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Wu W, Wang M, Deng Z, Xi M, Dong Y, Wang H, Zhang J, Wang C, Zhou Y, Xu Q. The miR-184-3p promotes rice black-streaked dwarf virus infection by suppressing Ken in Laodelphax striatellus (Fallén). PEST MANAGEMENT SCIENCE 2024; 80:1849-1858. [PMID: 38050810 DOI: 10.1002/ps.7917] [Citation(s) in RCA: 8] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/28/2023] [Revised: 11/02/2023] [Accepted: 12/05/2023] [Indexed: 12/06/2023]
Abstract
BACKGROUND MicroRNAs (miRNAs) play a key role in various biological processes by influencing the translation of target messenger RNAs (mRNAs) through post-transcriptional regulation. The miR-184-3p has been identified as an abundant conserved miRNA in insects. However, less is known about its functions in insect-plant virus interactions. RESULTS The function of miR-184-3p in regulation of plant viral infection in insects was investigated using a rice black-streaked dwarf virus (RBSDV) and Laodelphax striatellus (Fallén) interaction system. We found that the expression of miR-184-3p increased in L. striatellus after RBSDV infection. Injection of miR-184-3p mimics increased RBSDV accumulation, while treatment with miR-184-3p antagomirs inhibits the viral accumulation in L. striatellus. Ken, a zinc finger protein, was identified as a target of miR-184-3p. Knockdown of Ken increased the virus accumulation and promoted RBSDV transmission by L. striatellus. CONCLUSION This study demonstrates that RBSDV infection induces the expression of miR-184-3p in its insect vector L. striatellus. The miR-184-3p targets Ken to promote RBSDV accumulation and transmission. These findings provide a new insight into the function of the miRNAs in regulating plant viral infection in its insect vector. © 2023 Society of Chemical Industry.
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Affiliation(s)
- Wei Wu
- Key Laboratory of Food Quality and Safety of Jiangsu Province - State Key Laboratory Breeding Base, Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China
- Key laboratory of Acupuncture and Medicine Research of Ministry of Education, Nanjing University of Chinese Medicine, Nanjing, China
| | - Man Wang
- Key Laboratory of Food Quality and Safety of Jiangsu Province - State Key Laboratory Breeding Base, Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China
| | - Zhiting Deng
- Key Laboratory of Food Quality and Safety of Jiangsu Province - State Key Laboratory Breeding Base, Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China
- College of Life Sciences, Zhejiang Normal University, Jinhua, China
| | - Minmin Xi
- Key Laboratory of Food Quality and Safety of Jiangsu Province - State Key Laboratory Breeding Base, Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China
- College of Life Sciences, Zhejiang Normal University, Jinhua, China
| | - Yan Dong
- Key Laboratory of Food Quality and Safety of Jiangsu Province - State Key Laboratory Breeding Base, Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China
| | - Haitao Wang
- Key Laboratory of Food Quality and Safety of Jiangsu Province - State Key Laboratory Breeding Base, Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China
| | - Jianhua Zhang
- Key Laboratory of Food Quality and Safety of Jiangsu Province - State Key Laboratory Breeding Base, Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China
| | - Changchun Wang
- College of Life Sciences, Zhejiang Normal University, Jinhua, China
| | - Yijun Zhou
- Key Laboratory of Food Quality and Safety of Jiangsu Province - State Key Laboratory Breeding Base, Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China
| | - Qiufang Xu
- Key Laboratory of Food Quality and Safety of Jiangsu Province - State Key Laboratory Breeding Base, Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China
- College of Life Sciences, Anhui Normal University, Wuhu, China
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2
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Abel SM, Hong Z, Williams D, Ireri S, Brown MQ, Su T, Hung KY, Henke JA, Barton JP, Le Roch KG. Small RNA sequencing of field Culex mosquitoes identifies patterns of viral infection and the mosquito immune response. Sci Rep 2023; 13:10598. [PMID: 37391513 PMCID: PMC10313667 DOI: 10.1038/s41598-023-37571-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2023] [Accepted: 06/23/2023] [Indexed: 07/02/2023] Open
