| | - Ming Liao
- Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, China.,National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou, China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, China.,Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Guangzhou, China
| | - Wenbao Qi
- Key Laboratory of Zoonoses, Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, China.,National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou, China.,Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, China.,Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Guangzhou, China
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12
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Zhang R, Cheng M, Liu B, Yuan M, Chen D, Wang Y, Wu Z. DEAD-Box Helicase DDX6 Facilitated RIG-I-Mediated Type-I Interferon Response to EV71 Infection. Front Cell Infect Microbiol 2021; 11:725392. [PMID: 34485180 PMCID: PMC8414799 DOI: 10.3389/fcimb.2021.725392] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2021] [Accepted: 07/28/2021] [Indexed: 12/24/2022] Open
Abstract
Previous studies have shown that DEAD (Glu-Asp-Ala-Glu)-box RNA helicases play important roles in viral infection, either as cytosolic sensors of pathogenic molecules or as essential host factors against viral infection. In the current study, we found that DDX6, an RNA helicase belonging to the DEAD-box family of helicase, exhibited anti-Enterovirus 71 activity through augmenting RIG-I-mediated type-I IFN response. Moreover, DDX6 binds viral RNA to form an RNA-protein complex to positively regulate the RIG-I-mediated interferon response; however, EV71 has evolved a strategy to antagonize the antiviral effect of DDX6 by proteolytic degradation of the molecule through its non-structural protein 2A, a virus-encoded protease.
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Affiliation(s)
- Rui Zhang
- Center for Public Health Research, Medical School, Nanjing University, Nanjing, China
| | - Min Cheng
- Center for Public Health Research, Medical School, Nanjing University, Nanjing, China
| | - Bingxin Liu
- Center for Public Health Research, Medical School, Nanjing University, Nanjing, China
| | - Meng Yuan
- Center for Public Health Research, Medical School, Nanjing University, Nanjing, China
| | - Deyan Chen
- Center for Public Health Research, Medical School, Nanjing University, Nanjing, China
| | - Yujiong Wang
- School of Life Sciences, Ningxia University, Yinchuan, China
| | - Zhiwei Wu
- Center for Public Health Research, Medical School, Nanjing University, Nanjing, China
- School of Life Sciences, Ningxia University, Yinchuan, China
- Medical School and Jiangsu Key Laboratory of Molecular Medicine, Nanjing University, Nanjing, China
- State Key Lab of Analytical Chemistry for Life Science, Nanjing University, Nanjing, China
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13
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Batool M, Kim MS, Choi S. Structural insights into the distinctive RNA recognition and therapeutic potentials of RIG-I-like receptors. Med Res Rev 2021; 42:399-425. [PMID: 34287999 DOI: 10.1002/med.21845] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2020] [Revised: 06/11/2021] [Accepted: 07/04/2021] [Indexed: 12/12/2022]
Abstract
RNA viruses, including the coronavirus, develop a unique strategy to evade the host immune response by interrupting the normal function of cytosolic retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs). RLRs rapidly detect atypical nucleic acids, thereby triggering the antiviral innate immune signaling cascade and subsequently activates the interferons transcription and induction of other proinflammatory cytokines and chemokines. Nonetheless, these receptors are manipulated by viral proteins to subvert the host immune system and sustain the infectivity and replication potential of the virus. RIG-I senses the single-stranded, double-stranded, and short double-stranded RNAs and recognizes the key signature, a 5'-triphosphate moiety, at the blunt end of the viral RNA. Meanwhile, the melanoma differentiation-associated gene 5 (MDA5) is triggered by longer double stranded RNAs, messenger RNAs lacking 2'-O-methylation in their 5'-cap, and RNA aggregates. Therefore, structural insights into the nucleic-acid-sensing and downstream signaling mechanisms of these receptors hold great promise for developing effective antiviral therapeutic interventions. This review highlights the critical roles played by RLRs in viral infections as well as their ligand recognition mechanisms. In addition, we highlight the crosstalk between the toll-like receptors and RLRs and provide a comprehensive overview of RLR-associated diseases as well as the therapeutic potential of RLRs for the development of antiviral-drugs. Moreover, we believe that these RLR-based antivirals will serve as a step toward countering the recent coronavirus disease 2019 pandemic.
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Affiliation(s)
- Maria Batool
- Department of Molecular Science and Technology, Ajou University, Suwon, Korea
- S&K Therapeutics, Campus Plaza 418, Ajou University, Suwon, Korea
| | - Moon Suk Kim
- Department of Molecular Science and Technology, Ajou University, Suwon, Korea
| | - Sangdun Choi
- Department of Molecular Science and Technology, Ajou University, Suwon, Korea
- S&K Therapeutics, Campus Plaza 418, Ajou University, Suwon, Korea
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14
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Martín-Fernández JM, Fleischer A, Vallejo-Diez S, Palomino E, Sánchez-Gilabert A, Ruiz R, Bejarano Y, Llinàs P, Gayá A, Bachiller D. New Bicistronic TALENs Greatly Improve Genome Editing. ACTA ACUST UNITED AC 2021; 52:e104. [PMID: 32023363 DOI: 10.1002/cpsc.104] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Genome editing has become one of the most powerful tools in present-day stem cell and regenerative medicine research, but despite its rapid acceptance and widespread use, some elements of the technology still need improvement. In this unit, we present data regarding the use of a new, more efficient type of transcription activator-like effector nuclease (TALEN) for gene editing. Our group has generated bicistronic genes in which classical TALEN coding sequences are linked by 2A elements to different reporter molecules, such as fluorochromes (TALEN-F) or membrane receptors (TALEN-M). This structure results in two proteins transcribed from the same transcript, of which the second (the reporter) can be used as the target for selection by fluorescence-assisted cell sorting (FACS) or magnetic-activated cell sorting (MACS). The application of these new TALEN genes allows a rapid enrichment of cells in which both members of the TALEN pair are active, thus eliminating the need for lengthy selection in culture and laborious characterization of a large number of clones. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of new TALENs Basic Protocol 2: Genome editing using TALEN-F Alternate Protocol 1: Generation of TALEN-M Support Protocol 1: mRNA in vitro transcription (IVT) of TALEN-T2A-reporter expression vector Alternate Protocol 2: Editing of primary T cells using TALEN-M Basic Protocol 3: Verifying gene editing Support Protocol 2: Rapid expansion protocol for edited T-cells.
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Affiliation(s)
| | - Aarne Fleischer
- Karuna Good Cells Technologies SL, Vitoria-Gasteiz, Álava, Spain.,Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
| | - Sara Vallejo-Diez
- Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
| | - Esther Palomino
- Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
| | - Almudena Sánchez-Gilabert
- Karuna Good Cells Technologies SL, Vitoria-Gasteiz, Álava, Spain.,Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
| | - Raúl Ruiz
- Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
| | - Yazmine Bejarano
- Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain.,Current address: Centro de Investigación del Cáncer, Campus Miguel de Unamuno, Salamanca, Spain
| | - Pere Llinàs
- Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain.,Current address: Josep Carreras Leukaemia Research Institute (IJC), Ctra. de Can Ruti, Camí de les Escoles, Badalona, Spain
| | - Antoni Gayá
- Instituto de Investigación Sanitaria Illes Balears (IDISBA), Fundació Banc de Sang i Teixits de les Illes Balears (FBSTIB), Grupo de Terapia Celular e Ingenieria Tisular, Palma de Mallorca, Spain
| | - Daniel Bachiller
- Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA), Esporles, Spain
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15
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Faber E, Tshilwane SI, Kleef MV, Pretorius A. Virulent African horse sickness virus serotype 4 interferes with the innate immune response in horse peripheral blood mononuclear cells in vitro. INFECTION GENETICS AND EVOLUTION 2021; 91:104836. [PMID: 33798756 DOI: 10.1016/j.meegid.2021.104836] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/04/2020] [Revised: 03/09/2021] [Accepted: 03/29/2021] [Indexed: 12/17/2022]
Abstract
African horse sickness (AHS) is caused by African horse sickness virus (AHSV), a double stranded RNA (dsRNA) virus of the genus Orbivirus, family Reoviridae. For the development of new generation AHS vaccines or antiviral treatments, it is crucial to understand the host immune response against the virus and the immune evasion strategies the virus employs. To achieve this, the current study used transcriptome analysis of RNA sequences to characterize and compare the innate immune responses activated during the attenuated AHSV serotype 4 (attAHSV4) (in vivo) and the virulent AHSV4 (virAHSV4) (in vitro) primary and secondary immune responses in horse peripheral blood mononuclear cells (PBMC) after 24 h. The pro-inflammatory cytokine and chemokine responses were negatively regulated by anti-inflammatory cytokines, whereas the parallel type I and type III IFN responses were maintained downstream of nucleic acid sensing pattern recognition receptor (PRR) signalling pathways during the attAHSV4 primary and secondary immune responses. It appeared that after translation, virAHSV4 proteins were able to interfere with the C-terminal IRF association domain (IAD)-type 1 (IAD1) containing IRFs, which inhibited the expression of type I and type III IFNs downstream of PRR signalling during the virAHSV4 primary and secondary immune responses. Viral interference resulted in an impaired innate immune response that was not able to eliminate virAHSV4-infected PBMC and gave rise to prolonged expression of pro-inflammatory cytokines and chemokines during the virAHSV4 induced primary immune response. Indicating that virAHSV4 interference with the innate immune response may give rise to an excessive inflammatory response that causes immunopathology, which could be a major contributing factor to the pathogenesis of AHS in a naïve horse. Viral interference was overcome by the fast kinetics and increased effector responses of innate immune cells due to trained innate immunity and memory T cells and B cells during the virAHSV4 secondary immune response.
