|
1
|
Wu Y, Liu P, Zhou J, Fu M, Wang C, Xiong N, Ji W, Wang Z, Lin J, Yang Q. Virus-derived siRNA: Coronavirus and influenza virus trigger antiviral RNAi immunity in birds. Nucleic Acids Res 2025; 53:gkaf116. [PMID: 39988316 PMCID: PMC11840554 DOI: 10.1093/nar/gkaf116] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2024] [Revised: 01/30/2025] [Accepted: 02/05/2025] [Indexed: 02/25/2025] Open
Abstract
RNA interference (RNAi) is a key antiviral immune mechanism in eukaryotes. However, antiviral RNAi in vertebrates has only been observed in cells with poor interferon systems or in viral suppressors of RNAi (VSR) deficiency virus infections. Our research discovered that infecting macrophages with wild-type coronavirus (Infectious bronchitis virus, IBV) and influenza viruses (Avian influenza virus, AIV) can trigger RNAi antiviral immunity and produce a certain amount of virus-derived siRNA (vsiRNA). These vsiRNAs have an inhibitory effect on the virus and carry out targeted silencing along the Dicer-Ago2-vsiRNA axis. Notably, these vsiRNAs are distributed throughout the virus's entire genome, with a predilection for A/U at the 5' and 3' termini of vsiRNA. In addition, Dicer cleavage produces vsiRNA based on the RWM motif, where R represents A/G, W represents A/C, and M represents A/U. We also discovered that avian LGP2 and MDA5 proteins positively impact the expression of the Dicer protein and the Dicer subtype "DicerM." Most importantly, the PS-vsiRNA plasmid combined with nanomaterial polyetherimide (PEI) showed excellent anti-virus activity in specific-pathogen-free (SPF) chickens. These findings show that RNA viruses trigger the production of the vsiRNA in avian somatic cells, which is of great significance for the application of therapeutic vaccines.
Collapse
Affiliation(s)
- Yaotang Wu
- College of Veterinary Medicine, Nanjing Agricultural University, Wei gang 1, Nanjing, Jiangsu 210095, PR China
| | - Peng Liu
- College of Veterinary Medicine, Jiangsu Agri-Animal Husbandry Vocational College, Taizhou, Jiangsu 225300, PR China
| | - Jie Zhou
- College of Veterinary Medicine, Nanjing Agricultural University, Wei gang 1, Nanjing, Jiangsu 210095, PR China
| | - Mei Fu
- College of Veterinary Medicine, Nanjing Agricultural University, Wei gang 1, Nanjing, Jiangsu 210095, PR China
| | - Chenlu Wang
- College of Life Sciences, Nanjing Agricultural University, Wei gang 1, Nanjing, Jiangsu 210095, PR China
| | - Ningna Xiong
- College of Veterinary Medicine, Nanjing Agricultural University, Wei gang 1, Nanjing, Jiangsu 210095, PR China
| | - Wenxin Ji
- College of Veterinary Medicine, Nanjing Agricultural University, Wei gang 1, Nanjing, Jiangsu 210095, PR China
| | - Zhisheng Wang
- Institute of Veterinary Immunology and Engineering, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, PR China
| | - Jian Lin
- College of Veterinary Medicine, Nanjing Agricultural University, Wei gang 1, Nanjing, Jiangsu 210095, PR China
| | - Qian Yang
- College of Veterinary Medicine, Nanjing Agricultural University, Wei gang 1, Nanjing, Jiangsu 210095, PR China
| |
Collapse
|
|
2
|
Chen J, Mu J, Zhou K, Zhang Y, Zhang J, Shu T, Shang W, Ren Y, Xu XQ, Zhang L, Yuan S, Zhang D, Cai K, Qiu Y, Zhou X. Targeting viral suppressor of RNAi confers anti-coronaviral activity. Mol Ther 2025; 33:201-214. [PMID: 39663700 PMCID: PMC11764073 DOI: 10.1016/j.ymthe.2024.12.009] [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: 09/03/2024] [Revised: 11/02/2024] [Accepted: 12/06/2024] [Indexed: 12/13/2024] Open
Abstract
Infections caused by coronaviruses are persistent threats to human health in recent decades, necessitating the development of innovative anti-coronaviral therapies. RNA interference (RNAi) is a conserved cell-intrinsic antiviral mechanism in diverse eukaryotic organisms, including mammals. To counteract, many viruses encode viral suppressors of RNAi (VSRs) to evade antiviral RNAi, implying that targeting VSRs could be a promising strategy to develop antiviral therapies. Here, we designed a series of peptides specifically targeting the SARS-CoV-2-encoded VSR, nucleocapsid (N) protein. Among these peptides, one designated GL directly interacts with N protein and inactivates its VSR activity, which unlocks a potent RNAi response and effectively inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Moreover, GL exhibited RNAi-dependent antiviral effects not only against various SARS-CoV-2 variants, including Delta, Omicron BA.5, XBB, and JN.1, but also against other coronaviruses such as human coronavirus (HCoV)-229E, HCoV-OC43, and mouse hepatitis virus. The in vivo anti-coronaviral activity of GL was also confirmed. Our findings indicate that the VSR-targeting peptide GL has the potential to be further developed as a broad-spectrum anti-coronaviral treatment, highlighting the functional importance and therapeutic potential of antiviral RNAi.
Collapse
Affiliation(s)
- Jiyao Chen
- Joint Laboratory of Infectious Diseases and Health, Wuhan Institute of Virology & Wuhan Jinyintan Hospital, Wuhan Jinyintan Hospital, Wuhan, Hubei 430023, China; State Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
| | - JingFang Mu
- State Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
| | - Kangping Zhou
- Institute of Health Inspection and Testing, Hubei Provincial Center for Disease Control and Prevention, Wuhan, China
| | - Yuming Zhang
- State Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
| | - Jieling Zhang
- State Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, China
| | - Ting Shu
- State Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
| | - Weijuan Shang
- State Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
| | - Yujie Ren
- State Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
| | - Xi-Qiu Xu
- State Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
| | - Leike Zhang
- State Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
| | - Shuai Yuan
- State Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, China
| | - Dingyu Zhang
- Union Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, Hubei 430023, China.
| | - Kun Cai
- Institute of Health Inspection and Testing, Hubei Provincial Center for Disease Control and Prevention, Wuhan, China.
| | - Yang Qiu
- State Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
| | - Xi Zhou
- State Key Laboratory of Virology and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
| |
Collapse
|
|
3
|
Kulmann MI, Taborska E, Benköova B, Palus M, Drobek A, Horvat F, Pasulka J, Malik R, Salyova E, Hönig V, Pellerova M, Borsanyiova M, Nedvedova L, Stepanek O, Bopegamage S, Ruzek D, Svoboda P. Enhanced RNAi does not provide efficient innate antiviral immunity in mice. Nucleic Acids Res 2025; 53:gkae1288. [PMID: 39778869 PMCID: PMC11707545 DOI: 10.1093/nar/gkae1288] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2024] [Revised: 12/08/2024] [Accepted: 12/17/2024] [Indexed: 01/11/2025] Open
Abstract
In RNA interference (RNAi), long double-stranded RNA is cleaved by the Dicer endonuclease into small interfering RNAs (siRNAs), which guide degradation of complementary RNAs. While RNAi mediates antiviral innate immunity in plants and many invertebrates, vertebrates have adopted a sequence-independent response and their Dicer produces siRNAs inefficiently because it is adapted to process small hairpin microRNA precursors in the gene-regulating microRNA pathway. Mammalian endogenous RNAi is thus a rudimentary pathway of unclear significance. To investigate its antiviral potential, we modified the mouse Dicer locus to express a truncated variant (DicerΔHEL1) known to stimulate RNAi and we analyzed how DicerΔHEL1/wt mice respond to four RNA viruses: coxsackievirus B3 and encephalomyocarditis virus from Picornaviridae; tick-borne encephalitis virus from Flaviviridae; and lymphocytic choriomeningitis virus (LCMV) from Arenaviridae. Increased Dicer activity in DicerΔHEL1/wt mice did not elicit any antiviral effect, supporting an insignificant antiviral function of endogenous mammalian RNAi in vivo. However, we also observed that sufficiently high expression of DicerΔHEL1 suppressed LCMV in embryonic stem cells and in a transgenic mouse model. Altogether, mice with increased Dicer activity offer a new benchmark for identifying and studying viruses susceptible to mammalian RNAi in vivo.
Collapse
Affiliation(s)
- Marcos Iuri Roos Kulmann
- Laboratory of Epigenetic Regulations, Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 142 20, Prague, Czech Republic
| | - Eliska Taborska
- Laboratory of Epigenetic Regulations, Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 142 20, Prague, Czech Republic
| | - Brigita Benköova
- Faculty of Medicine, Enterovirus Laboratory, Institute of Microbiology, Slovak Medical University, Limbova 12, 83303Bratislava, Slovakia
| | - Martin Palus
- Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Branisovska 31, CZ-37005 Ceske Budejovice, Czech Republic
- Laboratory of Emerging Viral Infections, Veterinary Research Institute, Hudcova 70, CZ-62100 Brno, Czech Republic
| | - Ales Drobek
- Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 142 20, Prague, Czech Republic
| | - Filip Horvat
- Laboratory of Epigenetic Regulations, Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 142 20, Prague, Czech Republic
- Bioinformatics Group, Department of Biology, Faculty of Science, University of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia
| | - Josef Pasulka
- Laboratory of Epigenetic Regulations, Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 142 20, Prague, Czech Republic
| | - Radek Malik
- Laboratory of Epigenetic Regulations, Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 142 20, Prague, Czech Republic
| | - Eva Salyova
- Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 142 20, Prague, Czech Republic
| | - Vaclav Hönig
- Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Branisovska 31, CZ-37005 Ceske Budejovice, Czech Republic
- Laboratory of Emerging Viral Infections, Veterinary Research Institute, Hudcova 70, CZ-62100 Brno, Czech Republic
| | - Michaela Pellerova
- Faculty of Medicine, Enterovirus Laboratory, Institute of Microbiology, Slovak Medical University, Limbova 12, 83303Bratislava, Slovakia
| | - Maria Borsanyiova
- Faculty of Medicine, Enterovirus Laboratory, Institute of Microbiology, Slovak Medical University, Limbova 12, 83303Bratislava, Slovakia
| | - Lenka Nedvedova
- Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Branisovska 31, CZ-37005 Ceske Budejovice, Czech Republic
- Faculty of Science, University of South Bohemia, Branisovska 1645/31a, CZ-37005Ceske Budejovice, Czech Republic
| | - Ondrej Stepanek
- Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 142 20, Prague, Czech Republic
| | - Shubhada Bopegamage
- Faculty of Medicine, Enterovirus Laboratory, Institute of Microbiology, Slovak Medical University, Limbova 12, 83303Bratislava, Slovakia
| | - Daniel Ruzek
- Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Branisovska 31, CZ-37005 Ceske Budejovice, Czech Republic
- Laboratory of Emerging Viral Infections, Veterinary Research Institute, Hudcova 70, CZ-62100 Brno, Czech Republic
- Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, CZ-62500Brno, Czech Republic
| | - Petr Svoboda
- Laboratory of Epigenetic Regulations, Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 142 20, Prague, Czech Republic
| |
Collapse
|
|
4
|
Feng X, Guang S. Functions and applications of RNA interference and small regulatory RNAs. Acta Biochim Biophys Sin (Shanghai) 2024; 57:119-130. [PMID: 39578714 PMCID: PMC11802346 DOI: 10.3724/abbs.2024196] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2024] [Accepted: 09/03/2024] [Indexed: 11/24/2024] Open
Abstract
Small regulatory RNAs play a variety of crucial roles in eukaryotes, influencing gene regulation, developmental timing, antiviral defense, and genome integrity via a process termed RNA interference (RNAi). This process involves Argonaute/small RNA (AGO/sRNA) complexes that target transcripts via sequence complementarity and modulate gene expression and epigenetic modifications. RNAi is a highly conserved gene regulatory phenomenon that recognizes self- and non-self nucleic acids, thereby defending against invasive sequences. Since its discovery, RNAi has been widely applied in functional genomic studies and a range of practical applications. In this review, we focus on the current understanding of the biological roles of the RNAi pathway in transposon silencing, fertility, developmental regulation, immunity, stress responses, and acquired transgenerational inheritance. Additionally, we provide an overview of the applications of RNAi technology in biomedical research, agriculture, and therapeutics.
Collapse
Affiliation(s)
- Xuezhu Feng
- School of Basic Medical SciencesAnhui Medical UniversityHefei230032China
| | - Shouhong Guang
- Department of Obstetrics and Gynecologythe First Affiliated Hospital of USTCThe USTC RNA InstituteMinistry of Education Key Laboratory for Membraneless Organelles & Cellular DynamicsHefei National Research Center for Physical Sciences at the MicroscaleCenter for Advanced Interdisciplinary Science and Biomedicine of IHMSchool of Life SciencesDivision of Life Sciences and MedicineBiomedical Sciences and Health Laboratory of Anhui ProvinceUniversity of Science and Technology of ChinaHefei230027China
| |
Collapse
|
|
5
|
Gaucherand L, Baldaccini M, Pfeffer S. Beyond RNAi: How the Dicer protein modulates the antiviral innate immune response in mammalian cells: Mammalian Dicer could regulate the innate immune response in an RNAi-independent manner as a result of losing long dsRNA processive activity. Bioessays 2024; 46:e2400173. [PMID: 39248656 DOI: 10.1002/bies.202400173] [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: 07/11/2024] [Revised: 08/20/2024] [Accepted: 08/23/2024] [Indexed: 09/10/2024]
Abstract
While Dicer plays an important antiviral role through the RNAi pathway in plants and invertebrates, its contribution to antiviral immunity in vertebrates and more specifically mammals is more controversial. The apparent limited RNAi activity in mammalian cells has been attributed to the reduced long dsRNA processive activity of mammalian Dicer, as well as a functional incompatibility between the RNAi and IFN pathways. Why Dicer has lost this antiviral activity in the profit of the IFN pathway is still unclear. We propose that the primary direct antiviral activity of Dicer has been functionally replaced by other sensors in the IFN pathway, leading to its specialization toward microRNA maturation. As a result, Dicer can regulate the innate immune response and prevent basal activation of the IFN pathway in mammals. Here, we discuss this hypothesis, highlighting how the adaptation of the helicase domain of mammalian Dicer may be key to this process.