Abstract
Mosquito-borne disease remains a significant burden on global health. In the United States, the major threat posed by mosquitoes is transmission of arboviruses, including West Nile virus by mosquitoes of the Culex genus. Virus metagenomic analysis of mosquito small RNA using deep sequencing and advanced bioinformatic tools enables the rapid detection of viruses and other infecting organisms, both pathogenic and non-pathogenic to humans, without any precedent knowledge. In this study, we sequenced small RNA samples from over 60 pools of Culex mosquitoes from two major areas of Southern California from 2017 to 2019 to elucidate the virome and immune responses of Culex. Our results demonstrated that small RNAs not only allowed the detection of viruses but also revealed distinct patterns of viral infection based on location, Culex species, and time. We also identified miRNAs that are most likely involved in Culex immune responses to viruses and Wolbachia bacteria, and show the utility of using small RNA to detect antiviral immune pathways including piRNAs against some pathogens. Collectively, these findings show that deep sequencing of small RNA can be used for virus discovery and surveillance. One could also conceive that such work could be accomplished in various locations across the world and over time to better understand patterns of mosquito infection and immune response to many vector-borne diseases in field samples.
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Affiliation(s)
- Steven M Abel
- Department of Molecular, Cell and Systems Biology, Center for Infection Disease and Vector Research, University of California, Riverside, CA, 92521, USA
| | - Zhenchen Hong
- Department of Physics and Astronomy, University of California, Riverside, CA, 92521, USA
| | - Desiree Williams
- Department of Molecular, Cell and Systems Biology, Center for Infection Disease and Vector Research, University of California, Riverside, CA, 92521, USA
| | - Sally Ireri
- Department of Molecular, Cell and Systems Biology, Center for Infection Disease and Vector Research, University of California, Riverside, CA, 92521, USA
| | - Michelle Q Brown
- West Valley Mosquito & Vector Control District, Ontario, CA, 91761, USA
| | - Tianyun Su
- West Valley Mosquito & Vector Control District, Ontario, CA, 91761, USA
| | - Kim Y Hung
- Coachella Valley Mosquito & Vector Control District, Indio, CA, 92201, USA
| | - Jennifer A Henke
- Coachella Valley Mosquito & Vector Control District, Indio, CA, 92201, USA
| | - John P Barton
- Department of Physics and Astronomy, University of California, Riverside, CA, 92521, USA
| | - Karine G Le Roch
- Department of Molecular, Cell and Systems Biology, Center for Infection Disease and Vector Research, University of California, Riverside, CA, 92521, USA.
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Li X, Ma S, Gao T, Mai Y, Song Z, Yang J. The main battlefield of mRNA vaccine – Tumor immune microenvironment. Int Immunopharmacol 2022; 113:109367. [DOI: 10.1016/j.intimp.2022.109367] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2022] [Revised: 10/03/2022] [Accepted: 10/15/2022] [Indexed: 11/05/2022]
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4
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Lei L, Cheng A, Wang M, Jia R. The Influence of Host miRNA Binding to RNA Within RNA Viruses on Virus Multiplication. Front Cell Infect Microbiol 2022; 12:802149. [PMID: 35531344 PMCID: PMC9069554 DOI: 10.3389/fcimb.2022.802149] [Citation(s) in RCA: 15] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2021] [Accepted: 03/14/2022] [Indexed: 11/13/2022] Open
Abstract
microRNAs (miRNAs), non-coding RNAs about 22 nt long, regulate the post-transcription expression of genes to influence many cellular processes. The expression of host miRNAs is affected by virus invasion, which also affects virus replication. Increasing evidence has demonstrated that miRNA influences RNA virus multiplication by binding directly to the RNA virus genome. Here, the knowledge relating to miRNAs’ relationships between host miRNAs and RNA viruses are discussed.