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Affiliation(s)
- Erika Faber
- Agricultural Research Council - Onderstepoort Veterinary Research, Private Bag X5, Onderstepoort 0110, South Africa; Department of Veterinary Tropical Disease, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa.
| | - Selaelo Ivy Tshilwane
- School of Life Sciences, University of KwaZulu-Natal, Westville Campus, Durban 4000, South Africa
| | - Mirinda Van Kleef
- Agricultural Research Council - Onderstepoort Veterinary Research, Private Bag X5, Onderstepoort 0110, South Africa; Department of Veterinary Tropical Disease, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa
| | - Alri Pretorius
- Agricultural Research Council - Onderstepoort Veterinary Research, Private Bag X5, Onderstepoort 0110, South Africa; Department of Veterinary Tropical Disease, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa
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16
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Wang HT, Hur S. Substrate recognition by TRIM and TRIM-like proteins in innate immunity. Semin Cell Dev Biol 2021; 111:76-85. [PMID: 33092958 PMCID: PMC7572318 DOI: 10.1016/j.semcdb.2020.09.013] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2020] [Accepted: 09/28/2020] [Indexed: 12/23/2022]
Abstract
TRIM (Tripartite motif) and TRIM-like proteins have emerged as an important class of E3 ligases in innate immunity. Their functions range from activation or regulation of innate immune signaling pathway to direct detection and restriction of pathogens. Despite the importance, molecular mechanisms for many TRIM/TRIM-like proteins remain poorly characterized, in part due to challenges of identifying their substrates. In this review, we discuss several TRIM/TRIM-like proteins in RNA sensing pathways and viral restriction functions. We focus on those containing PRY-SPRY, the domain most frequently used for substrate recognition, and discuss emerging mechanisms that are commonly utilized by several TRIM/TRIM-like proteins to tightly control their interaction with the substrates.
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Affiliation(s)
- Hai-Tao Wang
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA
| | - Sun Hur
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA.
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17
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Kim GW, Siddiqui A. The role of N6-methyladenosine modification in the life cycle and disease pathogenesis of hepatitis B and C viruses. Exp Mol Med 2021; 53:339-345. [PMID: 33742132 PMCID: PMC8080661 DOI: 10.1038/s12276-021-00581-3] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2020] [Revised: 01/25/2021] [Accepted: 01/26/2021] [Indexed: 12/11/2022] Open
Abstract
N6-methyladenosine (m6A) is the most prevalent modification of mammalian cellular RNAs. m6A methylation is linked to epigenetic regulation of several aspects of gene expression, including RNA stability, splicing, nuclear export, RNA folding, and translational activity. m6A modification is reversibly catalyzed by methyltransferases (m6A writers) and demethylases (m6A erasers), and the dynamics of m6A-modified RNA are regulated by m6A-binding proteins (m6A readers). Recently, several studies have shown that m6A methylation sites have been identified in hepatitis B virus (HBV) transcripts and the hepatitis C virus (HCV) RNA genome. Here, we review the role of m6A modification in HBV/HCV replication and its contribution to liver disease pathogenesis. A better understanding of the functions of m6A methylation in the life cycles of HBV and HCV is required to establish the role of these modifications in liver diseases associated with these viral infections.
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Affiliation(s)
- Geon-Woo Kim
- Division of Infectious Diseases, Department of Medicine, University of California, San Diego, La Jolla, CA, 92093, USA.
| | - Aleem Siddiqui
- Division of Infectious Diseases, Department of Medicine, University of California, San Diego, La Jolla, CA, 92093, USA
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18
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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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19
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Borchardt EK, Martinez NM, Gilbert WV. Regulation and Function of RNA Pseudouridylation in Human Cells. Annu Rev Genet 2020; 54:309-336. [PMID: 32870730 DOI: 10.1146/annurev-genet-112618-043830] [Citation(s) in RCA: 136] [Impact Index Per Article: 27.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Recent advances in pseudouridine detection reveal a complex pseudouridine landscape that includes messenger RNA and diverse classes of noncoding RNA in human cells. The known molecular functions of pseudouridine, which include stabilizing RNA conformations and destabilizing interactions with varied RNA-binding proteins, suggest that RNA pseudouridylation could have widespread effects on RNA metabolism and gene expression. Here, we emphasize how much remains to be learned about the RNA targets of human pseudouridine synthases, their basis for recognizing distinct RNA sequences, and the mechanisms responsible for regulated RNA pseudouridylation. We also examine the roles of noncoding RNA pseudouridylation in splicing and translation and point out the potential effects of mRNA pseudouridylation on protein production, including in the context of therapeutic mRNAs.
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Affiliation(s)
- Erin K Borchardt
- Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, Yale University, New Haven, Connecticut 06520, USA; , ,
| | - Nicole M Martinez
- Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, Yale University, New Haven, Connecticut 06520, USA; , ,
| | - Wendy V Gilbert
- Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, Yale University, New Haven, Connecticut 06520, USA; , ,
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20
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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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21
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Johnson MB, Halman JR, Burmeister AR, Currin S, Khisamutdinov EF, Afonin KA, Marriott I. Retinoic acid inducible gene-I mediated detection of bacterial nucleic acids in human microglial cells. J Neuroinflammation 2020; 17:139. [PMID: 32357908 PMCID: PMC7195775 DOI: 10.1186/s12974-020-01817-1] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2020] [Accepted: 04/16/2020] [Indexed: 12/13/2022] Open
Abstract
Background Bacterial meningitis and meningoencephalitis are associated with devastating neuroinflammation. We and others have demonstrated the importance of glial cells in the initiation of immune responses to pathogens invading the central nervous system (CNS). These cells use a variety of pattern recognition receptors (PRRs) to identify common pathogen motifs and the cytosolic sensor retinoic acid inducible gene-1 (RIG-I) is known to serve as a viral PRR and initiator of interferon (IFN) responses. Intriguingly, recent evidence indicates that RIG-I also has an important role in the detection of bacterial nucleic acids, but such a role has not been investigated in glia. Methods In this study, we have assessed whether primary or immortalized human and murine glia express RIG-I either constitutively or following stimulation with bacteria or their products by immunoblot analysis. We have used capture ELISAs and immunoblot analysis to assess human microglial interferon regulatory factor 3 (IRF3) activation and IFN production elicited by bacterial nucleic acids and novel engineered nucleic acid nanoparticles. Furthermore, we have utilized a pharmacological inhibitor of RIG-I signaling and siRNA-mediated knockdown approaches to assess the relative importance of RIG-I in such responses. Results We demonstrate that RIG-I is constitutively expressed by human and murine microglia and astrocytes, and is elevated following bacterial infection in a pathogen and cell type-specific manner. Additionally, surface and cytosolic PRR ligands are also sufficient to enhance RIG-I expression. Importantly, our data demonstrate that bacterial RNA and DNA both trigger RIG-I-dependent IRF3 phosphorylation and subsequent type I IFN production in human microglia. This ability has been confirmed using our nucleic acid nanoparticles where we demonstrate that both RNA- and DNA-based nanoparticles can stimulate RIG-I-dependent IFN responses in these cells. Conclusions The constitutive and bacteria-induced expression of RIG-I by human glia and its ability to mediate IFN responses to bacterial RNA and DNA and nucleic acid nanoparticles raises the intriguing possibility that RIG-I may be a potential target for therapeutic intervention during bacterial infections of the CNS, and that the use of engineered nucleic acid nanoparticles that engage this sensor might be a method to achieve this goal.
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Affiliation(s)
- M Brittany Johnson
- Department of Biological Sciences, University of North Carolina at Charlotte, 9201 University City Blvd, Charlotte, NC, 28223, USA
| | - Justin R Halman
- Nanoscale Science Program, Department of Chemistry, University of North Carolina at Charlotte, Charlotte, NC, 28223, USA
| | - Amanda R Burmeister
- Center for Neurodegenerative Science, Van Andel Institute, Grand Rapids, MI, 49503, USA
| | - Saralynn Currin
- Department of Biological Sciences, University of North Carolina at Charlotte, 9201 University City Blvd, Charlotte, NC, 28223, USA
| | | | - Kirill A Afonin
- Nanoscale Science Program, Department of Chemistry, University of North Carolina at Charlotte, Charlotte, NC, 28223, USA
| | - Ian Marriott
- Department of Biological Sciences, University of North Carolina at Charlotte, 9201 University City Blvd, Charlotte, NC, 28223, USA.