Collapse
Affiliation(s)
- Léa Gaucherand
- Université de Strasbourg, Architecture et Réactivité de l'ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France
| | | | - Sébastien Pfeffer
- Université de Strasbourg, Architecture et Réactivité de l'ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France
| |
Collapse
|
|
6
|
Chen G, Han Q, Li WX, Hai R, Ding SW. Live-attenuated virus vaccine defective in RNAi suppression induces rapid protection in neonatal and adult mice lacking mature B and T cells. Proc Natl Acad Sci U S A 2024; 121:e2321170121. [PMID: 38630724 PMCID: PMC11046691 DOI: 10.1073/pnas.2321170121] [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: 12/01/2023] [Accepted: 03/15/2024] [Indexed: 04/19/2024] Open
Abstract
Global control of infectious diseases depends on the continuous development and deployment of diverse vaccination strategies. Currently available live-attenuated and killed virus vaccines typically take a week or longer to activate specific protection by the adaptive immunity. The mosquito-transmitted Nodamura virus (NoV) is attenuated in mice by mutations that prevent expression of the B2 viral suppressor of RNA interference (VSR) and consequently, drastically enhance in vivo production of the virus-targeting small-interfering RNAs. We reported recently that 2 d after immunization with live-attenuated VSR-disabled NoV (NoVΔB2), neonatal mice become fully protected against lethal NoV challenge and develop no detectable infection. Using Rag1-/- mice that produce no mature B and T lymphocytes as a model, here we examined the hypothesis that adaptive immunity is dispensable for the RNAi-based protective immunity activated by NoVΔB2 immunization. We show that immunization of both neonatal and adult Rag1-/- mice with live but not killed NoVΔB2 induces full protection against NoV challenge at 2 or 14 d postimmunization. Moreover, NoVΔB2-induced protective antiviral immunity is virus-specific and remains effective in adult Rag1-/- mice 42 and 90 d after a single-shot immunization. We conclude that immunization with the live-attenuated VSR-disabled RNA virus vaccine activates rapid and long-lasting protective immunity against lethal challenges by a distinct mechanism independent of the adaptive immunity mediated by B and T cells. Future studies are warranted to determine whether additional animal and human viruses attenuated by VSR inactivation induce similar protective immunity in healthy and adaptive immunity-compromised individuals.
Collapse
Affiliation(s)
- Gang Chen
- Department of Microbiology and Plant Pathology, University of California, Riverside, CA92521
| | - Qingxia Han
- Department of Microbiology and Plant Pathology, University of California, Riverside, CA92521
| | - Wan-Xiang Li
- Department of Microbiology and Plant Pathology, University of California, Riverside, CA92521
| | - Rong Hai
- Department of Microbiology and Plant Pathology, University of California, Riverside, CA92521
| | - Shou-Wei Ding
- Department of Microbiology and Plant Pathology, University of California, Riverside, CA92521
| |
Collapse
|
|
7
|
Baldaccini M, Gaucherand L, Chane-Woon-Ming B, Messmer M, Gucciardi F, Pfeffer S. The helicase domain of human Dicer prevents RNAi-independent activation of antiviral and inflammatory pathways. EMBO J 2024; 43:806-835. [PMID: 38287188 PMCID: PMC10907635 DOI: 10.1038/s44318-024-00035-2] [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: 10/04/2023] [Revised: 12/19/2023] [Accepted: 01/08/2024] [Indexed: 01/31/2024] Open
Abstract
In mammalian somatic cells, the relative contribution of RNAi and the type I interferon response during viral infection is unclear. The apparent inefficiency of antiviral RNAi might be due to self-limiting properties and mitigating co-factors of the key enzyme Dicer. In particular, the helicase domain of human Dicer appears to be an important restriction factor of its activity. Here, we study the involvement of several helicase-truncated mutants of human Dicer in the antiviral response. All deletion mutants display a PKR-dependent antiviral phenotype against certain viruses, and one of them, Dicer N1, acts in a completely RNAi-independent manner. Transcriptomic analyses show that many genes from the interferon and inflammatory response pathways are upregulated in Dicer N1 expressing cells. We show that some of these genes are controlled by NF-kB and that blocking this pathway abrogates the antiviral phenotype of Dicer N1. Our findings highlight the crosstalk between Dicer, PKR, and the NF-kB pathway, and suggest that human Dicer may have repurposed its helicase domain to prevent basal activation of antiviral and inflammatory pathways.
Collapse
Affiliation(s)
- Morgane Baldaccini
- Université de Strasbourg, Architecture et Réactivité de l'ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS, 67000, Strasbourg, France
| | - Léa Gaucherand
- Université de Strasbourg, Architecture et Réactivité de l'ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS, 67000, Strasbourg, France
| | - Béatrice Chane-Woon-Ming
- Université de Strasbourg, Architecture et Réactivité de l'ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS, 67000, Strasbourg, France
| | - Mélanie Messmer
- Université de Strasbourg, Architecture et Réactivité de l'ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS, 67000, Strasbourg, France
| | - Floriane Gucciardi
- Université de Strasbourg, Architecture et Réactivité de l'ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS, 67000, Strasbourg, France
| | - Sébastien Pfeffer
- Université de Strasbourg, Architecture et Réactivité de l'ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS, 67000, Strasbourg, France.
| |
Collapse
|
|
8
|
Driedonks TA, Nyberg LH, Conte A, Ma Z, Pekosz A, Duban E, Tonevitsky A, Sültmann H, Turchinovich A, Witwer KW. Viral and host small RNA transcriptome analysis of SARS-CoV-1 and SARS-CoV-2-infected human cells reveals novel viral short RNAs. Heliyon 2024; 10:e24570. [PMID: 38314306 PMCID: PMC10837498 DOI: 10.1016/j.heliyon.2024.e24570] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2023] [Revised: 12/18/2023] [Accepted: 01/10/2024] [Indexed: 02/06/2024] Open
Abstract
RNA viruses have been shown to express various short RNAs, some of which have regulatory roles during replication, transcription, and translation of viral genomes. However, short viral RNAs generated from SARS-CoV-1 and SARS-CoV-2 genomic RNAs remained largely unexplored, possibly due limitations of the widely used library preparation methods for small RNA deep sequencing and corresponding data processing. By analyzing publicly available small RNA sequencing datasets, we observed that human Calu-3 cells infected by SARS-CoV-1 or SARS-CoV-2 accumulate multiple previously unreported short viral RNAs. In addition, we verified the presence of the five most abundant SARS-CoV-2 short viral RNAs in SARS-CoV-2-infected human lung adenocarcinoma cells by quantitative PCR. Interestingly, the copy number of the observed SARS-CoV-2 short viral RNAs dramatically exceeded the expression of previously reported viral microRNAs in the same cells. We hypothesize that the reported SARS-CoV-2 short viral RNAs could serve as biomarkers for early infection stages due to their high abundance. Furthermore, unlike SARS-CoV-1, the SARS-CoV-2 infection induced significant (Benjamini-Hochberg-corrected p-value <0.05) deregulation of Y-RNA, transfer RNA, vault RNA, as well as more than 300 endogenous short RNAs that aligned predominantly to human protein-coding and long noncoding RNA transcripts. In particular, more than 20-fold upregulation of reads derived from Y-RNA (and several transfer RNAs) have been documented in RNA-seq datasets from SARS-CoV-2 infected cells. Finally, a significant proportion of short RNAs derived from full-length viral genomes also aligned to various human genome (hg38) sequences, suggesting opportunities to investigate regulatory roles of short viral RNAs during infection. Further characterization of the small RNA landscape of both viral and host genomes is clearly warranted to improve our understanding of molecular events related to infection and to design more efficient strategies for therapeutic interventions as well as early diagnosis.
Collapse
Affiliation(s)
- Tom A.P. Driedonks
- Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Lyle H. Nyberg
- Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| | - Abigail Conte
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Zexu Ma
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | - Andrew Pekosz
- W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA
| | | | | | - Holger Sültmann
- Division of Cancer Genome Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany
| | - Andrey Turchinovich
- Division of Cancer Genome Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany
- Heidelberg Biolabs GmbH, Heidelberg, Germany
- Department of Cardiovascular Research, European Center for Angioscience (ECAS), Heidelberg University, Heidelberg/Mannheim, Germany
| | - Kenneth W. Witwer
- Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
| |
Collapse
|
|
9
|
Zhang X, Li Y, Cao Y, Wu Y, Cheng G. The Role of Noncoding RNA in the Transmission and Pathogenicity of Flaviviruses. Viruses 2024; 16:242. [PMID: 38400018 PMCID: PMC10892091 DOI: 10.3390/v16020242] [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: 12/11/2023] [Revised: 01/28/2024] [Accepted: 01/31/2024] [Indexed: 02/25/2024] Open
Abstract
Noncoding RNAs (ncRNAs) constitute a class of RNA molecules that lack protein-coding capacity. ncRNAs frequently modulate gene expression through specific interactions with target proteins or messenger RNAs, thereby playing integral roles in a wide array of cellular processes. The Flavivirus genus comprises several significant members, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV), which have caused global outbreaks, resulting in high morbidity and mortality in human populations. The life cycle of arthropod-borne flaviviruses encompasses their transmission between hematophagous insect vectors and mammalian hosts. During this process, a complex three-way interplay occurs among the pathogen, vector, and host, with ncRNAs exerting a critical regulatory influence. ncRNAs not only constitute a crucial regulatory mechanism that has emerged from the coevolution of viruses and their hosts but also hold potential as antiviral targets for controlling flavivirus epidemics. This review introduces the biogenesis of flavivirus-derived ncRNAs and summarizes the regulatory roles of ncRNAs in viral replication, vector-mediated viral transmission, antiviral innate immunity, and viral pathogenicity. A profound comprehension of the interplay between ncRNAs and flaviviruses will help formulate efficacious prophylactic and therapeutic strategies against flavivirus-related diseases.
Collapse
Affiliation(s)
- Xianwen Zhang
- Shenzhen Bay Laboratory, Institute of Infectious Diseases, Shenzhen 518000, China
| | - Yuhan Li
- New Cornerstone Science Laboratory, Tsinghua University-Peking University Joint Center for Life Sciences, School of Basic Medical Sciences, Tsinghua University, Beijing 100084, China; (Y.L.); (Y.C.)
| | - Yingyi Cao
- New Cornerstone Science Laboratory, Tsinghua University-Peking University Joint Center for Life Sciences, School of Basic Medical Sciences, Tsinghua University, Beijing 100084, China; (Y.L.); (Y.C.)
| | - Ying Wu
- State Key Laboratory of Virology and Hubei Province Key Laboratory of Allergy and Immunology, Institute of Medical Virology, TaiKang Medical School, Wuhan University, Wuhan 430072, China;
| | - Gong Cheng
- New Cornerstone Science Laboratory, Tsinghua University-Peking University Joint Center for Life Sciences, School of Basic Medical Sciences, Tsinghua University, Beijing 100084, China; (Y.L.); (Y.C.)
- Institute of Pathogenic Organisms, Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China
- Southwest United Graduate School, Kunming 650092, China
| |
Collapse
|
|
10
|
Wang J, Li Y. Current advances in antiviral RNA interference in mammals. FEBS J 2024; 291:208-216. [PMID: 36652199 DOI: 10.1111/febs.16728] [Citation(s) in RCA: 11] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2022] [Revised: 11/09/2022] [Accepted: 01/16/2023] [Indexed: 01/19/2023]
Abstract
Mammals have potent innate immune systems that work together to fight against a variety of distinct viruses. In addition to interferon (IFN) response, which has been intensively studied, antiviral RNA interference (RNAi) is gradually being studied. However, previous studies indicated low Dicer activity on double-stranded RNA (dsRNA) substrates in vitro and that IFN response masks or inhibits antiviral RNAi in mammals. Therefore, whether or not the RNAi is functional for antiviral response in mammalian somatic cells is still an ongoing area of research. In this review, we will present the current advances in antiviral RNAi in mammals and focus on three fundamental questions critical to the intense debate about whether RNAi can function as an innate antiviral immunity in mammals.
Collapse
Affiliation(s)
- Jiaxin Wang
- CAS Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Yang Li
- CAS Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
| |
Collapse
|
|
11
|
Tang D, Liu Y, Wang C, Li L, Al-Farraj SA, Chen X, Yan Y. Invasion by exogenous RNA: cellular defense strategies and implications for RNA inference. MARINE LIFE SCIENCE & TECHNOLOGY 2023; 5:573-584. [PMID: 38045546 PMCID: PMC10689678 DOI: 10.1007/s42995-023-00209-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/02/2023] [Accepted: 11/02/2023] [Indexed: 12/05/2023]
Abstract
Exogenous RNA poses a continuous threat to genome stability and integrity across various organisms. Accumulating evidence reveals complex mechanisms underlying the cellular response to exogenous RNA, including endo-lysosomal degradation, RNA-dependent repression and innate immune clearance. Across a variety of mechanisms, the natural anti-sense RNA-dependent defensive strategy has been utilized both as a powerful gene manipulation tool and gene therapy strategy named RNA-interference (RNAi). To optimize the efficiency of RNAi silencing, a comprehensive understanding of the whole life cycle of exogenous RNA, from cellular entry to its decay, is vital. In this paper, we review recent progress in comprehending the recognition and elimination of foreign RNA by cells, focusing on cellular entrance, intracellular transportation, and immune-inflammatory responses. By leveraging these insights, we highlight the potential implications of these insights for advancing RNA interference efficiency, underscore the need for future studies to elucidate the pathways and fates of various exogenous RNA forms, and provide foundational information for more efficient RNA delivery methods in both genetic manipulation and therapy in different organisms.