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Affiliation(s)
- Lin Lei
- Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Anchun Cheng
- Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Mingshu Wang
- Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Renyong Jia
- Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
- *Correspondence: Renyong Jia,
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5
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Xu TL, Sun YW, Feng XY, Zhou XN, Zheng B. Development of miRNA-Based Approaches to Explore the Interruption of Mosquito-Borne Disease Transmission. Front Cell Infect Microbiol 2021; 11:665444. [PMID: 34235091 PMCID: PMC8256169 DOI: 10.3389/fcimb.2021.665444] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2021] [Accepted: 06/02/2021] [Indexed: 01/21/2023] Open
Abstract
MicroRNA (miRNA or miR)-based approaches to interrupt the transmission of mosquito-borne diseases have been explored since 2005. A review of these studies and areas in which to proceed is needed. In this review, significant progress is reviewed at the level of individual miRNAs, and miRNA diversification and relevant confounders are described in detail. Current miRNA studies in mosquitoes include four steps, namely, identifying miRNAs, validating miRNA-pathogen interactions, exploring action mechanisms, and performing preapplication investigations. Notably, regarding the Plasmodium parasite, mosquito miRNAs generally bind to mosquito immunity- or development-related mRNAs, indirectly regulating Plasmodium infection; However, regarding arboviruses, mosquito miRNAs can bind to the viral genome, directly modifying viral replication. Thus, during explorations of miRNA-based approaches, researchers need select an ideal miRNA for investigation based on the mosquito species, tissue, and mosquito-borne pathogen of interest. Additionally, strategies for miRNA-based approaches differ for arboviruses and protozoan parasites.
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Affiliation(s)
- Tie-Long Xu
- Evidence-Based Medicine Research Center, Jiangxi University of Chinese Medicine, Nanchang, China
- National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, and WHO Collaborating Center for Tropical Diseases, Shanghai, China
- Key Laboratory of Parasite and Vector Biology, Ministry of Public Health, Shanghai, China
| | - Ya-Wen Sun
- National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, and WHO Collaborating Center for Tropical Diseases, Shanghai, China
- Key Laboratory of Parasite and Vector Biology, Ministry of Public Health, Shanghai, China
| | - Xin-Yu Feng
- National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, and WHO Collaborating Center for Tropical Diseases, Shanghai, China
- Key Laboratory of Parasite and Vector Biology, Ministry of Public Health, Shanghai, China
- School of Global Health, Chinese Center for Tropical Diseases Research, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Xiao-Nong Zhou
- National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, and WHO Collaborating Center for Tropical Diseases, Shanghai, China
- Key Laboratory of Parasite and Vector Biology, Ministry of Public Health, Shanghai, China
- School of Global Health, Chinese Center for Tropical Diseases Research, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Bin Zheng
- National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, and WHO Collaborating Center for Tropical Diseases, Shanghai, China
- Key Laboratory of Parasite and Vector Biology, Ministry of Public Health, Shanghai, China
- School of Global Health, Chinese Center for Tropical Diseases Research, Shanghai Jiao Tong University School of Medicine, Shanghai, China
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6
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Infection of Aedes aegypti Mosquitoes with Midgut-Attenuated Sindbis Virus Reduces, but Does Not Eliminate, Disseminated Infection. J Virol 2021; 95:e0013621. [PMID: 33853958 DOI: 10.1128/jvi.00136-21] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Arboviruses are transmitted by specific vectors, and the reasons for this specificity are not fully understood. One contributing factor is the existence of tissue barriers within the vector such as the midgut escape barrier. We used microRNA (miRNA) targeting of Sindbis virus (SINV) to study how replication in midgut cells contributes to overcoming this barrier in the mosquito Aedes aegypti. SINV constructs were designed to be attenuated specifically in midgut cells by inserting binding sites for midgut-specific miRNAs into either the 3' untranslated region (MRE3'miRT) or the structural open reading frame (MRE-ORFmiRT) of the SINV genome. Both miRNA-targeted viruses replicated less efficiently than control viruses in the presence of these miRNAs. When mosquitoes were given infectious