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22
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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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Yang Q, Bai SY, Li LF, Li S, Zhang Y, Munir M, Qiu HJ. Human Hemoglobin Subunit Beta Functions as a Pleiotropic Regulator of RIG-I/MDA5-Mediated Antiviral Innate Immune Responses. J Virol 2019; 93:e00718-19. [PMID: 31167908 PMCID: PMC6675906 DOI: 10.1128/jvi.00718-19] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2019] [Accepted: 05/20/2019] [Indexed: 12/28/2022] Open
Abstract
Hemoglobin is an important oxygen-carrying protein and plays crucial roles in establishing host resistance against pathogens and in regulating innate immune responses. The hemoglobin subunit beta (HB) is an essential component of hemoglobin, and we have previously demonstrated that the antiviral role of the porcine HB (pHB) is mediated by promoting type I interferon pathways. Thus, considering the high homology between human HB (hHB) and pHB, we hypothesized that hHB also plays an important role in the antiviral innate immunity. In this study, we characterized hHB as a regulatory factor for the replication of RNA viruses by differentially regulating the RIG-I- and MDA5-mediated antiviral signaling pathways. Furthermore, we showed that hHB directly inhibited MDA5-mediated signaling by reducing the MDA5-double-stranded RNA (dsRNA) interaction. Additionally, hHB required hHB-induced reactive oxygen species (ROS) to promote RIG-I-mediated signaling through enhancement of K63-linked RIG-I ubiquitination. Taken together, our findings suggest that hHB is a pleiotropic regulator of RIG-I/MDA5-mediated antiviral responses and further highlight the importance of the intercellular microenvironment, including the redox state, in regulating antiviral innate immune responses.IMPORTANCE Hemoglobin, the most important oxygen-carrying protein, is involved in the regulation of innate immune responses. We have previously reported that the porcine hemoglobin subunit beta (HB) exerts antiviral activity through regulation of type I interferon production. However, the antiviral activities and the underlying mechanisms of HBs originating from other animals have been poorly understood. Here, we identified human HB (hHB) as a pleiotropic regulator of the replication of RNA viruses through regulation of RIG-I/MDA5-mediated signaling pathways. hHB enhances RIG-I-mediated antiviral responses by promoting RIG-I ubiquitination depending on the hHB-induced reactive oxygen species (ROS), while it blocks MDA5-mediated antiviral signaling by suppressing the MDA5-dsRNA interaction. Our results contribute to an understanding of the crucial roles of hHB in the regulation of the RIG-I/MDA5-mediated signaling pathways. We also provide novel insight into the correlation of the intercellular redox state with the regulation of antiviral innate immunity.
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Affiliation(s)
- Qian Yang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Si-Yu Bai
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Lian-Feng Li
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Su Li
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Yuexiu Zhang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Muhammad Munir
- Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, United Kingdom
| | - Hua-Ji Qiu
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
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Fan J, Cheng M, Chi X, Liu X, Yang W. A Human Long Non-coding RNA LncATV Promotes Virus Replication Through Restricting RIG-I-Mediated Innate Immunity. Front Immunol 2019; 10:1711. [PMID: 31379885 PMCID: PMC6658999 DOI: 10.3389/fimmu.2019.01711] [Citation(s) in RCA: 34] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2019] [Accepted: 07/08/2019] [Indexed: 12/03/2022] Open
Abstract
Pattern recognition receptors sense pathogen components and initiate the host antiviral innate immune response, such as inducing interferons (IFNs). Long non-coding RNAs (lncRNAs) are emerging regulators of multiple biological processes. However, their role in antiviral response, especially through regulating the human innate immune, is largely unexplored. Here we characterized that lncATV, a human specific lncRNA, was up-regulated upon type I/III IFN stimulations and virus infection. LncATV was cytoplasmic localized and relatively high expressed in human monocytes, erythroleukemia cells and hepatoma cells. Notably, lncATV knockdown significantly inhibited the replication of multiple RNA viruses, such as hepatitis C virus, Zika virus, Newcastle disease virus, and Sendai virus. Mechanistically, RIG-I antiviral signaling and IFN effective pathway were enhanced when lncATV expression was knocked down but inhibited by overexpressed lncATV. RNA immunoprecipitation results demonstrated an association between LncATV and RIG-I. Collectively, our findings reveal the functional role of a novel human specific lncATV as a regulatory lncRNA restricting virus associated innate immune response.
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Affiliation(s)
- Jingjing Fan
- NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Min Cheng
- NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Xiaojing Chi
- NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Xiuying Liu
- NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Wei Yang
- NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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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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Kasumba DM, Grandvaux N. Therapeutic Targeting of RIG-I and MDA5 Might Not Lead to the Same Rome. Trends Pharmacol Sci 2019; 40:116-127. [PMID: 30606502 PMCID: PMC7112877 DOI: 10.1016/j.tips.2018.12.003] [Citation(s) in RCA: 39] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2018] [Revised: 12/05/2018] [Accepted: 12/06/2018] [Indexed: 12/12/2022]
Abstract
RIG-I and MDA5 receptors are key sensors of pathogen-associated molecular pattern (PAMP)-containing viral RNA and transduce downstream signals to activate an antiviral and immunomodulatory response. Fifteen years of research have put them at the center of an ongoing hunt for novel pharmacological pan-antivirals, vaccine adjuvants, and antitumor strategies. Current knowledge testifies to the redundant, but also distinct, functions mediated by RIG-I and MDA5, opening opportunities for the use of specific and potent nucleic acid agonists. We critically discuss the evidence and remaining knowledge gaps that have an impact on the choice and design of optimal RNA ligands to achieve an appropriate immunostimulatory response, with limited adverse effects, for prophylactic and therapeutic interventions against viruses and cancer in humans.
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Affiliation(s)
- Dacquin M. Kasumba
- Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montréal, QC, Canada,Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, QC, Canada
| | - Nathalie Grandvaux
- Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montréal, QC, Canada; Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, Montréal, QC, Canada.
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Yang Y, Tu ZK, Liu XK, Zhang P. Mononuclear phagocyte system in hepatitis C virus infection. World J Gastroenterol 2018; 24:4962-4973. [PMID: 30510371 PMCID: PMC6262249 DOI: 10.3748/wjg.v24.i44.4962] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/07/2018] [Revised: 10/30/2018] [Accepted: 11/08/2018] [Indexed: 02/06/2023] Open
Abstract
The mononuclear phagocyte system (MPS), which consists of monocytes, dendritic cells (DCs), and macrophages, plays a vital role in the innate immune defense against pathogens. Hepatitis C virus (HCV) is efficient in evading the host immunity, thereby facilitating its development into chronic infection. Chronic HCV infection is the leading cause of end-stage liver diseases, liver cirrhosis, and hepatocellular carcinoma. Acquired immune response was regarded as the key factor to eradicate HCV. However, innate immunity can regulate the acquired immune response. Innate immunity-derived cytokines shape the adaptive immunity by regulating T-cell differentiation, which determines the outcome of acute HCV infection. Inhibition of HCV-specific T-cell responses is one of the most important strategies for immune system evasion. It is meaningful to illustrate the role of innate immune response in HCV infection. With the MPS being the important factor in innate immunity, therefore, understanding the role of the MPS in HCV infection will shed light on the pathophysiology of chronic HCV infection. In this review, we outline the impact of HCV infection on the MPS and cytokine production. We discuss how HCV is detected by the MPS and describe the function and impairment of MPS components in HCV infection.
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Affiliation(s)
- Yu Yang
- Department of Hepatobiliary and Pancreatic Surgery, The First Hospital of Jilin University, Changchun 130021, Jilin Province, China
| | - Zheng-Kun Tu
- Institute of Translational Medicine, The First Hospital of Jilin University, Changchun 130061, Jilin Province, China
| | - Xing-Kai Liu
- Department of Hepatobiliary and Pancreatic Surgery, The First Hospital of Jilin University, Changchun 130021, Jilin Province, China
| | - Ping Zhang
- Department of Hepatobiliary and Pancreatic Surgery, The First Hospital of Jilin University, Changchun 130021, Jilin Province, China
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29
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Chow KT, Wilkins C, Narita M, Green R, Knoll M, Loo YM, Gale M. Differential and Overlapping Immune Programs Regulated by IRF3 and IRF5 in Plasmacytoid Dendritic Cells. THE JOURNAL OF IMMUNOLOGY 2018; 201:3036-3050. [PMID: 30297339 DOI: 10.4049/jimmunol.1800221] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Received: 02/15/2018] [Accepted: 09/13/2018] [Indexed: 01/20/2023]
Abstract
We examined the signaling pathways and cell type-specific responses of IFN regulatory factor (IRF) 5, an immune-regulatory transcription factor. We show that the protein kinases IKKα, IKKβ, IKKε, and TANK-binding kinase 1 each confer IRF5 phosphorylation/dimerization, thus extending the family of IRF5 activator kinases. Among primary human immune cell subsets, we found that IRF5 is most abundant in plasmacytoid dendritic cells (pDCs). Flow cytometric cell imaging revealed that IRF5 is specifically activated by endosomal TLR signaling. Comparative analyses revealed that IRF3 is activated in pDCs uniquely through RIG-I-like receptor (RLR) signaling. Transcriptomic analyses of pDCs show that the partitioning of TLR7/IRF5 and RLR/IRF3 pathways confers differential gene expression and immune cytokine production in pDCs, linking IRF5 with immune regulatory and proinflammatory gene expression. Thus, TLR7/IRF5 and RLR-IRF3 partitioning serves to polarize pDC response outcome. Strategies to differentially engage IRF signaling pathways should be considered in the design of immunotherapeutic approaches to modulate or polarize the immune response for specific outcome.