Collapse
Affiliation(s)
- Danxu Tang
- Laboratory of Marine Protozoan Biodiversity and Evolution, Marine College, Shandong University, Weihai, 264209 China
- Key Laboratory of Evolution and Marine Biodiversity (Ministry of Education) and Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao, 266003 China
| | - Yan Liu
- Key Laboratory of Evolution and Marine Biodiversity (Ministry of Education) and Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao, 266003 China
| | - Chundi Wang
- Laboratory of Marine Protozoan Biodiversity and Evolution, Marine College, Shandong University, Weihai, 264209 China
| | - Lifang Li
- Laboratory of Marine Protozoan Biodiversity and Evolution, Marine College, Shandong University, Weihai, 264209 China
| | - Saleh A. Al-Farraj
- Zoology Department, College of Science, King Saud University, 11451 Riyadh, Saudi Arabia
| | - Xiao Chen
- Laboratory of Marine Protozoan Biodiversity and Evolution, Marine College, Shandong University, Weihai, 264209 China
- Suzhou Research Institute, Shandong University, Suzhou, 215123 China
| | - Ying Yan
- Key Laboratory of Evolution and Marine Biodiversity (Ministry of Education) and Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao, 266003 China
| |
Collapse
|
|
12
|
Wong B, Birtch R, Rezaei R, Jamieson T, Crupi MJF, Diallo JS, Ilkow CS. Optimal delivery of RNA interference by viral vectors for cancer therapy. Mol Ther 2023; 31:3127-3145. [PMID: 37735876 PMCID: PMC10638062 DOI: 10.1016/j.ymthe.2023.09.012] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2023] [Revised: 08/28/2023] [Accepted: 09/14/2023] [Indexed: 09/23/2023] Open
Abstract
In recent years, there has been a surge in the innovative modification and application of the viral vector-based gene therapy field. Significant and consistent improvements in the engineering, delivery, and safety of viral vectors have set the stage for their application as RNA interference (RNAi) delivery tools. Viral vector-based delivery of RNAi has made remarkable breakthroughs in the treatment of several debilitating diseases and disorders (e.g., neurological diseases); however, their novelty has yet to be fully applied and utilized for the treatment of cancer. This review highlights the most promising and emerging viral vector delivery tools for RNAi therapeutics while discussing the variables limiting their success and suitability for cancer therapy. Specifically, we outline different integrating and non-integrating viral platforms used for gene delivery, currently employed RNAi targets for anti-cancer effect, and various strategies used to optimize the safety and efficacy of these RNAi therapeutics. Most importantly, we provide great insight into what challenges exist in their application as cancer therapeutics and how these challenges can be effectively navigated to advance the field.
Collapse
Affiliation(s)
- Boaz Wong
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Rayanna Birtch
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Reza Rezaei
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Taylor Jamieson
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Mathieu J F Crupi
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Jean-Simon Diallo
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada
| | - Carolina S Ilkow
- Centre for Innovative Cancer Research, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada; Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H 8M5, Canada.
| |
Collapse
|
|
13
|
Yoth M, Maupetit-Méhouas S, Akkouche A, Gueguen N, Bertin B, Jensen S, Brasset E. Reactivation of a somatic errantivirus and germline invasion in Drosophila ovaries. Nat Commun 2023; 14:6096. [PMID: 37773253 PMCID: PMC10541861 DOI: 10.1038/s41467-023-41733-5] [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: 12/15/2022] [Accepted: 09/15/2023] [Indexed: 10/01/2023] Open
Abstract
Most Drosophila transposable elements are LTR retrotransposons, some of which belong to the genus Errantivirus and share structural and functional characteristics with vertebrate endogenous retroviruses. Like endogenous retroviruses, it is unclear whether errantiviruses retain some infectivity and transposition capacity. We created conditions where control of the Drosophila ZAM errantivirus through the piRNA pathway was abolished leading to its de novo reactivation in somatic gonadal cells. After reactivation, ZAM invaded the oocytes and severe fertility defects were observed. While ZAM expression persists in the somatic gonadal cells, the germline then set up its own adaptive genomic immune response by producing piRNAs against the constantly invading errantivirus, restricting invasion. Our results suggest that although errantiviruses are continuously repressed by the piRNA pathway, they may retain their ability to infect the germline and transpose, thus allowing them to efficiently invade the germline if they are expressed.
Collapse
Affiliation(s)
- Marianne Yoth
- iGReD, Université Clermont Auvergne, CNRS, INSERM, Faculté de Médecine, 63000, Clermont-Ferrand, France
| | | | - Abdou Akkouche
- iGReD, Université Clermont Auvergne, CNRS, INSERM, Faculté de Médecine, 63000, Clermont-Ferrand, France
| | - Nathalie Gueguen
- iGReD, Université Clermont Auvergne, CNRS, INSERM, Faculté de Médecine, 63000, Clermont-Ferrand, France
| | - Benjamin Bertin
- LIMAGRAIN EUROPE, Centre de recherche, 63720, Chappes, France
| | - Silke Jensen
- iGReD, Université Clermont Auvergne, CNRS, INSERM, Faculté de Médecine, 63000, Clermont-Ferrand, France.
| | - Emilie Brasset
- iGReD, Université Clermont Auvergne, CNRS, INSERM, Faculté de Médecine, 63000, Clermont-Ferrand, France.
| |
Collapse
|
|
14
|
Lopez-Orozco J, Fayad N, Khan JQ, Felix-Lopez A, Elaish M, Rohamare M, Sharma M, Falzarano D, Pelletier J, Wilson J, Hobman TC, Kumar A. The RNA Interference Effector Protein Argonaute 2 Functions as a Restriction Factor Against SARS-CoV-2. J Mol Biol 2023; 435:168170. [PMID: 37271493 PMCID: PMC10238125 DOI: 10.1016/j.jmb.2023.168170] [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: 10/10/2022] [Revised: 05/17/2023] [Accepted: 05/30/2023] [Indexed: 06/06/2023]
Abstract
Argonaute 2 (Ago2) is a key component of the RNA interference (RNAi) pathway, a gene-regulatory system that is present in most eukaryotes. Ago2 uses microRNAs (miRNAs) and small interfering RNAs (siRNAs) for targeting to homologous mRNAs which are then degraded or translationally suppressed. In plants and invertebrates, the RNAi pathway has well-described roles in antiviral defense, but its function in limiting viral infections in mammalian cells is less well understood. Here, we examined the role of Ago2 in replication of the betacoronavirus SARS-CoV-2, the etiologic agent of COVID-19. Microscopic analyses of infected cells revealed that a pool of Ago2 closely associates with viral replication sites and gene ablation studies showed that loss of Ago2 resulted in over 1,000-fold increase in peak viral titers. Replication of the alphacoronavirus 229E was also significantly increased in cells lacking Ago2. The antiviral activity of Ago2 was dependent on both its ability to bind small RNAs and its endonuclease function. Interestingly, in cells lacking Dicer, an upstream component of the RNAi pathway, viral replication was the same as in parental cells. This suggests that the antiviral activity of Ago2 is independent of Dicer processed miRNAs. Deep sequencing of infected cells by other groups identified several SARS-CoV-2-derived small RNAs that bind to Ago2. A mutant virus lacking the most abundant ORF7A-derived viral miRNA was found to be significantly less sensitive to Ago2-mediated restriction. This combined with our findings that endonuclease and small RNA-binding functions of Ago2 are required for its antiviral function, suggests that Ago2-small viral RNA complexes target nascent viral RNA produced at replication sites for cleavage. Further studies are required to elucidate the processing mechanism of the viral small RNAs that are used by Ago2 to limit coronavirus replication.
Collapse
Affiliation(s)
- Joaquin Lopez-Orozco
- Department of Cell Biology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Canada
| | - Nawell Fayad
- Department of Cell Biology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Canada
| | - Juveriya Qamar Khan
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada
| | - Alberto Felix-Lopez
- Department of Medical Microbiology & Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Canada
| | - Mohamed Elaish
- Department of Cell Biology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Canada
| | - Megha Rohamare
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada
| | - Maansi Sharma
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada
| | - Darryl Falzarano
- Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, Saskatoon, Canada; Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Canada
| | - Jerry Pelletier
- Department of Biochemistry, McGill University, Montreal, Canada
| | - Joyce Wilson
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada
| | - Tom C Hobman
- Department of Cell Biology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Canada; Department of Medical Microbiology & Immunology, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Canada; Li Ka Shing Institute of Virology, University of Alberta, Edmonton, Canada.
| | - Anil Kumar
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada.
| |
Collapse
|
|
15
|
Kong J, Bie Y, Ji W, Xu J, Lyu B, Xiong X, Qiu Y, Zhou X. Alphavirus infection triggers antiviral RNAi immunity in mammals. Cell Rep 2023; 42:112441. [PMID: 37104090 DOI: 10.1016/j.celrep.2023.112441] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2023] [Revised: 03/29/2023] [Accepted: 04/11/2023] [Indexed: 04/28/2023] Open
Abstract
RNA interference (RNAi) is a well-established antiviral immunity. However, for mammalian somatic cells, antiviral RNAi becomes evident only when viral suppressors of RNAi (VSRs) are disabled by mutations or VSR-targeting drugs, thereby limiting its scope as a mammalian immunity. We find that a wild-type alphavirus, Semliki Forest virus (SFV), triggers the Dicer-dependent production of virus-derived small interfering RNAs (vsiRNAs) in both mammalian somatic cells and adult mice. These SFV-vsiRNAs are located at a particular region within the 5' terminus of the SFV genome, Argonaute loaded, and active in conferring effective anti-SFV activity. Sindbis virus, another alphavirus, also induces vsiRNA production in mammalian somatic cells. Moreover, treatment with enoxacin, an RNAi enhancer, inhibits SFV replication dependent on RNAi response in vitro and in vivo and protects mice from SFV-induced neuropathogenesis and lethality. These findings show that alphaviruses trigger the production of active vsiRNA in mammalian somatic cells, highlighting the functional importance and therapeutic potential of antiviral RNAi in mammals.
Collapse
Affiliation(s)
- Jing Kong
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yuanyuan Bie
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Wenting Ji
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, China
| | - Jiuyue Xu
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Bao Lyu
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Xiaobei Xiong
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
| | - Yang Qiu
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China.
| | - Xi Zhou
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China; School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230027, China.
| |
Collapse
|
|
16
|
Aderounmu AM, Aruscavage PJ, Kolaczkowski B, Bass BL. Ancestral protein reconstruction reveals evolutionary events governing variation in Dicer helicase function. eLife 2023; 12:e85120. [PMID: 37068011 PMCID: PMC10159624 DOI: 10.7554/elife.85120] [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: 11/23/2022] [Accepted: 04/14/2023] [Indexed: 04/18/2023] Open
Abstract
Antiviral defense in ecdysozoan invertebrates requires Dicer with a helicase domain capable of ATP hydrolysis. But despite well-conserved ATPase motifs, human Dicer is incapable of ATP hydrolysis, consistent with a muted role in antiviral defense. To investigate this enigma, we used ancestral protein reconstruction to resurrect Dicer's helicase in animals and trace the evolutionary trajectory of ATP hydrolysis. Biochemical assays indicated ancient Dicer possessed ATPase function, that like extant invertebrate Dicers, is stimulated by dsRNA. Analyses revealed that dsRNA stimulates ATPase activity by increasing ATP affinity, reflected in Michaelis constants. Deuterostome Dicer-1 ancestor, while exhibiting lower dsRNA affinity, retained some ATPase activity; importantly, ATPase activity was undetectable in the vertebrate Dicer-1 ancestor, which had even lower dsRNA affinity. Reverting residues in the ATP hydrolysis pocket was insufficient to rescue hydrolysis, but additional substitutions distant from the pocket rescued vertebrate Dicer-1's ATPase function. Our work suggests Dicer lost ATPase function in the vertebrate ancestor due to loss of ATP affinity, involving motifs distant from the active site, important for coupling dsRNA binding to the active conformation. By competing with Dicer for viral dsRNA, RIG-I-like receptors important for interferon signaling may have allowed or actively caused loss of ATPase function.
Collapse
Affiliation(s)
| | | | - Bryan Kolaczkowski
- Department of Microbiology and Cell Science, University of FloridaGainesvilleUnited States
| | - Brenda L Bass
- Department of Biochemistry, University of UtahSalt Lake CityUnited States
| |
Collapse
|
|
17
|
Anobile DP, Poirier EZ. RNA interference, an emerging component of antiviral immunity in mammals. Biochem Soc Trans 2023; 51:137-146. [PMID: 36606711 DOI: 10.1042/bst20220385] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2022] [Revised: 12/12/2022] [Accepted: 12/13/2022] [Indexed: 01/07/2023]
Abstract
Antiviral RNA interference (RNAi) is an immune pathway that can, in certain conditions, protect mammalian cells against RNA viruses. It depends on the recognition and dicing of viral double-stranded RNA by a protein of the Dicer family, which leads to the production of viral small interfering RNAs (vsiRNAs) that sequence-specifically guide the degradation of cognate viral RNA. If the first line of defence against viruses relies on type-I and type-III interferons (IFN) in mammals, certain cell types such as stem cells, that are hyporesponsive for IFN, instead use antiviral RNAi via the expression of a specific antiviral Dicer. In certain conditions, antiviral RNAi can also contribute to the protection of differentiated cells. Indeed, abundant vsiRNAs are detected in infected cells and efficiently guide the degradation of viral RNA, especially in cells infected with viruses disabled for viral suppressors of RNAi (VSRs), which are virally encoded blockers of antiviral RNAi. The existence and importance of antiviral RNAi in differentiated cells has however been debated in the field, because data document mutual inhibition between IFN and antiviral RNAi. Recent developments include the engineering of a small molecule inhibitor of VSR to probe antiviral RNAi in vivo, as well as the detection of vsiRNAs inside extracellular vesicles in the serum of infected mice. It suggests that using more complex, in vivo models could allow to unravel the contribution of antiviral RNAi to immunity at the host level.
Collapse
Affiliation(s)
- Dario Pasquale Anobile
- Stem Cell Immunity Team, Institut Curie, PSL Research University, INSERM U932, Paris, France
| | - Enzo Z Poirier
- Stem Cell Immunity Team, Institut Curie, PSL Research University, INSERM U932, Paris, France
| |
Collapse
|
|
18
|
Iwama RE, Moran Y. Origins and diversification of animal innate immune responses against viral infections. Nat Ecol Evol 2023; 7:182-193. [PMID: 36635343 DOI: 10.1038/s41559-022-01951-4] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2022] [Accepted: 11/11/2022] [Indexed: 01/14/2023]
Abstract
Immune systems are of pivotal importance to any living organism on Earth, as they protect the organism against deleterious effects of viral infections. Though the current knowledge about these systems is still biased towards the immune response in vertebrates, some studies have focused on the identification and characterization of components of invertebrate antiviral immune systems. Two classic model organisms, the insect Drosophila melanogaster and the nematode Caenorhabditis elegans, were instrumental in the discovery of several important components of the innate immune system, such as the Toll-like receptors and the RNA interference pathway. However, these two model organisms provide only a limited view of the evolutionary history of the immune system, as they both are ecdysozoan protostomes. Recent functional studies in non-classic models such as unicellular holozoans (for example, choanoflagellates), lophotrochozoans (for example, oysters) and cnidarians (for example, sea anemones) have added crucial information for understanding the evolution of antiviral systems, as they revealed unexpected ancestral complexity. This Review aims to summarize this information and present the ancestral nature of the antiviral immune response in animals. We also discuss lineage-specific adaptations and future perspectives for the comparative study of the innate immune system that are essential for understanding its evolution.