blood meals containing miRNA-targeted viruses, only around 20% (MRE3'miRT) or 40% (MRE-ORFmiRT) of mosquitoes developed disseminated infection. In contrast, dissemination occurred in almost all mosquitoes fed control viruses. Deep sequencing of virus populations from individual mosquitoes ruled out selection for mutations in the inserted target sequences as the cause for dissemination in these mosquitoes. In mosquitoes that became infected with miRNA-targeted viruses, titers were equivalent to those of mosquitoes infected with control virus in both the midgut and the carcass, and there was no evidence of a threshold titer necessary for dissemination. Instead, it appeared that if infection was successfully established in the midgut, replication and dissemination were largely normal. Our results support the hypothesis that replication is an important factor in allowing SINV to overcome the midgut escape barrier but hint that other factors are also likely involved. IMPORTANCE When a mosquito ingests an arbovirus during a blood meal, the arbovirus must escape from the midgut of the vector and infect the salivary glands in order to be transmitted to a new host. We used tissue-specific miRNA targeting to examine the requirement for Sindbis virus (SINV) to replicate in midgut epithelium in order to cause disseminated infection in the mosquito Aedes aegypti. Our results indicate that specifically reducing the ability of SINV to replicate in the mosquito midgut reduces its overall ability to establish infection in the mosquito, but if infection is established, replication and dissemination occur normally. These results are consistent with an importance for replication in the midgut epithelium in aiding arboviruses in crossing the midgut barrier.
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7
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Haque A, Akçeşme FB, Pant AB. A review of Zika virus: hurdles toward vaccine development and the way forward. Antivir Ther 2019; 23:285-293. [PMID: 29300166 DOI: 10.3851/imp3215] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/08/2017] [Indexed: 10/18/2022]
Abstract
The Zika virus (ZIKV) epidemic has recently emerged as a public health threat due to its teratogenic nature and association with the serious neurological condition Guillain-Barré syndrome (GBS). To date, no approved antiviral therapeutics to treat, nor vaccines to prevent, ZIKV infection are available. In order to develop effective anti-ZIKV vaccines, improved animal models and a better understanding of immunological correlates of protection against ZIKV are required. In this paper, we discuss the recent progress in developing vaccines against ZIKV and the hurdles to overcome in making efficacious anti-ZIKV vaccines. Here, we propose strategies to make efficacious and safe vaccines against ZIKV by using novel approaches including molecular attenuation of viruses and TLR-based nanoparticle vaccines. The question of exacerbating dengue virus infection or causing GBS through the production of cross-reactive immunity targeting viral or host proteins have been addressed in this paper. Challenges in implementing immunogenic and protective ZIKV vaccine trials in immunodepressed target populations (for example, pregnant women) have also been discussed.
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Affiliation(s)
- Azizul Haque
- Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA
| | - Faruk Berat Akçeşme
- Department of Biostatistics and Medical Informatics at University of Medical Sciences, Üsküdar/İstanbul, Turkey
| | - Anudeep B Pant
- School of Public Health and Tropical Medicine at Tulane University, New Orleans, LA, USA
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8
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Stable and Highly Immunogenic MicroRNA-Targeted Single-Dose Live Attenuated Vaccine Candidate against Tick-Borne Encephalitis Constructed Using Genetic Backbone of Langat Virus. mBio 2019; 10:mBio.02904-18. [PMID: 31015334 PMCID: PMC6479010 DOI: 10.1128/mbio.02904-18] [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] [Indexed: 12/31/2022] Open
Abstract