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Affiliation(s)
- Kwan T Chow
- Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA 98109.,Department of Biomedical Sciences, City University of Hong Kong, Kowloon, Hong Kong Special Administrative Region; and
| | - Courtney Wilkins
- Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA 98109
| | - Miwako Narita
- Laboratory of Hematology and Oncology, Graduate School of Health Sciences, Niigata University, Niigata, Niigata Prefecture 950-2181, Japan
| | - Richard Green
- Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA 98109
| | - Megan Knoll
- Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA 98109
| | - Yueh-Ming Loo
- Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA 98109;
| | - Michael Gale
- Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington, Seattle, WA 98109;
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Vaidyanathan S, Azizian KT, Haque AKMA, Henderson JM, Hendel A, Shore S, Antony JS, Hogrefe RI, Kormann MSD, Porteus MH, McCaffrey AP. Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification. MOLECULAR THERAPY. NUCLEIC ACIDS 2018; 12:530-542. [PMID: 30195789 PMCID: PMC6076213 DOI: 10.1016/j.omtn.2018.06.010] [Citation(s) in RCA: 181] [Impact Index Per Article: 25.9] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/21/2018] [Revised: 06/21/2018] [Accepted: 06/22/2018] [Indexed: 12/25/2022]
Abstract
The Cas9/guide RNA (Cas9/gRNA) system is commonly used for genome editing. mRNA expressing Cas9 can induce innate immune responses, reducing Cas9 expression. First-generation Cas9 mRNAs were modified with pseudouridine and 5-methylcytosine to reduce innate immune responses. We combined four approaches to produce more active, less immunogenic second-generation Cas9 mRNAs. First, we developed a novel co-transcriptional capping method yielding natural Cap 1. Second, we screened modified nucleotides in Cas9 mRNA to identify novel modifications that increase Cas9 activity. Third, we depleted the mRNA of uridines to improve mRNA activity. Lastly, we tested high-performance liquid chromatography (HPLC) purification to remove double-stranded RNAs. The activity of these mRNAs was tested in cell lines and primary human CD34+ cells. Cytokines were measured in whole blood and mice. These approaches yielded more active and less immunogenic mRNA. Uridine depletion (UD) most impacted insertion or deletion (indel) activity. Specifically, 5-methoxyuridine UD induced indel frequencies as high as 88% (average ± SD = 79% ± 11%) and elicited minimal immune responses without needing HPLC purification. Our work suggests that uridine-depleted Cas9 mRNA modified with 5-methoxyuridine (without HPLC purification) or pseudouridine may be optimal for the broad use of Cas9 both in vitro and in vivo.
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Affiliation(s)
| | | | - A K M Ashiqul Haque
- Department of Pediatrics I, Pediatric Infectiology and Immunology, Translational Genomics and Gene Therapy in Pediatrics, University of Tuebingen, Tuebingen, Germany
| | | | - Ayal Hendel
- The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel
| | | | - Justin S Antony
- Department of Pediatrics I, Pediatric Infectiology and Immunology, Translational Genomics and Gene Therapy in Pediatrics, University of Tuebingen, Tuebingen, Germany
| | | | - Michael S D Kormann
- Department of Pediatrics I, Pediatric Infectiology and Immunology, Translational Genomics and Gene Therapy in Pediatrics, University of Tuebingen, Tuebingen, Germany
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TRIM21 Promotes Innate Immune Response to RNA Viral Infection through Lys27-Linked Polyubiquitination of MAVS. J Virol 2018; 92:JVI.00321-18. [PMID: 29743353 DOI: 10.1128/jvi.00321-18] [Citation(s) in RCA: 109] [Impact Index Per Article: 15.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2018] [Accepted: 04/27/2018] [Indexed: 12/24/2022] Open
Abstract
Human innate immunity responds to viral infection by activating the production of interferons (IFNs) and proinflammatory cytokines. The mitochondrial adaptor molecule MAVS plays a critical role in innate immune response to viral infection. In this study, we show that TRIM21 (tripartite motif-containing protein 21) interacts with MAVS to positively regulate innate immunity. Under viral infection, TRIM21 is upregulated through the IFN/JAK/STAT signaling pathway. Knockdown of TRIM21 dramatically impairs innate immune response to viral infection. Moreover, TRIM21 interacts with MAVS and catalyzes its K27-linked polyubiquitination, thereby promoting the recruitment of TBK1 to MAVS. Specifically, the PRY-SPRY domain of TRIM21 is the key domain for its interaction with MAVS, while the RING domain of TRIM21 facilitates the polyubiquitination chains of MAVS. In addition, the MAVS-mediated innate immune response is enhanced by both the PRY-SPRY and RING domains of TRIM21. Mutation analyses of all the lysine residues of MAVS further revealed that Lys325 of MAVS is catalyzed by TRIM21 for the K27-linked polyubiquitination. Overall, this study reveals a novel mechanism by which TRIM21 promotes the K27-linked polyubiquitination of MAVS to positively regulate innate immune response, thereby inhibiting viral infection.IMPORTANCE Activation of innate immunity is essential for host cells to restrict the spread of invading viruses and other pathogens. MAVS plays a critical role in innate immune response to RNA viral infection. In this study, we demonstrated that TRIM21 targets MAVS to positively regulate innate immunity. Notably, TRIM21 targets and catalyzes K27-linked polyubiquitination of MAVS and then promotes the recruitment of TBK1 to MAVS, leading to upregulation of innate immunity. Our study outlines a novel mechanism by which the IFN signaling pathway blocks RNA virus to escape immune elimination.
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32
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Liang Y, Cao X, Ding Q, Zhao Y, He Z, Zhong J. Hepatitis C virus NS4B induces the degradation of TRIF to inhibit TLR3-mediated interferon signaling pathway. PLoS Pathog 2018; 14:e1007075. [PMID: 29782532 PMCID: PMC5983870 DOI: 10.1371/journal.ppat.1007075] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2018] [Revised: 06/01/2018] [Accepted: 05/07/2018] [Indexed: 12/12/2022] Open
Abstract
Toll-like receptor 3 (TLR3) senses dsRNA intermediates produced during RNA virus replication to activate innate immune signaling pathways through adaptor protein TRIF. Many viruses have evolved strategies to block TLR3-mediated interferon signaling via targeting TRIF. Here we studied how hepatitis C virus (HCV) antagonizes the TLR3-mediated interferon signaling. We found that HCV-encoded NS4B protein inhibited TLR3-mediated interferon signaling by down-regulating TRIF protein level. Mechanism studies indicated that the downregulation of TRIF by NS4B was dependent on caspase8. NS4B transfection or HCV infection can activate caspase8 to promote TRIF degradation, leading to suppression of TLR3-mediated interferon signaling. Knockout of caspase8 can prevent TRIF degradation triggered by NS4B, thereby enhancing the TLR3-mediated interferon signaling activation in response to HCV infection. In conclusion, our work revealed a new mechanism for HCV to evade innate immune response by blocking the TLR3-mediated interferon signaling via NS4B-induced TRIF degradation.