Collapse
Affiliation(s)
- Rafael E Iwama
- Department of Ecology, Evolution and Behavior, Alexander Silberman Institute of Life Sciences, Faculty of Science, Hebrew University of Jerusalem, Jerusalem, Israel.
| | - Yehu Moran
- Department of Ecology, Evolution and Behavior, Alexander Silberman Institute of Life Sciences, Faculty of Science, Hebrew University of Jerusalem, Jerusalem, Israel.
| |
Collapse
|
|
19
|
Arai Y, Yamanaka I, Okamoto T, Isobe A, Nakai N, Kamimura N, Suzuki T, Daidoji T, Ono T, Nakaya T, Matsumoto K, Okuzaki D, Watanabe Y. Stimulation of interferon-β responses by aberrant SARS-CoV-2 small viral RNAs acting as retinoic acid-inducible gene-I agonists. iScience 2023; 26:105742. [PMID: 36507221 PMCID: PMC9726650 DOI: 10.1016/j.isci.2022.105742] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2022] [Revised: 07/03/2022] [Accepted: 12/02/2022] [Indexed: 12/12/2022] Open
Abstract
Patients with severe COVID-19 exhibit a cytokine storm characterized by greatly elevated levels of cytokines. Despite this, the interferon (IFN) response is delayed, contributing to disease progression. Here, we report that SARS-CoV-2 excessively generates small viral RNAs (svRNAs) encoding exact 5' ends of positive-sense genes in human cells in vitro and ex vivo, whereas endemic human coronaviruses (OC43 and 229E) produce significantly fewer similar svRNAs. SARS-CoV-2 5' end svRNAs are RIG-I agonists and induce the IFN-β response in the later stages of infection. The first 60-nt ends bearing duplex structures and 5'-triphosphates are responsible for immune-stimulation. We propose that RIG-I activation by accumulated SARS-CoV-2 5' end svRNAs may contribute to later drive over-exuberant IFN production. Additionally, the differences in the amounts of svRNAs produced and the corresponding IFN response among CoV strains suggest that lower svRNA production during replication may correlate with the weaker immune response seen in less pathogenic CoVs.
Collapse
Affiliation(s)
- Yasuha Arai
- Department of Infectious Diseases, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
| | - Itaru Yamanaka
- Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
| | - Toru Okamoto
- Institute for Advanced Co-Creation Studies, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
| | - Ayana Isobe
- Department of Infectious Diseases, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
| | - Naomi Nakai
- Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
| | - Naoko Kamimura
- Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
| | - Tatsuya Suzuki
- Institute for Advanced Co-Creation Studies, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan
| | - Tomo Daidoji
- Department of Infectious Diseases, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
| | - Takao Ono
- SANKEN, Osaka University, Osaka 567-0047, Japan
| | - Takaaki Nakaya
- Department of Infectious Diseases, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan
| | | | - Daisuke Okuzaki
- Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan,Single Cell Genomics, Human Immunology, WPI Immunology Frontier Research Center, Osaka University, Osaka 565-0871, Japan
| | - Yohei Watanabe
- Department of Infectious Diseases, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan,Corresponding author
| |
Collapse
|
|
20
|
Palatini U, Alfano N, Carballar RL, Chen XG, Delatte H, Bonizzoni M. Virome and nrEVEome diversity of Aedes albopictus mosquitoes from La Reunion Island and China. Virol J 2022; 19:190. [DOI: 10.1186/s12985-022-01918-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2022] [Accepted: 11/04/2022] [Indexed: 11/19/2022] Open
Abstract
Abstract
Background
Aedes albopictus is a public health threat for its worldwide spread and ability to transmit arboviruses. Understanding mechanisms of mosquito immunity can provide new tools to control arbovirus spread. The genomes of Aedes mosquitoes contain hundreds of nonretroviral endogenous viral elements (nrEVEs), which are enriched in piRNA clusters and produce piRNAs, with the potential to target cognate viruses. Recently, one nrEVE was shown to limit cognate viral infection through nrEVE-derived piRNAs. These findings suggest that nrEVEs constitute an archive of past viral infection and that the landscape of viral integrations may be variable across populations depending on their viral exposure.
Methods
We used bioinformatics and molecular approaches to identify known and novel (i.e. absent in the reference genome) viral integrations in the genome of wild collected Aedes albopictus mosquitoes and characterize their virome.
Results
We showed that the landscape of viral integrations is dynamic with seven novel viral integrations being characterized, but does not correlate with the virome, which includes both viral species known and unknown to infect mosquitoes. However, the small RNA coverage profile of nrEVEs and the viral genomic contigs we identified confirmed an interaction among these elements and the piRNA and siRNA pathways in mosquitoes.
Conclusions
Mosquitoes nrEVEs have been recently described as a new form of heritable, sequence-specific mechanism of antiviral immunity. Our results contribute to understanding the dynamic distribution of nrEVEs in the genomes of wild Ae. albopictus and their interaction with mosquito viruses.
Collapse
|
|
21
|
The Impact of Deleting Stem-Loop 1 of Epstein-Barr Virus-Encoded RNA 1 on Cell Proliferation. Viruses 2022; 14:v14112538. [PMID: 36423146 PMCID: PMC9696203 DOI: 10.3390/v14112538] [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: 10/10/2022] [Revised: 11/10/2022] [Accepted: 11/14/2022] [Indexed: 11/18/2022] Open
Abstract
Epstein-Barr virus-encoded RNAs (EBERs) are two small, noncoding, structurally conserved transcripts, constitutively expressed at >106 copies per EBV-infected cell. They have been shown to drive cell growth. However, the mechanism(s) involved in EBER-induced proliferation is not clear. In this study, we investigated the molecular mechanisms and structural impact of EBER1. Sequences of EBER1 stem-loops (SL) 1, 3, and 4 were deleted, creating three mutants: ∆SL1, ∆SL3, and ∆SL4. These mutants were cloned into pHebo plasmids and expressed in Jurkat cell lines. Cells transfected with wildtype EBER1 and pHebo were used as controls. Cell proliferation was monitored by microscopy and flow cytometry. Microarray, qPCR, and Western blotting were used to investigate the cell cycle markers. We found significantly higher cell proliferation in wildtype EBER1 cells compared to pHebo, ∆SL1, and ∆SL3, but not ∆SL4 mutants. There was also significant upregulation of S-phase and G2/M phase markers in wildtype EBER1 and ∆SL4 mutant. Furthermore, CDT1, a factor for DNA replication, was upregulated in wildtype EBER1 and ∆SL4 mutant. However, in ∆SL1 mutant, CDT1 was significantly downregulated and translocated to the cytoplasm. These data indicate that the structure of EBER1 is important in cell proliferation.
Collapse
|
|
22
|
Viral and Host Small RNA Response to SARS-CoV-2 Infection. MICROBIOLOGY RESEARCH 2022. [DOI: 10.3390/microbiolres13040056] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022] Open
Abstract
After two years into the pandemic of the coronavirus disease 2019 (COVID-19), it remains unclear how the host RNA interference (RNAi) pathway and host miRNAs regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and impact the development of COVID-19. In this study, we profiled small RNAs in SARS-CoV-2-infected human ACE2-expressing HEK293T cells and observed dysregulated host small RNA groups, including specific host miRNAs that are altered in response to SARS-CoV-2 infection. By comparing dysregulated miRNAs in different SARS-CoV-2-infected samples, we identified miRNA-210-3p, miRNA-30-5p, and miR-146a/b as key host miRNAs that may be involved in SARS-CoV-2 infection. Furthermore, by comparing virally derived small RNAs (vsmRNAs) in different SARS-CoV-2-infected samples, we observed multiple hot spots in the viral genome that are prone to generating vsmRNAs, and their biogenesis can be dependent on the antiviral isoform of Dicer. Moreover, we investigated the biogenesis of a recently identified SARS-CoV-2 viral miRNA encoded by ORF7a and found that it is differentially expressed in different infected cell lines or in the same cell line with different viral doses. Our results demonstrate the involvement of both host small RNAs and vsmRNAs in SARS-CoV-2 infection and identify these small RNAs as potential targets for anti-COVID-19 therapeutic development.
Collapse
|
|
23
|
Hotspot siRNA Confers Plant Resistance against Viral Infection. BIOLOGY 2022; 11:biology11050714. [PMID: 35625441 PMCID: PMC9138956 DOI: 10.3390/biology11050714] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/07/2022] [Revised: 05/02/2022] [Accepted: 05/04/2022] [Indexed: 11/16/2022]
Abstract
Simple Summary A hallmark of antiviral RNAi is the production of viral siRNA (vsiRNA). Profiling of vsiRNAs indicates that certain hotspot regions of viral genome or transcribed viral RNAs are more prone to RNAi-mediated cleavage. However, the biological relevance of hotspot vsiRNAs to the host innate defence remains to be elucidated. Here, we show that direct targeting a hotspot by synthetic vsiRNA confers plant resistance to virus infection. Hotspot and coldspot vsiRNAs, based on vsiRNA profile of the African cassava mosaic virus (ACMV), were synthesised. However, only the double-stranded hotspot vsiRNA protected plants from ACMV infection with undetectable levels of viral DNA replication and viral mRNA. We further demonstrated that the hotspot vsiRNA-mediated virus resistance had a threshold effect and required an active RDR6. These data show that hotspot vsiRNAs bear a functional significance on antiviral RNAi, suggesting that they may have the potential as exogenous protection agents for controlling destructive plant viral diseases. Abstract A hallmark of antiviral RNA interference (RNAi) is the production of viral small interfering RNA (vsiRNA). Profiling of vsiRNAs indicates that certain regions of viral RNA genome or transcribed viral RNA, dubbed vsiRNA hotspots, are more prone to RNAi-mediated cleavage for vsiRNA biogenesis. However, the biological relevance of hotspot vsiRNAs to the host innate defence against pathogens remains to be elucidated. Here, we show that direct targeting a hotspot by a synthetic vsiRNA confers host resistance to virus infection. Using Northern blotting and RNAseq, we obtained a profile of vsiRNAs of the African cassava mosaic virus (ACMV), a single-stranded DNA virus. Sense and anti-sense strands of small RNAs corresponding to a hotspot and a coldspot vsiRNA were synthesised. Co-inoculation of Nicotiana benthamiana with the double-stranded hotspot siRNA protected plants from ACMV infection, where viral DNA replication and accumulation of viral mRNA were undetectable. The sense or anti-sense strand of this hotspot vsiRNA, and the coldspot vsiRNA in both double-stranded and single-stranded formats possessed no activity in viral protection. We further demonstrated that the hotspot vsiRNA-mediated virus resistance had a threshold effect and required an active RDR6. These data show that hotspot vsiRNAs bear a functional significance on antiviral RNAi, suggesting that they may have the potential as an exogenous protection agent for controlling destructive viral diseases in plants.
Collapse
|
|
24
|
Liu X, Wen YZ, Huang ZL, Shen X, Wang JH, Luo YH, Chen WX, Lun ZR, Li HB, Qu LH, Shan H, Zheng LL. SARS-CoV-2 causes a significant stress response mediated by small RNAs in the blood of COVID-19 patients. MOLECULAR THERAPY. NUCLEIC ACIDS 2022; 27:751-762. [PMID: 35003892 PMCID: PMC8719421 DOI: 10.1016/j.omtn.2021.12.034] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/02/2021] [Accepted: 12/29/2021] [Indexed: 12/24/2022]
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a serious impact on the world. In this study, small RNAs from the blood of COVID-19 patients with moderate or severe symptoms were extracted for high-throughput sequencing and analysis. Interestingly, the levels of a special group of tRNA-derived small RNAs (tsRNAs) were found to be dramatically upregulated after SARS-CoV-2 infection, particularly in coronavirus disease 2019 (COVID-19) patients with severe symptoms. In particular, the 3′CCA tsRNAs from tRNA-Gly were highly consistent with the inflammation indicator C-reactive protein (CRP). In addition, we found that the majority of significantly changed microRNAs (miRNAs) were associated with endoplasmic reticulum (ER)/unfolded protein response (UPR) sensors, which may lead to the induction of proinflammatory cytokine and immune responses. This study found that SARS-CoV-2 infection caused significant changes in the levels of stress-associated small RNAs in patient blood and their potential functions. Our research revealed that the cells of COVID-19 patients undergo tremendous stress and respond, which can be reflected or regulated by small non-coding RNA (sncRNAs), thus providing potential thought for therapeutic intervention in COVID-19 by modulating small RNA levels or activities.
Collapse
Affiliation(s)
- Xi Liu
- Department of Infectious Diseases, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, P. R. China
| | - Yan-Zi Wen
- MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China
| | - Zi-Liang Huang
- MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China
| | - Xia Shen
- MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China.,Greater Bay Area Institute of Precision Medicine (Guangzhou), Fudan University, Guangzhou 511458, P. R. China.,Center for Global Health Research, Usher Institute, University of Edinburgh, Edinburgh EH8 9AG, UK
| | - Jun-Hao Wang
- MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China
| | - Yi-Hai Luo
- MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China
| | - Wen-Xin Chen
- MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China
| | - Zhao-Rong Lun
- MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China
| | - Hui-Bin Li
- MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China
| | - Liang-Hu Qu
- MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China
| | - Hong Shan
- Guangdong Provincial Engineering Research Center of Molecular Imaging, the Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, P. R. China.,Guangdong Provincial Key Laboratory of Biomedical Imaging, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, P. R. China.,Department of Interventional Medicine, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, P. R. China
| | - Ling-Ling Zheng
- MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P. R. China
| |
Collapse
|
|
25
|
Pawlica P, Yario TA, White S, Wang J, Moss WN, Hui P, Vinetz JM, Steitz JA. SARS-CoV-2 expresses a microRNA-like small RNA able to selectively repress host genes. Proc Natl Acad Sci U S A 2021; 118:e2116668118. [PMID: 34903581 PMCID: PMC8719879 DOI: 10.1073/pnas.2116668118] [Citation(s) in RCA: 51] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/28/2021] [Indexed: 12/13/2022] Open
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease (COVID-19), continues to be a pressing health concern. In this study, we investigated the impact of SARS-CoV-2 infection on host microRNA (miRNA) populations in three human lung-derived cell lines, as well as in nasopharyngeal swabs from SARS-CoV-2-infected individuals. We did not detect any major and consistent differences in host miRNA levels after SARS-CoV-2 infection. However, we unexpectedly discovered a viral miRNA-like small RNA, named CoV2-miR-O7a (for SARS-CoV-2 miRNA-like ORF7a-derived small RNA). Its abundance ranges from low to moderate as compared to host miRNAs and it associates with Argonaute proteins-core components of the RNA interference pathway. We identify putative targets for CoV2-miR-O7a, including Basic Leucine Zipper ATF-Like Transcription Factor 2 (BATF2), which participates in interferon signaling. We demonstrate that CoV2-miR-O7a production relies on cellular machinery, yet is independent of Drosha protein, and is enhanced by the presence of a strong and evolutionarily conserved hairpin formed within the ORF7a sequence.