Tick-borne encephalitis virus (TBEV) is one of the most medically important tick-borne pathogens of the Old World. Despite decades of active research, efforts to develop of TBEV live attenuated virus (LAV) vaccines with acceptable safety and immunogenicity characteristics have not been successful. Here we report the development and evaluation of a highly attenuated and immunogenic microRNA-targeted TBEV LAV. Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is one of the most medically important tick-borne pathogens of the Old World. Despite decades of active research, attempts to develop of a live attenuated virus (LAV) vaccine against TBEV with acceptable safety and immunogenicity characteristics have not been successful. To overcome this impasse, we generated a chimeric TBEV that was highly immunogenic in nonhuman primates (NHPs). The chimeric virus contains the prM/E genes of TBEV, which are expressed in the genetic background of an antigenically closely related, but less pathogenic member of the TBEV complex—Langat virus (LGTV), strain T-1674. The neurovirulence of this chimeric virus was subsequently controlled by robust targeting of the viral genome with multiple copies of central nervous system-enriched microRNAs (miRNAs). This miRNA-targeted T/1674-mirV2 virus was highly stable in Vero cells and was not pathogenic in various mouse models of infection or in NHPs. Importantly, in NHPs, a single dose of the T/1674-mirV2 virus induced TBEV-specific neutralizing antibody (NA) levels comparable to those seen with a three-dose regimen of an inactivated TBEV vaccine, currently available in Europe. Moreover, our vaccine candidate provided complete protection against a stringent wild-type TBEV challenge in mice and against challenge with a parental (not miRNA-targeted) chimeric TBEV/LGTV in NHPs. Thus, this highly attenuated and immunogenic T/1674-mirV2 virus is a promising LAV vaccine candidate against TBEV and warrants further preclinical evaluation of its neurovirulence in NHPs prior to entering clinical trials in humans.
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9
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Niranjan R, Muthukumaravel S, Jambulingam P. The Involvement of Neuroinflammation in Dengue Viral Disease: Importance of Innate and Adaptive Immunity. Neuroimmunomodulation 2019; 26:111-118. [PMID: 31352457 DOI: 10.1159/000501209] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/18/2019] [Accepted: 05/23/2019] [Indexed: 11/19/2022] Open
Abstract
Neuroinflammation (inflammation in brain) has been known to play an important role in the development of dengue virus disease. Recently, studies from both clinical and experimental models suggest the involvement of neuroinflammation in dengue viral disease. Studies in clinical setup demonstrated that, microglial cells are actively involved in the patients having dengue virus infection, showing involvement of innate immune response in neuroinflammation. It was further proved that, clinical isolates of dengue-2 virus were able to initiate the pathologic response when injected in the mice brain. Natural killer cells were also found to play a crucial role to activate adaptive immune response. Notably, CXCL10/IFN-inducible protein 10 and CXCR3 are involved in dengue virus-mediated pathogenesis and play an important role in the development of dengue virus-mediated paralysis. In a latest report, it was seen that intracranial injection of dengue virus increases the CD8+ T-cell infiltration in brain, showing an important mechanism of neuroinflammation during the dengue virus infection. A similar study has described that, when DENV-3 is injected into the mice, it enhances the infiltration of CD8+ and CD4+ T cells as well as neutrophils. Cells immune-reactive against NS3 antigen were found throughout the brain. In conclusion, we focus on the various molecular mechanisms which contribute to the basic understanding about the role of neuroinflammation in dengue fever. These mechanisms will help in better understanding dengue pathophysiology and thus help in the development of possible therapeutics.
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Affiliation(s)
- Rituraj Niranjan
- Unit of Microbiology and Molecular Biology, ICMR-Vector Control Research Center, Puducherry, India,
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10
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Fay EJ, Langlois RA. MicroRNA-Attenuated Virus Vaccines. Noncoding RNA 2018; 4:E25. [PMID: 30279330 PMCID: PMC6316615 DOI: 10.3390/ncrna4040025] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2018] [Revised: 09/25/2018] [Accepted: 09/28/2018] [Indexed: 12/18/2022] Open
Abstract
Live-attenuated vaccines are the most effective way to establish robust, long-lasting immunity against viruses. However, the possibility of reversion to wild type replication and pathogenicity raises concerns over the safety of these vaccines. The use of host-derived microRNAs (miRNAs) to attenuate viruses has been accomplished in an array of biological contexts. The broad assortment of effective tissue- and species-specific miRNAs, and the ability to target a virus with multiple miRNAs, allow for targeting to be tailored to the virus of interest. While escape is always a concern, effective strategies have been developed to improve the safety and stability of miRNA-attenuated viruses. In this review, we discuss the various approaches that have been used to engineer miRNA-attenuated viruses, the steps that have been taken to improve their safety, and the potential use of these viruses as vaccines.