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Affiliation(s)
- Yisha Liang
- CAS Key Laboratory of Molecular Virology and Immunology, Unit of Viral Hepatitis, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
- ShanghaiTech University, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Xuezhi Cao
- CAS Key Laboratory of Molecular Virology and Immunology, Unit of Viral Hepatitis, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
| | - Qiang Ding
- CAS Key Laboratory of Molecular Virology and Immunology, Unit of Viral Hepatitis, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
| | - Yanan Zhao
- CAS Key Laboratory of Molecular Virology and Immunology, Unit of Viral Hepatitis, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Zhenliang He
- CAS Key Laboratory of Molecular Virology and Immunology, Unit of Viral Hepatitis, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Jin Zhong
- CAS Key Laboratory of Molecular Virology and Immunology, Unit of Viral Hepatitis, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
- ShanghaiTech University, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
- * E-mail:
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Jiang M, Zhang S, Yang Z, Lin H, Zhu J, Liu L, Wang W, Liu S, Liu W, Ma Y, Zhang L, Cao X. Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response. Cell 2018; 173:906-919.e13. [DOI: 10.1016/j.cell.2018.03.064] [Citation(s) in RCA: 157] [Impact Index Per Article: 22.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2017] [Revised: 02/17/2018] [Accepted: 03/26/2018] [Indexed: 12/25/2022]
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Dustin LB. Innate and Adaptive Immune Responses in Chronic HCV Infection. Curr Drug Targets 2018; 18:826-843. [PMID: 26302811 DOI: 10.2174/1389450116666150825110532] [Citation(s) in RCA: 47] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2015] [Revised: 07/25/2015] [Accepted: 07/27/2015] [Indexed: 12/14/2022]
Abstract
Hepatitis C virus (HCV) remains a public health problem of global importance, even in the era of potent directly-acting antiviral drugs. In this chapter, I discuss immune responses to acute and chronic HCV infection. The outcome of HCV infection is influenced by viral strategies that limit or delay the initiation of innate antiviral responses. This delay may enable HCV to establish widespread infection long before the host mounts effective T and B cell responses. HCV's genetic agility, resulting from its high rate of replication and its error prone replication mechanism, enables it to evade immune recognition. Adaptive immune responses fail to keep up with changing viral epitopes. Neutralizing antibody epitopes may be hidden by decoy structures, glycans, and lipoproteins. T cell responses fail due to changing epitope sequences and due to exhaustion, a phenomenon that may have evolved to limit immune-mediated pathology. Despite these difficulties, innate and adaptive immune mechanisms do impact HCV replication. Immune-mediated clearance of infection is possible, occurring in 20-50% of people who contract the disease. New developments raise hopes for effective immunological interventions to prevent or treat HCV infection.
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Affiliation(s)
- Lynn B Dustin
- University of Oxford, Nuffield Department of Orthopaedics, Rheumatology, and Musculoskeletal Sciences, Kennedy Institute of Rheumatology, Peter Medawar Building for Pathogen Research, South Parks Road, Oxford OX1 3SY, United Kingdom
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Hopcraft SE, Damania B. Tumour viruses and innate immunity. Philos Trans R Soc Lond B Biol Sci 2018; 372:rstb.2016.0267. [PMID: 28893934 DOI: 10.1098/rstb.2016.0267] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/12/2017] [Indexed: 12/13/2022] Open
Abstract
Host cells sense viral infection through pattern recognition receptors (PRRs), which detect pathogen-associated molecular patterns (PAMPs) and stimulate an innate immune response. PRRs are localized to several different cellular compartments and are stimulated by viral proteins and nucleic acids. PRR activation initiates signal transduction events that ultimately result in an inflammatory response. Human tumour viruses, which include Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus, human papillomavirus, hepatitis C virus, hepatitis B virus, human T-cell lymphotropic virus type 1 and Merkel cell polyomavirus, are detected by several different PRRs. These viruses engage in a variety of mechanisms to evade the innate immune response, including downregulating PRRs, inhibiting PRR signalling, and disrupting the activation of transcription factors critical for mediating the inflammatory response, among others. This review will describe tumour virus PAMPs and the PRRs responsible for detecting viral infection, PRR signalling pathways, and the mechanisms by which tumour viruses evade the host innate immune system.This article is part of the themed issue 'Human oncogenic viruses'.
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Affiliation(s)
- Sharon E Hopcraft
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.,Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Blossom Damania
- Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA .,Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
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Guo S, Li H, Ma M, Fu J, Dong Y, Guo P. Size, Shape, and Sequence-Dependent Immunogenicity of RNA Nanoparticles. MOLECULAR THERAPY. NUCLEIC ACIDS 2017; 9:399-408. [PMID: 29246318 PMCID: PMC5701797 DOI: 10.1016/j.omtn.2017.10.010] [Citation(s) in RCA: 81] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/09/2017] [Revised: 10/14/2017] [Accepted: 10/14/2017] [Indexed: 02/01/2023]
Abstract
RNA molecules have emerged as promising therapeutics. Like all other drugs, the safety profile and immune response are important criteria for drug evaluation. However, the literature on RNA immunogenicity has been controversial. Here, we used the approach of RNA nanotechnology to demonstrate that the immune response of RNA nanoparticles is size, shape, and sequence dependent. RNA triangle, square, pentagon, and tetrahedron with same shape but different sizes, or same size but different shapes were used as models to investigate the immune response. The levels of pro-inflammatory cytokines induced by these RNA nanoarchitectures were assessed in macrophage-like cells and animals. It was found that RNA polygons without extension at the vertexes were immune inert. However, when single-stranded RNA with a specific sequence was extended from the vertexes of RNA polygons, strong immune responses were detected. These immunostimulations are sequence specific, because some other extended sequences induced little or no immune response. Additionally, larger-size RNA square induced stronger cytokine secretion. 3D RNA tetrahedron showed stronger immunostimulation than planar RNA triangle. These results suggest that the immunogenicity of RNA nanoparticles is tunable to produce either a minimal immune response that can serve as safe therapeutic vectors, or a strong immune response for cancer immunotherapy or vaccine adjuvants.
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Affiliation(s)
- Sijin Guo
- Center for RNA Nanobiotechnology and Nanomedicine, The Ohio State University, Columbus, OH 43210, USA; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University, Columbus, OH 43210, USA
| | - Hui Li
- Center for RNA Nanobiotechnology and Nanomedicine, The Ohio State University, Columbus, OH 43210, USA; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University, Columbus, OH 43210, USA
| | - Mengshi Ma
- Center for Research on Environmental Disease, College of Medicine, Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, KY 40506, USA
| | - Jian Fu
- Center for Research on Environmental Disease, College of Medicine, Department of Toxicology and Cancer Biology, University of Kentucky, Lexington, KY 40506, USA
| | - Yizhou Dong
- Center for RNA Nanobiotechnology and Nanomedicine, The Ohio State University, Columbus, OH 43210, USA; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University, Columbus, OH 43210, USA; Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA; NCI Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.
| | - Peixuan Guo
- Center for RNA Nanobiotechnology and Nanomedicine, The Ohio State University, Columbus, OH 43210, USA; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry, The Ohio State University, Columbus, OH 43210, USA; College of Medicine, The Ohio State University, Columbus, OH 43210, USA; Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, OH 43210, USA; NCI Comprehensive Cancer Center, The Ohio State University, Columbus, OH 43210, USA.
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Oh S, Kessler JA. Design, Assembly, Production, and Transfection of Synthetic Modified mRNA. Methods 2017; 133:29-43. [PMID: 29080741 DOI: 10.1016/j.ymeth.2017.10.008] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2017] [Revised: 10/11/2017] [Accepted: 10/12/2017] [Indexed: 10/18/2022] Open
Abstract
Proteins are drivers of cell functions and are targets of many therapies. Exogenous protein expression techniques, therefore, have been essential for research and medicine. The most common method for exogenous protein expression relies on DNA-based viral or non-viral vectors. However, DNA-based vectors have the potential to integrate into the host genome and cause permanent mutations. RNA-based vectors solve this shortcoming. In particular, synthetic modified mRNA provides non-viral, integration-free, zero-footprint method for expressing proteins. Modified mRNA can direct cell fate specification and cellular reprogramming faster and more efficiently than other methods. Furthermore, when simultaneously express multiple different proteins, mRNA vectors allow for greater flexibility and control over stoichiometric ratios, dose titrations, and complete silencing of expressions. Additionally, modified mRNAs have been shown to be viable and safe as therapeutic agents for gene therapy and vaccine, providing an alternative approach to address diseases. Despite these advantages, technical challenge, mRNA instability, and host immunogenicity have caused significant barriers to widespread use of this technology. The comprehensive method presented here addresses all of these shortcomings. This stepwise protocol describes every step necessary for the synthesis of modified mRNA from any coding DNA sequence of interest. The meticulously detailed protocol enables the users to make alterations to each component of modified mRNA for even more significant customization, allowing the researchers to apply this technology to a wide range of uses. This non-cytotoxic synthetic modified mRNA can be used for protein expression, regulation of cell reprogramming or differentiation, and drug delivery.
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Affiliation(s)
- Sanders Oh
- Department of Neurology, Northwestern University, Chicago, IL, USA.
| | - John A Kessler
- Department of Neurology, Northwestern University, Chicago, IL, USA
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Granot Y, Peer D. Delivering the right message: Challenges and opportunities in lipid nanoparticles-mediated modified mRNA therapeutics-An innate immune system standpoint. Semin Immunol 2017; 34:68-77. [PMID: 28890238 DOI: 10.1016/j.smim.2017.08.015] [Citation(s) in RCA: 107] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/10/2017] [Revised: 08/29/2017] [Accepted: 08/30/2017] [Indexed: 12/11/2022]
Abstract
mRNA molecules hold tremendous potential as a tool for gene therapy of a wide range of diseases. However, the main hurdle in implementation of mRNA for therapeutics, the systemic delivery of mRNA molecules to target cells, remains a challenge. A feasible solution for this challenge relies in the rapidly evolving field of nucleic acid-loaded nanocarriers and specifically in the established family of lipid-based nanoparticles (LNPs). Herein, we will discuss the main factors, which determine the fate of modified mRNA (mmRNA)-loaded LNPs in-vivo, and will focus on their interactions with the innate immune system as a main consideration in the design of lipid-based mmRNA delivery platforms.