Collapse
Affiliation(s)
- Paulina Pawlica
- Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06536;
| | - Therese A Yario
- Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06536
- HHMI, Yale University School of Medicine, New Haven, CT 06536
| | - Sylvia White
- Department of Pathology, Yale University School of Medicine, New Haven, CT 06536
| | - Jianhui Wang
- Department of Pathology, Yale University School of Medicine, New Haven, CT 06536
| | - Walter N Moss
- Roy J. Carver Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011
| | - Pei Hui
- Department of Pathology, Yale University School of Medicine, New Haven, CT 06536
| | - Joseph M Vinetz
- Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520
| | - Joan A Steitz
- Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06536;
- HHMI, Yale University School of Medicine, New Haven, CT 06536
| |
Collapse
|
|
26
|
Abstract
One of the first layers of protection that metazoans put in place to defend themselves against viruses rely on the use of proteins containing DExD/H-box helicase domains. These members of the duplex RNA–activated ATPase (DRA) family act as sensors of double-stranded RNA (dsRNA) molecules, a universal marker of viral infections. DRAs can be classified into 2 subgroups based on their mode of action: They can either act directly on the dsRNA, or they can trigger a signaling cascade. In the first group, the type III ribonuclease Dicer plays a key role to activate the antiviral RNA interference (RNAi) pathway by cleaving the viral dsRNA into small interfering RNAs (siRNAs). This represents the main innate antiviral immune mechanism in arthropods and nematodes. Even though Dicer is present and functional in mammals, the second group of DRAs, containing the RIG-I-like RNA helicases, appears to have functionally replaced RNAi and activate type I interferon (IFN) response upon dsRNA sensing. However, recent findings tend to blur the frontier between these 2 mechanisms, thereby highlighting the crucial and diverse roles played by RNA helicases in antiviral innate immunity. Here, we will review our current knowledge of the importance of these key proteins in viral infection, with a special focus on the interplay between the 2 main types of response that are activated by dsRNA.
Collapse
Affiliation(s)
- Morgane Baldaccini
- Université de Strasbourg, Architecture et Réactivité de l’ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France
| | - Sébastien Pfeffer
- Université de Strasbourg, Architecture et Réactivité de l’ARN, Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France
- * E-mail:
| |
Collapse
|
|
27
|
Fang Y, Liu Z, Qiu Y, Kong J, Fu Y, Liu Y, Wang C, Quan J, Wang Q, Xu W, Yin L, Cui J, Xu Y, Curry S, Jiang S, Lu L, Zhou X. Inhibition of viral suppressor of RNAi proteins by designer peptides protects from enteroviral infection in vivo. Immunity 2021; 54:2231-2244.e6. [PMID: 34555337 DOI: 10.1016/j.immuni.2021.08.027] [Citation(s) in RCA: 31] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2020] [Revised: 04/27/2021] [Accepted: 08/23/2021] [Indexed: 12/15/2022]
Abstract
RNA interference (RNAi) is the major antiviral mechanism in plants and invertebrates, but the absence of detectable viral (v)siRNAs in mammalian cells upon viral infection has questioned the functional relevance of this pathway in mammalian immunity. We designed a series of peptides specifically targeting enterovirus A71 (EV-A71)-encoded protein 3A, a viral suppressor of RNAi (VSR). These peptides abrogated the VSR function of EV-A71 in infected cells and resulted in the accumulation of vsiRNAs and reduced viral replication. These vsiRNAs were functional, as evidenced by RISC-loading and silencing of target RNAs. The effects of VSR-targeting peptides (VTPs) on infection with EV-A71 as well as another enterovirus, Coxsackievirus-A16, were ablated upon deletion of Dicer1 or AGO2, core components of the RNAi pathway. In vivo, VTP treatment protected mice against lethal EV-A71 challenge, with detectable vsiRNAs. Our findings provide evidence for the functional relevance of RNAi in mammalian immunity and present a therapeutic strategy for infectious disease.
Collapse
Affiliation(s)
- Yuan Fang
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China
| | - Zezhong Liu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences and Shanghai Public Health Clinical Center, Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Yang Qiu
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China
| | - Jing Kong
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China
| | - Yuhong Fu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences and Shanghai Public Health Clinical Center, Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Yujie Liu
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China
| | - Chong Wang
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China; Guangzhou Institute of Pediatrics, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, 510120, China
| | - Jia Quan
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China
| | - Qian Wang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences and Shanghai Public Health Clinical Center, Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Wei Xu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences and Shanghai Public Health Clinical Center, Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Lei Yin
- State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China
| | - Jie Cui
- CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, CAS, Shanghai 200031, China
| | - Yi Xu
- Guangzhou Institute of Pediatrics, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou, 510120, China
| | - Stephen Curry
- Department of Life Sciences, Imperial College London, London SW7 2AZ, UK
| | - Shibo Jiang
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences and Shanghai Public Health Clinical Center, Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China
| | - Lu Lu
- Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences and Shanghai Public Health Clinical Center, Shanghai Institute of Infectious Disease and Biosecurity, Shanghai Medical College, Fudan University, Shanghai 200032, China.
| | - Xi Zhou
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan 430071, China.
| |
Collapse
|
|
28
|
Rapozzi V, Juarranz A, Habib A, Ihan A, Strgar R. Is haem the real target of COVID-19? Photodiagnosis Photodyn Ther 2021; 35:102381. [PMID: 34119708 PMCID: PMC8192263 DOI: 10.1016/j.pdpdt.2021.102381] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2021] [Revised: 05/25/2021] [Accepted: 06/01/2021] [Indexed: 02/08/2023]
Abstract
Although a vaccination campaign has been launched in many countries, the COVID-19 pandemic is not under control. The main concern is the emergence of new variants of SARS-CoV-2; therefore, it is important to find approaches to prevent or reduce the virulence and pathogenicity of the virus. Currently, the mechanism of action of SARS-CoV-2 is not fully understood. Considering the clinical effects that occur during the disease, attacking the human respiratory and hematopoietic systems, and the changes in biochemical parameters (including decreases in haemoglobin [Hb] levels and increases in serum ferritin), it is clear that iron metabolism is involved. SARS-CoV-2 induces haemolysis and interacts with Hb molecules via ACE2, CD147, CD26, and other receptors located on erythrocytes and/or blood cell precursors that produce dysfunctional Hb. A molecular docking study has reported a potential link between the virus and the beta chain of haemoglobin and attack on haem. Considering that haem is involved in miRNA processing by binding to the DGCR8-DROSHA complex, we hypothesised that the virus may check this mechanism and thwart the antiviral response.
Collapse
Affiliation(s)
| | - Angeles Juarranz
- Department of Biology, University Autonoma of Madrid, Madrid 28049, Spain
| | - Ahsan Habib
- Department of Chemistry, University of Dhaka, Dhaka 1000, Bangladesh
| | - Alojz Ihan
- Institute for Microbiology and Immunology, Medical Faculty of Ljubljana, Slovenia
| | - Rebeka Strgar
- Institution of Applicative Biophotonics, Technological Park Ljubljana, Slovenia
| |
Collapse
|
|
29
|
Zhang Y, Xu Y, Dai Y, Li Z, Wang J, Ye Z, Ren Y, Wang H, Li WX, Lu J, Ding SW, Li Y. Efficient Dicer processing of virus-derived double-stranded RNAs and its modulation by RIG-I-like receptor LGP2. PLoS Pathog 2021; 17:e1009790. [PMID: 34343211 PMCID: PMC8362961 DOI: 10.1371/journal.ppat.1009790] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Revised: 08/13/2021] [Accepted: 07/09/2021] [Indexed: 02/06/2023] Open
Abstract
The interferon-regulated antiviral responses are essential for the induction of both innate and adaptive immunity in mammals. Production of virus-derived small-interfering RNAs (vsiRNAs) to restrict virus infection by RNA interference (RNAi) is a recently identified mammalian immune response to several RNA viruses, which cause important human diseases such as influenza and Zika virus. However, little is known about Dicer processing of viral double-stranded RNA replicative intermediates (dsRNA-vRIs) in mammalian somatic cells. Here we show that infected somatic cells produced more influenza vsiRNAs than cellular microRNAs when both were produced by human Dicer expressed de novo, indicating that dsRNA-vRIs are not poor Dicer substrates as previously proposed according to in vitro Dicer processing of synthetic long dsRNA. We report the first evidence both for canonical vsiRNA production during wild-type Nodamura virus infection and direct vsiRNA sequestration by its RNAi suppressor protein B2 in two strains of suckling mice. Moreover, Sindbis virus (SINV) accumulation in vivo was decreased by prior production of SINV-targeting vsiRNAs triggered by infection and increased by heterologous expression of B2 in cis from SINV genome, indicating an antiviral function for the induced RNAi response. These findings reveal that unlike artificial long dsRNA, dsRNA-vRIs made during authentic infection of mature somatic cells are efficiently processed by Dicer into vsiRNAs to direct antiviral RNAi. Interestingly, Dicer processing of dsRNA-vRIs into vsiRNAs was inhibited by LGP2 (laboratory of genetics and physiology 2), which was encoded by an interferon-stimulated gene (ISG) shown recently to inhibit Dicer processing of artificial long dsRNA in cell culture. Our work thus further suggests negative modulation of antiviral RNAi by a known ISG from the interferon response. The function and mechanism of the interferon-regulated antiviral responses have been extensively characterized. Recent studies have demonstrated induction of antiviral RNA interference (RNAi) in somatic cells against several mammalian RNA viruses rendered incapable of RNAi suppression. However, little is known about Dicer-mediated production of virus-derived small-interfering RNAs (vsiRNAs) in these cells active in the type I interferon response. Here we show that the dsRNA precursors of influenza vsiRNAs were processed more efficiently than cellular precursor microRNA hairpins by wild-type human Dicer expressed de novo in Dicer-knockout somatic cells. We found that infection of two strains of suckling mice with wild-type Nodamura virus (NoV) was associated with production of silencing-active vsiRNAs and direct sequestration of duplex vsiRNAs by its RNAi suppressor protein B2. Our findings from in vivo infection with Sindbis virus recombinants expressing NoV B2 or carrying a vsiRNA-targeted insert provide evidence for an antiviral function of the induced RNAi response. Interestingly, NoV infection induces expression of RIG-I-like receptor LGP2 to inhibit vsiRNA biogenesis and promote virulent infection in suckling mice. Our findings together reveal efficient Dicer processing of vsiRNA precursors in interferon-competent somatic cells and suckling mice in contrast to synthetic long dsRNA examined previously by in vitro dicing.
Collapse
Affiliation(s)
- Yuqiang Zhang
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Yan Xu
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Yunpeng Dai
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Zhe Li
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Jiaxing Wang
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Zhi Ye
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Yanxin Ren
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Hua Wang
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Wan-xiang Li
- Department of Microbiology & Plant Pathology, University of California, Riverside, California, United States of America
| | - Jinfeng Lu
- Department of Microbiology & Plant Pathology, University of California, Riverside, California, United States of America
- * E-mail: (LJ); (S-WD); (YL)
| | - Shou-Wei Ding
- Department of Microbiology & Plant Pathology, University of California, Riverside, California, United States of America
- * E-mail: (LJ); (S-WD); (YL)
| | - Yang Li
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
- CAS Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
- * E-mail: (LJ); (S-WD); (YL)
| |
Collapse
|
|
30
|
Montavon TC, Baldaccini M, Lefèvre M, Girardi E, Chane-Woon-Ming B, Messmer M, Hammann P, Chicher J, Pfeffer S. Human DICER helicase domain recruits PKR and modulates its antiviral activity. PLoS Pathog 2021; 17:e1009549. [PMID: 33984068 PMCID: PMC8118307 DOI: 10.1371/journal.ppat.1009549] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Accepted: 04/08/2021] [Indexed: 11/18/2022] Open
Abstract
The antiviral innate immune response mainly involves type I interferon (IFN) in mammalian cells. The contribution of the RNA silencing machinery remains to be established, but several recent studies indicate that the ribonuclease DICER can generate viral siRNAs in specific conditions. It has also been proposed that type I IFN and RNA silencing could be mutually exclusive antiviral responses. In order to decipher the implication of DICER during infection of human cells with alphaviruses such as the Sindbis virus and Semliki forest virus, we determined its interactome by proteomics analysis. We show that DICER specifically interacts with several double-stranded RNA binding proteins and RNA helicases during viral infection. In particular, proteins such as DHX9, ADAR-1 and the protein kinase RNA-activated (PKR) are enriched with DICER in virus-infected cells. We demonstrate that the helicase domain of DICER is essential for this interaction and that its deletion confers antiviral properties to this protein in an RNAi-independent, PKR-dependent, manner.