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Affiliation(s)
- Elizabeth J Fay
- Biochemistry, Molecular Biology, and Biophysics Graduate Program, University of Minnesota, Minneapolis, MN 55455, USA.
- Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA.
| | - Ryan A Langlois
- Biochemistry, Molecular Biology, and Biophysics Graduate Program, University of Minnesota, Minneapolis, MN 55455, USA.
- Center for Immunology, University of Minnesota, Minneapolis, MN 55455, USA.
- Department of Microbiology and Immunology, University of Minnesota, Minneapolis, MN 55455, USA.
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11
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Leggewie M, Schnettler E. RNAi-mediated antiviral immunity in insects and their possible application. Curr Opin Virol 2018; 32:108-114. [DOI: 10.1016/j.coviro.2018.10.004] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2018] [Revised: 10/16/2018] [Accepted: 10/23/2018] [Indexed: 01/09/2023]
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12
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Aguado LC, Jordan TX, Hsieh E, Blanco-Melo D, Heard J, Panis M, Vignuzzi M, tenOever BR. Homologous recombination is an intrinsic defense against antiviral RNA interference. Proc Natl Acad Sci U S A 2018. [PMID: 30209219 DOI: 10.1073/pnas.181022911] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/25/2023] Open
Abstract
RNA interference (RNAi) is the major antiviral defense mechanism of plants and invertebrates, rendering the capacity to evade it a defining factor in shaping the viral landscape. Here we sought to determine whether different virus replication strategies provided any inherent capacity to evade RNAi in the absence of an antagonist. Through the exploitation of host microRNAs, we recreated an RNAi-like environment in vertebrates and directly compared the capacity of positive- and negative-stranded RNA viruses to cope with this selective pressure. Applying this defense against four distinct viral families revealed that the capacity to undergo homologous recombination was the defining attribute that enabled evasion of this defense. Independent of gene expression strategy, positive-stranded RNA viruses that could undergo strand switching rapidly excised genomic material, while negative-stranded viruses were effectively targeted and cleared upon RNAi-based selection. These data suggest a dynamic relationship between host antiviral defenses and the biology of virus replication in shaping pathogen prevalence.
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Affiliation(s)
- Lauren C Aguado
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Tristan X Jordan
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Emily Hsieh
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Daniel Blanco-Melo
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - John Heard
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Maryline Panis
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029
| | - Marco Vignuzzi
- Viral Populations and Pathogenesis Unit, Institute Pasteur, 75015 Paris, France
| | - Benjamin R tenOever
- Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029;
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13
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Homologous recombination is an intrinsic defense against antiviral RNA interference. Proc Natl Acad Sci U S A 2018; 115:E9211-E9219. [PMID: 30209219 DOI: 10.1073/pnas.1810229115] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
RNA interference (RNAi) is the major antiviral defense mechanism of plants and invertebrates, rendering the capacity to evade it a defining factor in shaping the viral landscape. Here we sought to determine whether different virus replication strategies provided any inherent capacity to evade RNAi in the absence of an antagonist. Through the exploitation of host microRNAs, we recreated an RNAi-like environment in vertebrates and directly compared the capacity of positive- and negative-stranded RNA viruses to cope with this selective pressure. Applying this defense against four distinct viral families revealed that the capacity to undergo homologous recombination was the defining attribute that enabled evasion of this defense. Independent of gene expression strategy, positive-stranded RNA viruses that could undergo strand switching rapidly excised genomic material, while negative-stranded viruses were effectively targeted and cleared upon RNAi-based selection. These data suggest a dynamic relationship between host antiviral defenses and the biology of virus replication in shaping pathogen prevalence.