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Affiliation(s)
- Yasmin Granot
- Laboratory of Precision NanoMedicine, Dept. of Cell Research & Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv 69978, Israel; Dept. of Materials Sciences and Engineering, Iby and Aladar Fleischman Faculty of Engineering, Tel Aviv 69978, Israel; Center for Nanoscience and Nanotechnology, Tel Aviv 69978, Israel; Cancer Biology Research Center, Tel Aviv University, Tel Aviv 69978, Israel
| | - Dan Peer
- Laboratory of Precision NanoMedicine, Dept. of Cell Research & Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv 69978, Israel; Dept. of Materials Sciences and Engineering, Iby and Aladar Fleischman Faculty of Engineering, Tel Aviv 69978, Israel; Center for Nanoscience and Nanotechnology, Tel Aviv 69978, Israel; Cancer Biology Research Center, Tel Aviv University, Tel Aviv 69978, Israel.
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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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40
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Hei L, Zhong J. Laboratory of genetics and physiology 2 (LGP2) plays an essential role in hepatitis C virus infection-induced interferon responses. Hepatology 2017; 65:1478-1491. [PMID: 28090671 DOI: 10.1002/hep.29050] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/06/2016] [Revised: 12/19/2016] [Accepted: 01/09/2017] [Indexed: 12/24/2022]
Abstract
UNLABELLED Retinoic acid-inducible gene I (RIG-I)-like receptors are cytosolic pattern recognition receptors (PRRs) that detect non-self-RNA and activate downstream interferon (IFN) signaling. One of the RIG-I-like receptors, laboratory of genetics and physiology 2 (LGP2), was originally thought to be a negative feedback regulator in the RIG-I signaling pathway, but growing evidence indicates that LGP2 is one cofactor of melanoma differentiation-associated protein 5 (MDA5) in MDA5-mediated IFN signaling activation. Our previous work showed that MDA5 was the major PRR to sense hepatitis C virus (HCV) infection in hepatocytes, but the role of LGP2 in HCV infection-induced IFN signaling has not been elucidated. In this study, we reported that LGP2 was a positive regulator of HCV infection-induced IFN signaling. Knockout of LGP2 in hepatocytes significantly diminished IFN production in response to HCV infection, but not to HCV 3'untranslated region RNA transfection. Mechanistic studies showed that LGP2 exerted its function at a step upstream of MDA5 in the IFN signaling. HCV infection promoted the molecular interaction between LGP2 and MDA5, which, in turn, enhanced MDA5/HCV RNA association. Finally, we demonstrated that the ATPase activity of LGP2 was critical for assisting MDA5/HCV RNA interaction and activating IFN signaling during HCV infection. CONCLUSION Our work demonstrated that LGP2 plays an essential role in activating IFN signaling against HCV infection by promoting MDA5 recognition of HCV pathogen-associated molecular patterns. (Hepatology 2017;65:1478-1491).
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Affiliation(s)
- Lei Hei
- Unit of Viral Hepatitis, CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Jin Zhong
- Unit of Viral Hepatitis, CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
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41
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Zhang X, Zhu C, Wang T, Jiang H, Ren Y, Zhang Q, Wu K, Liu F, Liu Y, Wu J. GP73 represses host innate immune response to promote virus replication by facilitating MAVS and TRAF6 degradation. PLoS Pathog 2017; 13:e1006321. [PMID: 28394926 PMCID: PMC5398727 DOI: 10.1371/journal.ppat.1006321] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2016] [Revised: 04/20/2017] [Accepted: 03/28/2017] [Indexed: 12/31/2022] Open
Abstract
Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases and hepatocellular carcinoma (HCC) and Golgi protein 73 (GP73) is a serum biomarker for liver diseases and HCC. However, the mechanism underlying GP73 regulates HCV infection is largely unknown. Here, we revealed that GP73 acts as a novel negative regulator of host innate immunity to facilitate HCV infection. GP73 expression is activated and correlated with interferon-beta (IFN-β) production during HCV infection in patients’ serum, primary human hepatocytes (PHHs) and human hepatoma cells through mitochondrial antiviral signaling protein (MAVS), TNF receptor-associated factor 6 (TRAF6) and mitogen-activated protein kinase kinase/extracellular regulated protein kinase (MEK/ERK) pathway. Detailed studies revealed that HCV infection activates MAVS that in turn recruits TRAF6 via TRAF-interacting-motifs (TIMs), and TRAF6 subsequently directly recruits GP73 to MAVS via coiled-coil domain. After binding with MAVS and TRAF6, GP73 promotes MAVS and TRAF6 degradation through proteasome-dependent pathway. Moreover, GP73 attenuates IFN-β promoter, IFN-stimulated response element (ISRE) and nuclear factor κB (NF-κB) promoter and down-regulates IFN-β, IFN-λ1, interleukin-6 (IL-6) and IFN-stimulated gene 56 (ISG56), leading to the repression of host innate immunity. Finally, knock-down of GP73 down-regulates HCV infection and replication in Huh7-MAVSR cells and primary human hepatocytes (PHHs), but such repression is rescued by GP73m4 (a mutant GP73 resists to GP73-shRNA#4) in Huh7-MAVSR cells, suggesting that GP73 facilitates HCV infection. Taken together, we demonstrated that GP73 acts as a negative regulator of innate immunity to facilitate HCV infection by interacting with MAVS/TRAF6 and promoting MAVS/TRAF6 degradation. This study provides new insights into the mechanism of HCV infection and pathogenesis, and suggests that GP73 is a new potential antiviral target in the prevention and treatment of HCV associated diseases. Golgi protein 73 (GP73) is a serum biomarker for liver diseases and hepatocellular carcinoma (HCC). In this study, the authors reveal that GP73 acts as a novel negative regulator of host innate immunity to facilitate hepatitis C virus (HCV) infection. GP73 expression is activated and correlated with IFN-β production during HCV infection in patients’ serum, primary human hepatocytes (PHHs) and human hepatoma cells through mitochondrial antiviral signaling protein (MAVS), TNF receptor-associated factor 6 (TRAF6) and MEK/ERK pathway. They further demonstrate that during viral infection, MAVS recruits TRAF6 that subsequently directly binds with GP73. After binding with MAVS and TRAF6, GP73 promotes MAVS and TRAF6 degradation. Moreover, GP73 attenuates IFN-β promoter, IFN-stimulated response element (ISRE) and NF-κB promoter and down-regulates IFN-β, IFN-λ1, interleukin-6 (IL-6) and IFN-stimulated gene 56 (ISG56), leading to the repression of host innate immunity and the facilitation of virus infection. These results reveal a novel mechanism by which GP73 acts as a novel negative regulator of host innate immunity to facilitate virus infection and also provide new insights into the therapeutic design of anti-HCV drugs.
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Affiliation(s)
- Xuewu Zhang
- State Key Laboratory of Virology and College of Life Sciences, Wuhan University, Wuhan, P. R. China
| | - Chengliang Zhu
- State Key Laboratory of Virology and College of Life Sciences, Wuhan University, Wuhan, P. R. China
| | - Tianci Wang
- State Key Laboratory of Virology and College of Life Sciences, Wuhan University, Wuhan, P. R. China
| | - Hui Jiang
- State Key Laboratory of Virology and College of Life Sciences, Wuhan University, Wuhan, P. R. China
| | - Yahui Ren
- State Key Laboratory of Virology and College of Life Sciences, Wuhan University, Wuhan, P. R. China
| | - Qi Zhang
- State Key Laboratory of Virology and College of Life Sciences, Wuhan University, Wuhan, P. R. China
| | - Kailang Wu
- State Key Laboratory of Virology and College of Life Sciences, Wuhan University, Wuhan, P. R. China
| | - Fang Liu
- State Key Laboratory of Virology and College of Life Sciences, Wuhan University, Wuhan, P. R. China
- * E-mail: (JW); (YL); (FL)
| | - Yingle Liu
- State Key Laboratory of Virology and College of Life Sciences, Wuhan University, Wuhan, P. R. China
- * E-mail: (JW); (YL); (FL)
| | - Jianguo Wu
- State Key Laboratory of Virology and College of Life Sciences, Wuhan University, Wuhan, P. R. China
- * E-mail: (JW); (YL); (FL)
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Differential Induction of Immunogenic Cell Death and Interferon Expression in Cancer Cells by Structured ssRNAs. Mol Ther 2017; 25:1295-1305. [PMID: 28372998 DOI: 10.1016/j.ymthe.2017.03.014] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2016] [Revised: 03/06/2017] [Accepted: 03/07/2017] [Indexed: 12/24/2022] Open
Abstract
Activation of the RNA-sensing pattern recognition receptor (PRR) in cancer cells leads to cell death and cytokine expression. This cancer cell death releases tumor antigens and damage-associated molecular patterns (DAMPs) that induce anti-tumor immunity. However, these cytokines and DAMPs also cause adverse inflammatory and thrombotic complications that can limit the overall therapeutic benefits of PRR-targeting anti-cancer therapies. To overcome this problem, we generated and evaluated two novel and distinct ssRNA molecules (immunogenic cell-killing RNA [ICR]2 and ICR4). ICR2 and ICR4 differentially stimulated cell death and PRR signaling pathways and induced different patterns of cytokine expression in cancer and innate immune cells. Interestingly, DAMPs released from ICR2- and ICR4-treated cancer cells had distinct patterns of stimulation of innate immune receptors and coagulation. Finally, ICR2 and ICR4 inhibited in vivo tumor growth as effectively as poly(I:C). ICR2 and ICR4 are potential therapeutic agents that differentially induce cell death, immune stimulation, and coagulation when introduced into tumors.