Collapse
Affiliation(s)
- Thomas C Montavon
- Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR9002, Strasbourg, France
| | - Morgane Baldaccini
- Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR9002, Strasbourg, France
| | - Mathieu Lefèvre
- Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR9002, Strasbourg, France
| | - Erika Girardi
- Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR9002, Strasbourg, France
| | - Béatrice Chane-Woon-Ming
- Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR9002, Strasbourg, France
| | - Mélanie Messmer
- Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR9002, Strasbourg, France
| | - Philippe Hammann
- Université de Strasbourg, CNRS, Institut de Biologie Moléculaire et Cellulaire, Plateforme Protéomique Strasbourg-Esplanade, Strasbourg, France
| | - Johana Chicher
- Université de Strasbourg, CNRS, Institut de Biologie Moléculaire et Cellulaire, Plateforme Protéomique Strasbourg-Esplanade, Strasbourg, France
| | - Sébastien Pfeffer
- Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR9002, Strasbourg, France
| |
Collapse
|
|
31
|
Markiewicz L, Drazkowska K, Sikorski PJ. Tricks and threats of RNA viruses - towards understanding the fate of viral RNA. RNA Biol 2021; 18:669-687. [PMID: 33618611 PMCID: PMC8078519 DOI: 10.1080/15476286.2021.1875680] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2020] [Revised: 12/22/2020] [Accepted: 01/09/2021] [Indexed: 12/24/2022] Open
Abstract
Human innate cellular defence pathways have evolved to sense and eliminate pathogens, of which, viruses are considered one of the most dangerous. Their relatively simple structure makes the identification of viral invasion a difficult task for cells. In the course of evolution, viral nucleic acids have become one of the strongest and most reliable early identifiers of infection. When considering RNA virus recognition, RNA sensing is the central mechanism in human innate immunity, and effectiveness of this sensing is crucial for triggering an appropriate antiviral response. Although human cells are armed with a variety of highly specialized receptors designed to respond only to pathogenic viral RNA, RNA viruses have developed an array of mechanisms to avoid being recognized by human interferon-mediated cellular defence systems. The repertoire of viral evasion strategies is extremely wide, ranging from masking pathogenic RNA through end modification, to utilizing sophisticated techniques to deceive host cellular RNA degrading enzymes, and hijacking the most basic metabolic pathways in host cells. In this review, we aim to dissect human RNA sensing mechanisms crucial for antiviral immune defences, as well as the strategies adopted by RNA viruses to avoid detection and degradation by host cells. We believe that understanding the fate of viral RNA upon infection, and detailing the molecular mechanisms behind virus-host interactions, may be helpful for developing more effective antiviral strategies; which are urgently needed to prevent the far-reaching consequences of widespread, highly pathogenic viral infections.
Collapse
|
|
32
|
Zeng J, Luo Z, Dong S, Xie X, Liang X, Yan Y, Liang Q, Zhao Z. Functional Mapping of AGO-Associated Zika Virus-Derived Small Interfering RNAs in Neural Stem Cells. Front Cell Infect Microbiol 2021; 11:628887. [PMID: 33718276 PMCID: PMC7946837 DOI: 10.3389/fcimb.2021.628887] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2020] [Accepted: 01/22/2021] [Indexed: 11/23/2022] Open
Abstract
Viral interfering RNA (viRNA) has been identified from several viral genomes via directly deep RNA sequencing of the virus-infected cells, including zika virus (ZIKV). Once produced by endoribonuclease Dicer, viRNAs are loaded onto the Argonaute (AGO) family proteins of the RNA-induced silencing complexes (RISCs) to pair with their RNA targets and initiate the cleavage of target genes. However, the identities of functional ZIKV viRNAs and their viral RNA targets remain largely unknown. Our recent study has shown that ZIKV capsid protein interacted with Dicer and antagonized its endoribonuclease activity, which requires its histidine residue at the 41st amino acid. Accordingly, the engineered ZIKV-H41R loss-of-function (LOF) mutant virus no longer suppresses Dicer enzymatic activity nor inhibits miRNA biogenesis in NSCs. By combining AGO-associated RNA sequencing, deep sequencing analysis in ZIKV-infected human neural stem cells (NSCs), and miRanda target scanning, we defined 29 ZIKV derived viRNA profiles in NSCs, and established a complex interaction network between the viRNAs and their viral targets. More importantly, we found that viRNA production from the ZIKV mRNA is dependent on Dicer function and is a limiting factor for ZIKV virulence in NSCs. As a result, much higher levels of viRNAs generated from the ZIKV-H41R virus-infected NSCs. Therefore, our mapping of viRNAs to their RNA targets paves a way to further investigate how viRNAs play the role in anti-viral mechanisms, and perhaps other unknown biological functions.
Collapse
Affiliation(s)
- Jianxiong Zeng
- Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States.,AngelicaMadlangbayanKeck School of Medicine, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, United States
| | - Zhifei Luo
- Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA, United States
| | - Shupeng Dong
- Department of Immunology and Microbiology, School of Medicine, Shanghai Institute of Immunology, Shanghai Jiao Tong University, Shanghai, China.,Research Center of Translational Medicine, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China
| | - Xiaochun Xie
- Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States.,AngelicaMadlangbayanKeck School of Medicine, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, United States
| | - Xinyan Liang
- Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States.,AngelicaMadlangbayanKeck School of Medicine, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, United States
| | - Youzhen Yan
- Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States.,AngelicaMadlangbayanKeck School of Medicine, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, United States
| | - Qiming Liang
- Department of Immunology and Microbiology, School of Medicine, Shanghai Institute of Immunology, Shanghai Jiao Tong University, Shanghai, China.,Research Center of Translational Medicine, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China
| | - Zhen Zhao
- Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States.,AngelicaMadlangbayanKeck School of Medicine, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA, United States
| |
Collapse
|
|
33
|
Zhang Y, Li Z, Ye Z, Xu Y, Wang B, Wang C, Dai Y, Lu J, Lu B, Zhang W, Li Y. The activation of antiviral RNA interference not only exists in neural progenitor cells but also in somatic cells in mammals. Emerg Microbes Infect 2021; 9:1580-1589. [PMID: 32576094 PMCID: PMC7473182 DOI: 10.1080/22221751.2020.1787798] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The RNA interference (RNAi) pathway directs an important antiviral immunity mechanism in plants and invertebrates. Recently, we and others have demonstrated that the antiviral RNAi response is also conserved in mammals, at least to five distinct RNA viruses, including Zika virus (ZIKV). ZIKV may preferentially infect neuronal progenitor cells (NPCs) in the developing foetal brain. Ex vivo ZIKV infection induces RNAi-mediated antiviral response in human NPCs, but not in the more differentiated NPCs or somatic cells. However, litter is known about the in vivo property or function of the virus-derived small-interfering RNAs (vsiRNAs) targeting ZIKV. Here we report a surprising observation: different from ex vivo observations, viral small RNAs (vsRNAs) targeting ZIKV were produced in vivo upon infection in both central neuron system (CNS) and muscle tissues. In addition, our findings demonstrate the production of canonical vsiRNAs in murine CNS upon antiviral RNAi activation by Sindbis virus (SINV), suggesting the possibility of antiviral immune strategy applied by mammals in the CNS.
Collapse
Affiliation(s)
- Yuqiang Zhang
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Zhe Li
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Zhi Ye
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Yan Xu
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Binbin Wang
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Congcong Wang
- State Key Laboratory of Medical Neurobiology, School of Life Sciences, Fudan University, Shanghai, China
| | - Yunpeng Dai
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Jinfeng Lu
- Institute for Genomic Medicine, Columbia University Irving Medical Center, New York, NY, USA
| | - Boxun Lu
- State Key Laboratory of Medical Neurobiology, School of Life Sciences, Fudan University, Shanghai, China
| | - Wanju Zhang
- Department of Pathogen Diagnosis and Biosafety, Shanghai Public Health Clinical Center, Fudan University, Shanghai, China
| | - Yang Li
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| |
Collapse
|
|
34
|
Ren Y, Li X, Tian Z, Xu Y, Zhang R, Li Y. Zebrafish as an animal model for the antiviral RNA interference pathway. J Gen Virol 2021; 102. [PMID: 33507144 DOI: 10.1099/jgv.0.001552] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The zebrafish (Danio rerio) possesses evolutionarily conserved innate and adaptive immunity as a mammal and has recently become a popular vertebrate model to exploit infection and immunity. Antiviral RNA interference (RNAi) has been illuminated in various model organisms, including Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans and mice. However, to date, there is no report on the antiviral RNAi pathway of zebrafish. Here, we have evaluated the possible use of zebrafish to study antiviral RNAi with Sindbis virus (SINV), vesicular stomatitis virus (VSV) and Nodamura virus (NoV). We find that SINVs and NoVs induce the production of virus-derived small interfering RNAs (vsiRNAs), the hallmark of antiviral RNAi, with a preference for a length of 22 nucleotides, after infection of larval zebrafish. Meanwhile, the suppressor of RNAi (VSR) protein, NoV B2, may affect the accumulation of the NoV in zebrafish. Furthermore, taking advantage of the fact that zebrafish argonaute-2 (Ago2) protein is naturally deficient in cleavage compared with that of mammals, we provide evidence that the slicing activity of human Ago2 can virtually inhibit the accumulation of RNA virus after being ectopically expressed in larval zebrafish. Thus, zebrafish may be a unique model organism to study the antiviral RNAi pathway.
Collapse
Affiliation(s)
- Yanxin Ren
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200438, PR China
| | - Xueyu Li
- School of Basic Medical Sciences, Wuhan University, Wuhan, PR China
| | - Zhonghui Tian
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200438, PR China
| | - Yan Xu
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200438, PR China
| | - Ruilin Zhang
- School of Basic Medical Sciences, Wuhan University, Wuhan, PR China
| | - Yang Li
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200438, PR China
| |
Collapse
|
|
35
|
miRNA-141 and its target long non-coding RNA HOTAIR as diagnostic marker in hepatocellular carcinoma on top of hepatitis C virus. GENE REPORTS 2020. [DOI: 10.1016/j.genrep.2020.100807] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
|
|
36
|
Gong P, Li X, Wu W, Cao L, Zhao P, Li X, Ren B, Li J, Zhang X. A Novel MicroRNA From the Translated Region of the Giardiavirus rdrp Gene Governs Virus Copy Number in Giardia duodenalis. Front Microbiol 2020; 11:569412. [PMID: 33329426 PMCID: PMC7719678 DOI: 10.3389/fmicb.2020.569412] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2020] [Accepted: 10/21/2020] [Indexed: 12/14/2022] Open
Abstract
Giardia duodenalis is an important zoonotic parasite that can cause human and animal diarrhea. Giardiavirus (GLV) is a double-stranded RNA virus in Totiviridae family, which specifically infects trophozoites of the primitive protozoan parasite G. duodenalis. However, the GLV infectious and the pathogenicity of the G. duodenalis still remain to be confirmed. The GLV genome is 6,277 bp, which encodes two proteins (Gag and Gag-Pol). The expression of Gag-Pol protein is regulated by a-1 ribosomal frameshift. In this report, we identified a novel microRNA (GLV miRNA1) from the GLV. Split ligation northern results showed that GLV miRNA1 is a special expression product of GLV, and the precursor was also identified by primer extension. Antisense sequence of the GLV miRNA1 could increase the copy number of virus in G. duodenalis. It suggests that GLV miRNA1 governs the copy number of Giardiavirus in G. duodenalis. Most importantly, the GLV miRNA1 lies at the translated region of the rdrp gene, which is the first case that microRNA locates in the translated region of a known protein. It may be implying a novel phenomenon for miRNA biogenesis.
Collapse
Affiliation(s)
- Pengtao Gong
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Xianhe Li
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Wei Wu
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Lili Cao
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China.,Jilin Academy of Animal Husbandry and Veterinary Medicine, Changchun, China
| | - Panpan Zhao
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Xin Li
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Baoyan Ren
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Jianhua Li
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Xichen Zhang
- Key Laboratory of Zoonosis Research, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| |
Collapse
|
|
37
|
Han Q, Chen G, Wang J, Jee D, Li WX, Lai EC, Ding SW. Mechanism and Function of Antiviral RNA Interference in Mice. mBio 2020; 11:e03278-19. [PMID: 32753500 PMCID: PMC7407090 DOI: 10.1128/mbio.03278-19] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Accepted: 07/02/2020] [Indexed: 12/18/2022] Open
Abstract
Distinct mammalian RNA viruses trigger Dicer-mediated production of virus-derived small-interfering RNAs (vsiRNA) and encode unrelated proteins to suppress vsiRNA biogenesis. However, the mechanism and function of the mammalian RNA interference (RNAi) response are poorly understood. Here, we characterized antiviral RNAi in a mouse model of infection with Nodamura virus (NoV), a mosquito-transmissible positive-strand RNA virus encoding a known double-stranded RNA (dsRNA)-binding viral suppressor of RNAi (VSR), the B2 protein. We show that inhibition of NoV RNA replication by antiviral RNAi in mouse embryonic fibroblasts (MEFs) requires Dicer-dependent vsiRNA biogenesis and Argonaute-2 slicer activity. We found that VSR-B2 of NoV enhances viral RNA replication in wild-type but not RNAi-defective MEFs such as Argonaute-2 catalytic-dead MEFs and Dicer or Argonaute-2 knockout MEFs, indicating that VSR-B2 acts mainly by suppressing antiviral RNAi in the differentiated murine cells. Consistently, VSR-B2 expression in MEFs has no detectable effect on the induction of interferon-stimulated genes or the activation of global RNA cleavages by RNase L. Moreover, we demonstrate that NoV infection of adult mice induces production of abundant vsiRNA active to guide RNA slicing by Argonaute-2. Notably, VSR-B2 suppresses the biogenesis of both vsiRNA and the slicing-competent vsiRNA-Argonaute-2 complex without detectable inhibition of Argonaute-2 slicing guided by endogenous microRNA, which dramatically enhances viral load and promotes lethal NoV infection in adult mice either intact or defective in the signaling by type I, II, and III interferons. Together, our findings suggest that the mouse RNAi response confers essential protective antiviral immunity in both the presence and absence of the interferon response.IMPORTANCE Innate immune sensing of viral nucleic acids in mammals triggers potent antiviral responses regulated by interferons known to antagonize the induction of RNA interference (RNAi) by synthetic long double-stranded RNA (dsRNA). Here, we show that Nodamura virus (NoV) infection in adult mice activates processing of the viral dsRNA replicative intermediates into small interfering RNAs (siRNAs) active to guide RNA slicing by Argonaute-2. Genetic studies demonstrate that NoV RNA replication in mouse embryonic fibroblasts is inhibited by the RNAi pathway and enhanced by the B2 viral RNAi suppressor only in RNAi-competent cells. When B2 is rendered nonexpressing or nonfunctional, the resulting mutant viruses become nonpathogenic and are cleared in adult mice either intact or defective in the signaling by type I, II, and III interferons. Our findings suggest that mouse antiviral RNAi is active and necessary for the in vivo defense against viral infection in both the presence and absence of the interferon response.