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Ireton RC, Wilkins C, Gale M. RNA PAMPs as Molecular Tools for Evaluating RIG-I Function in Innate Immunity. Methods Mol Biol 2017; 1656:119-129. [PMID: 28808965 DOI: 10.1007/978-1-4939-7237-1_6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Pathogen recognition receptors (PRR)s and their cognate pathogen-associated molecular pattern (PAMP) represent the basis of innate immune activation and immune response induction driven by the host-pathogen interaction that occurs during microbial infection in humans and other animals. For RNA virus infection such as hepatitis C virus (HCV) and others, specific motifs within viral RNA mark it as nonself and visible to the host as a PAMP through interaction with RIG-I-like receptors including retinoic inducible gene-I (RIG-I). Here, we present methods for producing and using HCV PAMP RNA as a molecular tool to study RIG-I and its signaling pathway, both in vitro and in vivo, in innate immune regulation.
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Affiliation(s)
- Renee C Ireton
- Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington School of Medicine, E383, 750 Republican Street, Seattle, WA, 98109, USA
| | - Courtney Wilkins
- Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington School of Medicine, E383, 750 Republican Street, Seattle, WA, 98109, USA
| | - Michael Gale
- Department of Immunology, Center for Innate Immunity and Immune Disease, University of Washington School of Medicine, E383, 750 Republican Street, Seattle, WA, 98109, USA.
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Vabret N, Bhardwaj N, Greenbaum BD. Sequence-Specific Sensing of Nucleic Acids. Trends Immunol 2016; 38:53-65. [PMID: 27856145 DOI: 10.1016/j.it.2016.10.006] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2016] [Revised: 10/14/2016] [Accepted: 10/14/2016] [Indexed: 12/25/2022]
Abstract
Innate immune cells are endowed with many nucleic acid receptors, but the role of sequence in the detection of foreign organisms remains unclear. Can sequence patterns influence recognition? In addition, how can we infer those patterns from sequence data? Here, we detail recent computational and experimental evidence associated with sequence-specific sensing. We review the mechanisms underlying the detection and discrimination of foreign sequences from self. We also describe quantitative approaches used to infer the stimulatory capacity of a given pathogen nucleic acid species, and the influence of sequence-specific sensing on host-pathogen coevolution, including endogenous sequences of foreign origin. Finally, we speculate how further studies of sequence-specific sensing will be useful to improve vaccine design, gene therapy and cancer treatment.
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Affiliation(s)
- Nicolas Vabret
- Tisch Cancer Institute, Departments of Medicine, Hematology, and Medical Oncology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Departments of Oncological Sciences and Pathology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
| | - Nina Bhardwaj
- Tisch Cancer Institute, Departments of Medicine, Hematology, and Medical Oncology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
| | - Benjamin D Greenbaum
- Tisch Cancer Institute, Departments of Medicine, Hematology, and Medical Oncology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Departments of Oncological Sciences and Pathology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
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45
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Pugh C, Kolaczkowski O, Manny A, Korithoski B, Kolaczkowski B. Resurrecting ancestral structural dynamics of an antiviral immune receptor: adaptive binding pocket reorganization repeatedly shifts RNA preference. BMC Evol Biol 2016; 16:241. [PMID: 27825296 PMCID: PMC5101713 DOI: 10.1186/s12862-016-0818-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2016] [Accepted: 10/28/2016] [Indexed: 02/07/2023] Open
Abstract
Background Although resurrecting ancestral proteins is a powerful tool for understanding the molecular-functional evolution of gene families, nearly all studies have examined proteins functioning in relatively stable biological processes. The extent to which more dynamic systems obey the same ‘rules’ governing stable processes is unclear. Here we present the first detailed investigation of the functional evolution of the RIG-like receptors (RLRs), a family of innate immune receptors that detect viral RNA in the cytoplasm. Results Using kinetic binding assays and molecular dynamics simulations of ancestral proteins, we demonstrate how a small number of adaptive protein-coding changes repeatedly shifted the RNA preference of RLRs throughout animal evolution by reorganizing the shape and electrostatic distribution across the RNA binding pocket, altering the hydrogen bond network between the RLR and its RNA target. In contrast to observations of proteins involved in metabolism and development, we find that RLR-RNA preference ‘flip flopped’ between two functional states, and shifts in RNA preference were not always coupled to gene duplications or speciation events. We demonstrate at least one reversion of RLR-RNA preference from a derived to an ancestral function through a novel structural mechanism, indicating multiple structural implementations of similar functions. Conclusions Our results suggest a model in which frequent shifts in selection pressures imposed by an evolutionary arms race preclude the long-term functional optimization observed in stable biological systems. As a result, the evolutionary dynamics of immune receptors may be less constrained by structural epistasis and historical contingency. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0818-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Charles Pugh
- Department of Microbiology & Cell Science and Institute for Food and Agricultural Sciences, University of Florida, Gainesville, USA
| | - Oralia Kolaczkowski
- Department of Microbiology & Cell Science and Institute for Food and Agricultural Sciences, University of Florida, Gainesville, USA
| | - Austin Manny
- Department of Microbiology & Cell Science and Institute for Food and Agricultural Sciences, University of Florida, Gainesville, USA
| | - Bryan Korithoski
- Department of Microbiology & Cell Science and Institute for Food and Agricultural Sciences, University of Florida, Gainesville, USA
| | - Bryan Kolaczkowski
- Department of Microbiology & Cell Science and Institute for Food and Agricultural Sciences, University of Florida, Gainesville, USA. .,Genetics Institute, University of Florida, Gainesville, USA.
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RNAs Containing Modified Nucleotides Fail To Trigger RIG-I Conformational Changes for Innate Immune Signaling. mBio 2016; 7:mBio.00833-16. [PMID: 27651356 PMCID: PMC5030355 DOI: 10.1128/mbio.00833-16] [Citation(s) in RCA: 177] [Impact Index Per Article: 19.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Invading pathogen nucleic acids are recognized and bound by cytoplasmic (retinoic acid-inducible gene I [RIG-I]-like) and membrane-bound (Toll-like) pattern recognition receptors to activate innate immune signaling. Modified nucleotides, when present in RNA molecules, diminish the magnitude of these signaling responses. However, mechanisms explaining the blunted signaling have not been elucidated. In this study, we used several independent biological assays, including inhibition of virus replication, RIG-I:RNA binding assays, and limited trypsin digestion of RIG-I:RNA complexes, to begin to understand how RNAs containing modified nucleotides avoid or suppress innate immune signaling. The experiments were based on a model innate immune activating RNA molecule, the polyU/UC RNA domain of hepatitis C virus, which was transcribed in vitro with canonical nucleotides or with one of eight modified nucleotides. The approach revealed signature assay responses associated with individual modified nucleotides or classes of modified nucleotides. For example, while both N-6-methyladenosine (m6A) and pseudouridine nucleotides correlate with diminished signaling, RNA containing m6A modifications bound RIG-I poorly, while RNA containing pseudouridine bound RIG-I with high affinity but failed to trigger the canonical RIG-I conformational changes associated with robust signaling. These data advance understanding of RNA-mediated innate immune signaling, with additional relevance for applying nucleotide modifications to RNA therapeutics. The innate immune system provides the first response to virus infections and must distinguish between host and pathogen nucleic acids to mount a protective immune response without activating autoimmune responses. While the presence of nucleotide modifications in RNA is known to correlate with diminished innate immune signaling, the underlying mechanisms have not been explored. The data reported here are important for defining mechanistic details to explain signaling suppression by RNAs containing modified nucleotides. The results suggest that RNAs containing modified nucleotides interrupt signaling at early steps of the RIG-I-like innate immune activation pathway and also that nucleotide modifications with similar chemical structures can be organized into classes that suppress or evade innate immune signaling steps. These data contribute to defining the molecular basis for innate immune signaling suppression by RNAs containing modified nucleotides. The results have important implications for designing therapeutic RNAs that evade innate immune detection.
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Zhang Y, El-Far M, Dupuy FP, Abdel-Hakeem MS, He Z, Procopio FA, Shi Y, Haddad EK, Ancuta P, Sekaly RP, Said EA. HCV RNA Activates APCs via TLR7/TLR8 While Virus Selectively Stimulates Macrophages Without Inducing Antiviral Responses. Sci Rep 2016; 6:29447. [PMID: 27385120 PMCID: PMC4935957 DOI: 10.1038/srep29447] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2016] [Accepted: 06/20/2016] [Indexed: 02/08/2023] Open
Abstract
The innate and adaptive immune systems fail to control HCV infection in the majority of infected individuals. HCV is an ssRNA virus, which suggests a role for Toll-like receptors (TLRs) 7 and 8 in initiating the anti-viral response. Here we demonstrate that HCV genomic RNA harbours specific sequences that initiate an anti-HCV immune response through TLR7 and TLR8 in various antigen presenting cells. Conversely, HCV particles are detected by macrophages, but not by monocytes and DCs, through a TLR7/8 dependent mechanism; this leads to chloroquine sensitive production of pro-inflammatory cytokines including IL-1β, while the antiviral type I Interferon response is not triggered in these cells. Antibodies to DC-SIGN, a c-type lectin selectively expressed by macrophages but not pDCs or mDCs, block the production of cytokines. Novel anti-HCV vaccination strategies should target the induction of TLR7/8 stimulation in APCs in order to establish potent immune responses against HCV.
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Affiliation(s)
- Yuwei Zhang
- Centre de recherche du centre Hospitalier de l'Université de Montréal (CRCHUM), Hôpital Saint-Luc, Québec H2X 0A9, Canada.,Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada.,Vaccine and Gene Therapy Institute-Florida (VGTI-FL), Port Saint Lucie, Florida 3498, USA
| | - Mohamed El-Far
- Centre de recherche du centre Hospitalier de l'Université de Montréal (CRCHUM), Hôpital Saint-Luc, Québec H2X 0A9, Canada.,Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada
| | - Franck P Dupuy
- Centre de recherche du centre Hospitalier de l'Université de Montréal (CRCHUM), Hôpital Saint-Luc, Québec H2X 0A9, Canada.,Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada.,Vaccine and Gene Therapy Institute-Florida (VGTI-FL), Port Saint Lucie, Florida 3498, USA.,Research Institute of the McGill University Health Centre, Montreal, Quebec, Canada
| | - Mohamed S Abdel-Hakeem
- Centre de recherche du centre Hospitalier de l'Université de Montréal (CRCHUM), Hôpital Saint-Luc, Québec H2X 0A9, Canada.,Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Kasr El-Aini, Cairo 11562, Egypt
| | - Zhong He
- Centre de recherche du centre Hospitalier de l'Université de Montréal (CRCHUM), Hôpital Saint-Luc, Québec H2X 0A9, Canada.,Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada.,Vaccine and Gene Therapy Institute-Florida (VGTI-FL), Port Saint Lucie, Florida 3498, USA
| | - Francesco Andrea Procopio
- Centre de recherche du centre Hospitalier de l'Université de Montréal (CRCHUM), Hôpital Saint-Luc, Québec H2X 0A9, Canada.,Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada.,Vaccine and Gene Therapy Institute-Florida (VGTI-FL), Port Saint Lucie, Florida 3498, USA
| | - Yu Shi
- Centre de recherche du centre Hospitalier de l'Université de Montréal (CRCHUM), Hôpital Saint-Luc, Québec H2X 0A9, Canada.,Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada.,Vaccine and Gene Therapy Institute-Florida (VGTI-FL), Port Saint Lucie, Florida 3498, USA
| | - Elias K Haddad
- Centre de recherche du centre Hospitalier de l'Université de Montréal (CRCHUM), Hôpital Saint-Luc, Québec H2X 0A9, Canada.,Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada.,Vaccine and Gene Therapy Institute-Florida (VGTI-FL), Port Saint Lucie, Florida 3498, USA
| | - Petronela Ancuta
- Centre de recherche du centre Hospitalier de l'Université de Montréal (CRCHUM), Hôpital Saint-Luc, Québec H2X 0A9, Canada.,Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada
| | - Rafick-Pierre Sekaly
- Centre de recherche du centre Hospitalier de l'Université de Montréal (CRCHUM), Hôpital Saint-Luc, Québec H2X 0A9, Canada.,Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada.,Vaccine and Gene Therapy Institute-Florida (VGTI-FL), Port Saint Lucie, Florida 3498, USA.,Case Western Reserve University, Cleveland, Ohio, USA
| | - Elias A Said
- Centre de recherche du centre Hospitalier de l'Université de Montréal (CRCHUM), Hôpital Saint-Luc, Québec H2X 0A9, Canada.,Département de Microbiologie, Infectiologie et Immunologie, Faculté de Médecine, Université de Montréal, Montréal, Québec H3T 1J4, Canada.,Department of Microbiology and Immunology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, the Sultanate of Oman
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48
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Innate immunity against hepatitis C virus. Curr Opin Immunol 2016; 42:98-104. [PMID: 27366996 DOI: 10.1016/j.coi.2016.06.009] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2016] [Revised: 06/09/2016] [Accepted: 06/15/2016] [Indexed: 12/24/2022]
Abstract
Hepatitis C virus (HCV) infection tends persistent and causes chronic liver diseases, including inflammation, cirrhosis and hepatocellular carcinoma. Innate immune responses triggered by HCV infection, particularly the production of interferons and pro-inflammatory cytokines, shape the early host antiviral defense, and orchestrate subsequent HCV-specific adaptive immunity. Host has evolved multifaceted means to sense HCV infection to induce innate immune responses, whereas HCV has also developed elaborate strategies to evade immune attack. Recent studies in the field have provided many new insights into the interplay of HCV and innate immunity. In this review, we summarized these recent advances, focusing on pathogen recognition by innate sensors, newly discovered anti-HCV innate effectors and new viral strategies to evade innate immunity.
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Abstract
The co-evolution of viruses with their hosts has led to the emergence of viral pathogens that are adept at evading or actively suppressing host immunity. Pattern recognition receptors (PRRs) are key components of antiviral immunity that detect conserved molecular features of viral pathogens and initiate signalling that results in the expression of antiviral genes. In this Review, we discuss the strategies that viruses use to escape immune surveillance by key intracellular sensors of viral RNA or DNA, with a focus on RIG-I-like receptors (RLRs), cyclic GMP-AMP synthase (cGAS) and interferon-γ (IFNγ)-inducible protein 16 (IFI16). Such viral strategies include the sequestration or modification of viral nucleic acids, interference with specific post-translational modifications of PRRs or their adaptor proteins, the degradation or cleavage of PRRs or their adaptors, and the sequestration or relocalization of PRRs. An understanding of viral immune-evasion mechanisms at the molecular level may guide the development of vaccines and antivirals.
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Affiliation(s)
- Ying Kai Chan
- grid.38142.3c000000041936754XDepartment of Microbiology and Immunobiology, Harvard Medical School, Boston, 02115 Massachusetts USA
| | - Michaela U. Gack
- grid.170205.10000 0004 1936 7822Department of Microbiology, The University of Chicago, Chicago, 60637 Illinois USA
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50
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Alagia A, Eritja R. siRNA and RNAi optimization. WILEY INTERDISCIPLINARY REVIEWS-RNA 2016; 7:316-29. [PMID: 26840434 DOI: 10.1002/wrna.1337] [Citation(s) in RCA: 56] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/28/2015] [Revised: 12/17/2015] [Accepted: 12/18/2015] [Indexed: 12/12/2022]
Abstract
The discovery and examination of the posttranscriptional gene regulatory mechanism known as RNA interference (RNAi) contributed to the identification of small interfering RNA (siRNA) and the comprehension of its enormous potential for clinical purposes. Theoretically, the ability of specific target gene downregulation makes the RNAi pathway an appealing solution for several diseases. Despite numerous hurdles resulting from the inherent properties of siRNA molecule and proper delivery to the target tissue, more than 50 RNA-based drugs are currently under clinical testing. In this work, we analyze the recent literature in the optimization of siRNA molecules. In detail, we focused on describing the most recent advances of siRNA field aimed at optimize siRNA pharmacokinetic properties. Special attention has been given in describing the impact of RNA modifications in the potential off-target effects (OTEs) such as saturation of the RNAi machinery, passenger strand-mediated silencing, immunostimulation, and miRNA-like OTEs as well as to recent developments on the delivery issue. The novel delivery systems and modified siRNA provide significant steps toward the development of reliable siRNA molecules for therapeutic use. WIREs RNA 2016, 7:316-329. doi: 10.1002/wrna.1337 For further resources related to this article, please visit the WIREs website.
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Affiliation(s)
- Adele Alagia
- Chemical and Biomolecular Nanotechnology, CIBER-BBN, Institute for Advanced Chemistry of Catalonia, IQAC-CSIC, Barcelona, Spain
| | - Ramon Eritja
- Chemical and Biomolecular Nanotechnology, CIBER-BBN, Institute for Advanced Chemistry of Catalonia, IQAC-CSIC, Barcelona, Spain
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