Collapse
Affiliation(s)
- Qingxia Han
- Department of Microbiology and Plant Pathology, University of California, Riverside, Riverside, California, USA
| | - Gang Chen
- Department of Microbiology and Plant Pathology, University of California, Riverside, Riverside, California, USA
| | - Jinyan Wang
- Department of Microbiology and Plant Pathology, University of California, Riverside, Riverside, California, USA
| | - David Jee
- Department of Developmental Biology, Sloan Kettering Institute, New York, New York, USA
| | - Wan-Xiang Li
- Department of Microbiology and Plant Pathology, University of California, Riverside, Riverside, California, USA
| | - Eric C Lai
- Department of Developmental Biology, Sloan Kettering Institute, New York, New York, USA
| | - Shou-Wei Ding
- Department of Microbiology and Plant Pathology, University of California, Riverside, Riverside, California, USA
| |
Collapse
|
|
38
|
Abstract
The two biological evidences to endorse the antiviral activity of RNA interference (RNAi) are biogenesis of viral-siRNA (v-siRNA) by the host and encoding of RNAi-suppressor protein by viral genome. It has been recently established that mammals and mammalian cell lines mount antiviral RNAi to defend themselves against the invading viruses. The large part of viral pathogenicity is also due to the RNAi suppressor proteins. In this context it is only natural to ask what kinds of RNAi suppressors are encoded by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the central character of the present pandemic. The following mini review addresses this question.
Collapse
|
|
39
|
I. Sardi S, H. Carvalho R, C. Pacheco LG, P. d. Almeida JP, M. d. A. Belitardo EM, S. Pinheiro C, S. Campos G, R. G. R. Aguiar E. High-Quality Resolution of the Outbreak-Related Zika Virus Genome and Discovery of New Viruses Using Ion Torrent-Based Metatranscriptomics. Viruses 2020; 12:v12070782. [PMID: 32708079 PMCID: PMC7411838 DOI: 10.3390/v12070782] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2020] [Revised: 05/15/2020] [Accepted: 05/20/2020] [Indexed: 01/13/2023] Open
Abstract
Arboviruses, including the Zika virus, have recently emerged as one of the most important threats to human health. The use of metagenomics-based approaches has already proven valuable to aid surveillance of arboviral infections, and the ability to reconstruct complete viral genomes from metatranscriptomics data is key to the development of new control strategies for these diseases. Herein, we used RNA-based metatranscriptomics associated with Ion Torrent deep sequencing to allow for the high-quality reconstitution of an outbreak-related Zika virus (ZIKV) genome (10,739 nt), with extended 5'-UTR and 3'-UTR regions, using a newly-implemented bioinformatics approach. Besides allowing for the assembly of one of the largest complete ZIKV genomes to date, our strategy also yielded high-quality complete genomes of two arthropod-infecting viruses co-infecting C6/36 cell lines, namely: Alphamesonivirus 1 strain Salvador (20,194 nt) and Aedes albopictus totivirus-like (4618 nt); the latter likely represents a new viral species. Altogether, our results demonstrate that our bioinformatics approach associated with Ion Torrent sequencing allows for the high-quality reconstruction of known and unknown viral genomes, overcoming the main limitation of RNA deep sequencing for virus identification.
Collapse
Affiliation(s)
- Silvia I. Sardi
- Laboratory of Virology, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Bahia 40.110-100, Brazil; (S.I.S.); (R.H.C.); (G.S.C.)
| | - Rejane H. Carvalho
- Laboratory of Virology, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Bahia 40.110-100, Brazil; (S.I.S.); (R.H.C.); (G.S.C.)
| | - Luis G. C. Pacheco
- Post-Graduate Program in Biotechnology, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Bahia 40.110-100, Brazil; (L.G.C.P.); (C.S.P.)
| | - João P. P. d. Almeida
- Department of Biochemistry and Immunology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte (UFMG), Minas Gerais 31270-901, Brazil;
| | - Emilia M. M. d. A. Belitardo
- Post-Graduate Program in Immunology, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Bahia 40.110-100, Brazil;
| | - Carina S. Pinheiro
- Post-Graduate Program in Biotechnology, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Bahia 40.110-100, Brazil; (L.G.C.P.); (C.S.P.)
| | - Gúbio S. Campos
- Laboratory of Virology, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Bahia 40.110-100, Brazil; (S.I.S.); (R.H.C.); (G.S.C.)
| | - Eric R. G. R. Aguiar
- Post-Graduate Program in Biotechnology, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Salvador, Bahia 40.110-100, Brazil; (L.G.C.P.); (C.S.P.)
- Virus Bioinformatics Laboratory, Department of Biological Science (DCB), Center of Biotechnology and Genetics (CBG), State University of Santa Cruz (UESC), Rodovia Ilhéus-Itabuna km 16, Ilhéus, Bahia 45652-900, Brazil
- Correspondence:
| |
Collapse
|
|
40
|
Nitschko V, Kunzelmann S, Fröhlich T, Arnold GJ, Förstemann K. Trafficking of siRNA precursors by the dsRBD protein Blanks in Drosophila. Nucleic Acids Res 2020; 48:3906-3921. [PMID: 32025726 PMCID: PMC7144943 DOI: 10.1093/nar/gkaa072] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2019] [Revised: 01/21/2020] [Accepted: 02/03/2020] [Indexed: 01/03/2023] Open
Abstract
RNA interference targets aberrant transcripts with cognate small interfering RNAs, which derive from double-stranded RNA precursors. Several functional screens have identified Drosophila blanks/lump (CG10630) as a facilitator of RNAi, yet its molecular function has remained unknown. The protein carries two dsRNA binding domains (dsRBD) and blanks mutant males have a spermatogenesis defect. We demonstrate that blanks selectively boosts RNAi triggered by dsRNA of nuclear origin. Blanks binds dsRNA via its second dsRBD in vitro, shuttles between nucleus and cytoplasm and the abundance of siRNAs arising at many sites of convergent transcription is reduced in blanks mutants. Since features of nascent RNAs - such as introns and transcription beyond the polyA site – contribute to the small RNA pool, we propose that Blanks binds dsRNA formed by cognate nascent RNAs in the nucleus and fosters its export to the cytoplasm for dicing. We refer to the resulting small RNAs as blanks exported siRNAs (bepsiRNAs). While bepsiRNAs were fully dependent on RNA binding to the second dsRBD of blanks in transgenic flies, male fertility was not. This is consistent with a previous report that linked fertility to the first dsRBD of Blanks. The role of blanks in spermatogenesis appears thus unrelated to its role in dsRNA export.
Collapse
Affiliation(s)
- Volker Nitschko
- Genzentrum & Department Biochemie, Ludwig-Maximilians-Universität, 81377 München, Germany
| | - Stefan Kunzelmann
- Genzentrum & Department Biochemie, Ludwig-Maximilians-Universität, 81377 München, Germany
| | - Thomas Fröhlich
- Laboratory of Functional Genome Analysis, Ludwig-Maximilians-Universität, 81377 München, Germany
| | - Georg J Arnold
- Laboratory of Functional Genome Analysis, Ludwig-Maximilians-Universität, 81377 München, Germany
| | - Klaus Förstemann
- Genzentrum & Department Biochemie, Ludwig-Maximilians-Universität, 81377 München, Germany
| |
Collapse
|
|
41
|
From the Argonauts Mythological Sailors to the Argonautes RNA-Silencing Navigators: Their Emerging Roles in Human-Cell Pathologies. Int J Mol Sci 2020; 21:ijms21114007. [PMID: 32503341 PMCID: PMC7312461 DOI: 10.3390/ijms21114007] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2020] [Revised: 06/01/2020] [Accepted: 06/01/2020] [Indexed: 12/27/2022] Open
Abstract
Regulation of gene expression has emerged as a fundamental element of transcript homeostasis. Key effectors in this process are the Argonautes (AGOs), highly specialized RNA-binding proteins (RBPs) that form complexes, such as the RNA-Induced Silencing Complex (RISC). AGOs dictate post-transcriptional gene-silencing by directly loading small RNAs and repressing their mRNA targets through small RNA-sequence complementarity. The four human highly-conserved family-members (AGO1, AGO2, AGO3, and AGO4) demonstrate multi-faceted and versatile roles in transcriptome’s stability, plasticity, and functionality. The post-translational modifications of AGOs in critical amino acid residues, the nucleotide polymorphisms and mutations, and the deregulation of expression and interactions are tightly associated with aberrant activities, which are observed in a wide spectrum of pathologies. Through constantly accumulating information, the AGOs’ fundamental engagement in multiple human diseases has recently emerged. The present review examines new insights into AGO-driven pathology and AGO-deregulation patterns in a variety of diseases such as in viral infections and propagations, autoimmune diseases, cancers, metabolic deficiencies, neuronal disorders, and human infertility. Altogether, AGO seems to be a crucial contributor to pathogenesis and its targeting may serve as a novel and powerful therapeutic tool for the successful management of diverse human diseases in the clinic.
Collapse
|
|
42
|
Prasad AN, Ronk AJ, Widen SG, Wood TG, Basler CF, Bukreyev A. Ebola Virus Produces Discrete Small Noncoding RNAs Independently of the Host MicroRNA Pathway Which Lack RNA Interference Activity in Bat and Human Cells. J Virol 2020; 94:e01441-19. [PMID: 31852785 PMCID: PMC7158719 DOI: 10.1128/jvi.01441-19] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2019] [Accepted: 12/06/2019] [Indexed: 02/07/2023] Open
Abstract
The question as to whether RNA viruses produce bona fide microRNAs (miRNAs) during infection has been the focus of intense research and debate. Recently, several groups using computational prediction methods have independently reported possible miRNA candidates produced by Ebola virus (EBOV). Additionally, efforts to detect these predicted RNA products in samples from infected animals and humans have produced positive results. However, these studies and their conclusions are predicated on the assumption that these RNA products are actually processed through, and function within, the miRNA pathway. In the present study, we performed the first rigorous assessment of the ability of filoviruses to produce miRNA products during infection of both human and bat cells. Using next-generation sequencing, we detected several candidate miRNAs from both EBOV and the closely related Marburg virus (MARV). Focusing our validation efforts on EBOV, we found evidence contrary to the idea that these small RNA products function as miRNAs. The results of our study are important because they highlight the potential pitfalls of relying on computational methods alone for virus miRNA discovery.IMPORTANCE Here, we report the discovery, via deep sequencing, of numerous noncoding RNAs (ncRNAs) derived from both EBOV and MARV during infection of both bat and human cell lines. In addition to identifying several novel ncRNAs from both viruses, we identified two EBOV ncRNAs in our sequencing data that were near-matches to computationally predicted viral miRNAs reported in the literature. Using molecular and immunological techniques, we assessed the potential of EBOV ncRNAs to function as viral miRNAs. Importantly, we found little evidence supporting this hypothesis. Our work is significant because it represents the first rigorous assessment of the potential for EBOV to encode viral miRNAs and provides evidence contrary to the existing paradigm regarding the biological role of computationally predicted EBOV ncRNAs. Moreover, our work highlights further avenues of research regarding the nature and function of EBOV ncRNAs.
Collapse
Affiliation(s)
- Abhishek N Prasad
- Department of Pathology, The University of Texas Medical Branch, Galveston, Texas, USA
- Galveston National Laboratory, The University of Texas Medical Branch, Galveston, Texas, USA
- The University of Texas Medical Branch, Galveston, Texas, USA
| | - Adam J Ronk
- Department of Pathology, The University of Texas Medical Branch, Galveston, Texas, USA
- Galveston National Laboratory, The University of Texas Medical Branch, Galveston, Texas, USA
- The University of Texas Medical Branch, Galveston, Texas, USA
| | - Steven G Widen
- Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, Texas, USA
| | - Thomas G Wood
- Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, Texas, USA
| | - Christopher F Basler
- Center of Microbial Pathogenesis, Institute of Biomedical Sciences, Georgia State University, Atlanta, Georgia, USA
| | - Alexander Bukreyev
- Department of Pathology, The University of Texas Medical Branch, Galveston, Texas, USA
- Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas, USA
- Galveston National Laboratory, The University of Texas Medical Branch, Galveston, Texas, USA
- The University of Texas Medical Branch, Galveston, Texas, USA
| |
Collapse
|
|
43
|
Adiliaghdam F, Basavappa M, Saunders TL, Harjanto D, Prior JT, Cronkite DA, Papavasiliou N, Jeffrey KL. A Requirement for Argonaute 4 in Mammalian Antiviral Defense. Cell Rep 2020; 30:1690-1701.e4. [PMID: 32049003 PMCID: PMC7039342 DOI: 10.1016/j.celrep.2020.01.021] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2019] [Revised: 09/09/2019] [Accepted: 01/06/2020] [Indexed: 01/07/2023] Open
Abstract
While interferon (IFN) responses are critical for mammalian antiviral defense, induction of antiviral RNA interference (RNAi) is evident. To date, individual functions of the mammalian RNAi and micro RNA (miRNA) effector proteins Argonautes 1-4 (AGO1-AGO4) during virus infection remain undetermined. AGO2 was recently implicated in mammalian antiviral defense, so we examined antiviral activity of AGO1, AGO3, or AGO4 in IFN-competent immune cells. Only AGO4-deficient cells are hyper-susceptible to virus infection. AGO4 antiviral function is both IFN dependent and IFN independent, since AGO4 promotes IFN but also maintains antiviral capacity following prevention of IFN signaling or production. We identified AGO-loaded virus-derived short interfering RNAs (vsiRNAs), a molecular marker of antiviral RNAi, in macrophages infected with influenza or influenza lacking the IFN and RNAi suppressor NS1, which are uniquely diminished without AGO4. Importantly, AGO4-deficient influenza-infected mice have significantly higher burden and viral titers in vivo. Together, our data assign an essential role for AGO4 in mammalian antiviral defense.
Collapse
Affiliation(s)
- Fatemeh Adiliaghdam
- Division of Gastroenterology and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
| | - Megha Basavappa
- Division of Gastroenterology and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
| | - Tahnee L Saunders
- Division of Gastroenterology and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
| | - Dewi Harjanto
- Division of Immune Diversity, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
| | - John T Prior
- Division of Gastroenterology and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
| | - D Alexander Cronkite
- Division of Gastroenterology and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
| | - Nina Papavasiliou
- Division of Immune Diversity, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
| | - Kate L Jeffrey
- Division of Gastroenterology and Center for the Study of Inflammatory Bowel Disease, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
| |
Collapse
|
|
44
|
Abstract
Protection against microbial infection in eukaryotes is provided by diverse cellular and molecular mechanisms. Here, we present a comparative view of the antiviral activity of virus-derived small interfering RNAs in fungi, plants, invertebrates and mammals, detailing the mechanisms for their production, amplification and activity. We also highlight the recent discovery of viral PIWI-interacting RNAs in animals and a new role for mobile host and pathogen small RNAs in plant defence against eukaryotic pathogens. In turn, viruses that infect plants, insects and mammals, as well as eukaryotic pathogens of plants, have evolved specific virulence proteins that suppress RNA interference (RNAi). Together, these advances suggest that an antimicrobial function of the RNAi pathway is conserved across eukaryotic kingdoms.
Collapse
|
|
45
|
No evidence for viral small RNA production and antiviral function of Argonaute 2 in human cells. Sci Rep 2019; 9:13752. [PMID: 31551491 PMCID: PMC6760161 DOI: 10.1038/s41598-019-50287-w] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2019] [Accepted: 09/09/2019] [Indexed: 12/22/2022] Open
Abstract
RNA interference (RNAi) has strong antiviral activity in a range of animal phyla, but the extent to which RNAi controls virus infection in chordates, and specifically mammals remains incompletely understood. Here we analyze the antiviral activity of RNAi against a number of positive-sense RNA viruses using Argonaute-2 deficient human cells. In line with absence of virus-derived siRNAs, Sindbis virus, yellow fever virus, and encephalomyocarditis virus replicated with similar kinetics in wildtype cells and Argonaute-2 deficient cells. Coxsackievirus B3 (CVB3) carrying mutations in the viral 3A protein, previously proposed to be a virus-encoded suppressor of RNAi in another picornavirus, human enterovirus 71, had a strong replication defect in wildtype cells. However, this defect was not rescued in Argonaute-2 deficient cells, arguing against a role of CVB3 3A as an RNAi suppressor. In agreement, neither infection with wildtype nor 3A mutant CVB3 resulted in small RNA production with the hallmarks of canonical vsiRNAs. Together, our results argue against strong antiviral activity of RNAi under these experimental conditions, but do not exclude that antiviral RNAi may be functional under other cellular, experimental, or physiological conditions in mammals.
Collapse
|
|
46
|
Lee KM, Gong YN, Shih SR. Methods for detection and study of virus-derived small RNAs produced from the intramolecular base-pairing region of the picornavirus genome. Methods 2019; 183:4-12. [PMID: 31493516 DOI: 10.1016/j.ymeth.2019.08.011] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2019] [Revised: 08/26/2019] [Accepted: 08/30/2019] [Indexed: 10/26/2022] Open
Abstract
There is conclusive evidential support for the existence of virus-derived small RNA (vsRNA) in mammals. Two types of vsRNA have been reported from picornaviruses. The first is virus-derived short-interfering RNA (vsiRNA) that is processed from viral double-stranded RNA intermediates during RNA replication. The other is small RNA derived from the highly base-paired single-stranded genomic region, e.g. the internal ribosome entry site (IRES) of picornaviruses. vsiRNA interacts with the Argonaute protein to control viral RNA replication through the process of RNA interference. However, the function of structure-based vsRNA is largely unknown. We previously identified vsRNA1 generated from the enterovirus-A71 (EV-A71) IRES region by the endogenous enzyme Dicer. Exogenous vsRNA1 can inhibit IRES activity both in vivo and in vitro, hence viral replication is inhibited. Here we describe key methods used to characterize vsRNA, including annotation by next-generation sequencing, abundance measurement by Northern blotting, determination of Dicer-dependence by gel-shift assay and in vitro cleavage assay, and the inhibitory effect on IRES activity via in vitro translation assay.
Collapse
Affiliation(s)
- Kuo-Ming Lee
- Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan
| | - Yu-Nong Gong
- Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Department of Laboratory Medicine, Linkou Chang Gung Memorial Hospital, Taoyuan, Taiwan
| | - Shin-Ru Shih
- Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Department of Laboratory Medicine, Linkou Chang Gung Memorial Hospital, Taoyuan, Taiwan; Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Research Center for Chinese Herbal Medicine, Research Center for Food and Cosmetic Safety, and Graduate Institute of Health Industry Technology, College of Human Ecology, Chang Gung University of Science and Technology, Taoyuan, Taiwan.
| |
Collapse
|
|
47
|
Mukhopadhyay U, Chanda S, Patra U, Mukherjee A, Komoto S, Chawla-Sarkar M. Biphasic regulation of RNA interference during rotavirus infection by modulation of Argonaute2. Cell Microbiol 2019; 21:e13101. [PMID: 31424151 PMCID: PMC7162324 DOI: 10.1111/cmi.13101] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2019] [Revised: 06/29/2019] [Accepted: 08/16/2019] [Indexed: 12/20/2022]
Abstract
RNA interference (RNAi) is an evolutionary ancient innate immune response in plants, nematodes, and arthropods providing natural protection against viral infection. Viruses have also gained counter‐defensive measures by producing virulence determinants called viral‐suppressors‐of‐RNAi (VSRs). Interestingly, in spite of dominance of interferon‐based immunity over RNAi in somatic cells of higher vertebrates, recent reports are accumulating in favour of retention of the antiviral nature of RNAi in mammalian cells. The present study focuses on the modulation of intracellular RNAi during infection with rotavirus (RV), an enteric virus with double‐stranded RNA genome. Intriguingly, a time point‐dependent bimodal regulation of RNAi was observed in RV‐infected cells, where short interfering RNA (siRNA)‐based RNAi was rendered non‐functional during early hours of infection only to be reinstated fully beyond that early infection stage. Subsequent investigations revealed RV nonstructural protein 1 to serve as a putative VSR by associating with and triggering degradation of Argonaute2 (AGO2), the prime effector of siRNA‐mediated RNAi, via ubiquitin–proteasome pathway. The proviral significance of AGO2 degradation was further confirmed when ectopic overexpression of AGO2 significantly reduced RV infection. Cumulatively, the current study presents a unique modulation of host RNAi during RV infection, highlighting the importance of antiviral RNAi in mammalian cells.
Collapse
Affiliation(s)
- Urbi Mukhopadhyay
- Division of Virology, National Institute of Cholera and Enteric Diseases, Kolkata, India
| | - Shampa Chanda
- Department of Biotechnology, GITAM Institute of Science, Visakhapatnam, India
| | - Upayan Patra
- Division of Virology, National Institute of Cholera and Enteric Diseases, Kolkata, India
| | - Anupam Mukherjee
- Division of Virology, National Institute of Cholera and Enteric Diseases, Kolkata, India
| | - Satoshi Komoto
- Department of Virology and Parasitology, School of Medicine, Fujita Health University, Aichi, Japan
| | - Mamta Chawla-Sarkar
- Division of Virology, National Institute of Cholera and Enteric Diseases, Kolkata, India
| |
Collapse
|
|
48
|
Antiviral RNAi in Insects and Mammals: Parallels and Differences. Viruses 2019; 11:v11050448. [PMID: 31100912 PMCID: PMC6563508 DOI: 10.3390/v11050448] [Citation(s) in RCA: 61] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2019] [Revised: 05/14/2019] [Accepted: 05/15/2019] [Indexed: 12/26/2022] Open
Abstract
The RNA interference (RNAi) pathway is a potent antiviral defense mechanism in plants and invertebrates, in response to which viruses evolved suppressors of RNAi. In mammals, the first line of defense is mediated by the type I interferon system (IFN); however, the degree to which RNAi contributes to antiviral defense is still not completely understood. Recent work suggests that antiviral RNAi is active in undifferentiated stem cells and that antiviral RNAi can be uncovered in differentiated cells in which the IFN system is inactive or in infections with viruses lacking putative viral suppressors of RNAi. In this review, we describe the mechanism of RNAi and its antiviral functions in insects and mammals. We draw parallels and highlight differences between (antiviral) RNAi in these classes of animals and discuss open questions for future research.
Collapse
|
|
49
|
Maillard PV, van der Veen AG, Poirier EZ, Reis e Sousa C. Slicing and dicing viruses: antiviral RNA interference in mammals. EMBO J 2019; 38:e100941. [PMID: 30872283 PMCID: PMC6463209 DOI: 10.15252/embj.2018100941] [Citation(s) in RCA: 78] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2018] [Revised: 01/17/2019] [Accepted: 01/25/2019] [Indexed: 12/15/2022] Open
Abstract
To protect against the harmful consequences of viral infections, organisms are equipped with sophisticated antiviral mechanisms, including cell-intrinsic means to restrict viral replication and propagation. Plant and invertebrate cells utilise mostly RNA interference (RNAi), an RNA-based mechanism, for cell-intrinsic immunity to viruses while vertebrates rely on the protein-based interferon (IFN)-driven innate immune system for the same purpose. The RNAi machinery is conserved in vertebrate cells, yet whether antiviral RNAi is still active in mammals and functionally relevant to mammalian antiviral defence is intensely debated. Here, we discuss cellular and viral factors that impact on antiviral RNAi and the contexts in which this system might be at play in mammalian resistance to viral infection.
Collapse
Affiliation(s)
- Pierre V Maillard
- Division of Infection and Immunity, University College London, London, UK
| | | | - Enzo Z Poirier
- Immunobiology Laboratory, The Francis Crick Institute, London, UK
| | | |
Collapse
|
|
50
|
Xu YP, Qiu Y, Zhang B, Chen G, Chen Q, Wang M, Mo F, Xu J, Wu J, Zhang RR, Cheng ML, Zhang NN, Lyu B, Zhu WL, Wu MH, Ye Q, Zhang D, Man JH, Li XF, Cui J, Xu Z, Hu B, Zhou X, Qin CF. Zika virus infection induces RNAi-mediated antiviral immunity in human neural progenitors and brain organoids. Cell Res 2019; 29:265-273. [PMID: 30814679 PMCID: PMC6461993 DOI: 10.1038/s41422-019-0152-9] [Citation(s) in RCA: 123] [Impact Index Per Article: 20.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2018] [Accepted: 01/29/2019] [Indexed: 12/27/2022] Open
Abstract
The re-emergence of Zika virus (ZIKV) in the Western Hemisphere has resulted in global public health crisis since 2015. ZIKV preferentially infects and targets human neural progenitor cells (hNPCs) and causes fetal microcephaly upon maternal infection. hNPCs not only play critical roles during fetal brain development, but also persist in adult brain throughout life. Yet the mechanism of innate antiviral immunity in hNPCs remains largely unknown. Here, we show that ZIKV infection triggers the abundant production of virus-derived small interfering RNAs in hNPCs, but not in the more differentiated progenies or somatic cells. Ablation of key RNAi machinery components significantly enhances ZIKV replication in hNPCs. Furthermore, enoxacin, a broad-spectrum antibiotic that is known as an RNAi enhancer, exerts potent anti-ZIKV activity in hNPCs and other RNAi-competent cells. Strikingly, enoxacin treatment completely prevents ZIKV infection and circumvents ZIKV-induced microcephalic phenotypes in brain organoid models that recapitulate human fetal brain development. Our findings highlight the physiological importance of RNAi-mediated antiviral immunity during the early stage of human brain development, uncovering a novel strategy to combat human congenital viral infections through enhancing RNAi.
Collapse
Affiliation(s)
- Yan-Peng Xu
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, 100071, Beijing, China
| | - Yang Qiu
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan, 430071, Hubei, China
| | - Boya Zhang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, CAS, 100101, Beijing, China
- University of Chinese Academy of Sciences, 100049, Beijing, China
| | - Guilai Chen
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, CAS, 100101, Beijing, China
- University of Chinese Academy of Sciences, 100049, Beijing, China
| | - Qi Chen
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, 100071, Beijing, China
| | - Miao Wang
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan, 430071, Hubei, China
| | - Fan Mo
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, CAS, 100101, Beijing, China
- University of Chinese Academy of Sciences, 100049, Beijing, China
| | - Jiuyue Xu
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan, 430071, Hubei, China
- University of Chinese Academy of Sciences, 100049, Beijing, China
| | - Jin Wu
- State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, 100850, Beijing, China
| | - Rong-Rong Zhang
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, 100071, Beijing, China
- School of Basic Sciences, Anhui Medical University, Hefei, 230032, Anhui, China
| | - Meng-Li Cheng
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, 100071, Beijing, China
- School of Basic Sciences, Anhui Medical University, Hefei, 230032, Anhui, China
| | - Na-Na Zhang
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, 100071, Beijing, China
| | - Bao Lyu
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan, 430071, Hubei, China
- College of Life Sciences, Wuhan University, Wuhan, 430072, Hubei, China
| | - Wen-Liang Zhu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, CAS, 100101, Beijing, China
- University of Chinese Academy of Sciences, 100049, Beijing, China
| | - Meng-Hua Wu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, CAS, 100101, Beijing, China
- University of Chinese Academy of Sciences, 100049, Beijing, China
| | - Qing Ye
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, 100071, Beijing, China
| | - Da Zhang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, CAS, 100101, Beijing, China
- University of Chinese Academy of Sciences, 100049, Beijing, China
| | - Jiang-Hong Man
- State Key Laboratory of Proteomics, Institute of Basic Medical Sciences, National Center of Biomedical Analysis, 100850, Beijing, China
| | - Xiao-Feng Li
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, 100071, Beijing, China
| | - Jie Cui
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan, 430071, Hubei, China
- University of Chinese Academy of Sciences, 100049, Beijing, China
| | - Zhiheng Xu
- University of Chinese Academy of Sciences, 100049, Beijing, China
- State Key Laboratory of Molecular Developmental Biology, Center for Excellence in Brain Science and Intelligence Technology, Institute of Genetics and Developmental Biology, CAS, 100101, Beijing, China
| | - Baoyang Hu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, CAS, 100101, Beijing, China.
- University of Chinese Academy of Sciences, 100049, Beijing, China.
| | - Xi Zhou
- State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences (CAS), Wuhan, 430071, Hubei, China.
- University of Chinese Academy of Sciences, 100049, Beijing, China.
- College of Life Sciences, Wuhan University, Wuhan, 430072, Hubei, China.
| | - Cheng-Feng Qin
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, 100071, Beijing, China.
- School of Basic Sciences, Anhui Medical University, Hefei, 230032, Anhui, China.
| |
Collapse
|