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14
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Dhungel B, Ramlogan-Steel CA, Steel JC. MicroRNA-Regulated Gene Delivery Systems for Research and Therapeutic Purposes. Molecules 2018; 23:E1500. [PMID: 29933586 PMCID: PMC6099389 DOI: 10.3390/molecules23071500] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2018] [Revised: 06/18/2018] [Accepted: 06/20/2018] [Indexed: 12/18/2022] Open
Abstract
Targeted gene delivery relies on the ability to limit the expression of a transgene within a defined cell/tissue population. MicroRNAs represent a class of highly powerful and effective regulators of gene expression that act by binding to a specific sequence present in the corresponding messenger RNA. Involved in almost every aspect of cellular function, many miRNAs have been discovered with expression patterns specific to developmental stage, lineage, cell-type, or disease stage. Exploiting the binding sites of these miRNAs allows for construction of targeted gene delivery platforms with a diverse range of applications. Here, we summarize studies that have utilized miRNA-regulated systems to achieve targeted gene delivery for both research and therapeutic purposes. Additionally, we identify criteria that are important for the effectiveness of a particular miRNA for such applications and we also discuss factors that have to be taken into consideration when designing miRNA-regulated expression cassettes.
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Affiliation(s)
- Bijay Dhungel
- Gallipoli Medical Research Institute, Greenslopes Private Hospital, 102 Newdegate Street, Brisbane, QLD 4120, Australia.
- Faculty of Medicine, University of Queensland, 288 Herston Road, Herston, Brisbane, QLD 4006, Australia.
- University of Queensland Diamantina Institute, Translational Research Institute, 37 Kent Street, Woolloongabba, QLD 4102, Australia.
| | - Charmaine A Ramlogan-Steel
- Faculty of Medicine, University of Queensland, 288 Herston Road, Herston, Brisbane, QLD 4006, Australia.
- Layton Vision Foundation, Translational Research Institute, 37 Kent Street, Woolloongabba, QLD 4102, Australia.
| | - Jason C Steel
- Faculty of Medicine, University of Queensland, 288 Herston Road, Herston, Brisbane, QLD 4006, Australia.
- OcuGene, Translational Research Institute, 37 Kent Street, Woolloongabba, QLD 4102, Australia.
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15
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Decreased accumulation of subgenomic RNA in human cells infected with vaccine candidate DEN4Δ30 increases viral susceptibility to type I interferon. Vaccine 2018; 36:3460-3467. [PMID: 29752023 DOI: 10.1016/j.vaccine.2018.04.087] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2018] [Revised: 04/19/2018] [Accepted: 04/20/2018] [Indexed: 11/20/2022]
Abstract
The NIH has developed live attenuated dengue virus (DENV) vaccine candidates by deletion of 30 nucleotides (Δ30) from the untranslated region of the viral genome. Although this attenuation strategy has proven to be effective in generating safe and immunogenic vaccine strains, the molecular mechanism of attenuation is largely unknown. To examine the mediators of the observed attenuation phenotype, differences in translation efficiency, genome replication, cytotoxicity, and type I interferon susceptibility were compared between wild type parental DENV and DENVΔ30 attenuated vaccine candidates. We observed that decreased accumulation of subgenomic RNA (sfRNA) from the vaccine candidates in infected human cells causes increased type I IFN susceptibility and propose this as one of the of attenuation mechanisms produced by the 3' UTR Δ30 mutation.
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16
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microRNA profiles and functions in mosquitoes. PLoS Negl Trop Dis 2018; 12:e0006463. [PMID: 29718912 PMCID: PMC5951587 DOI: 10.1371/journal.pntd.0006463] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2017] [Revised: 05/14/2018] [Accepted: 04/19/2018] [Indexed: 11/19/2022] Open
Abstract
Mosquitoes are incriminated as vectors for many crippling diseases, including malaria, West Nile fever, Dengue fever, and other neglected tropical diseases (NTDs). microRNAs (miRNAs) can interact with multiple target genes to elicit biological functions in the mosquitoes. However, characterization and function of individual miRNAs and their potential targets have not been fully determined to date. We conducted a systematic review of published literature following PRISMA guidelines. We summarize the information about miRNAs in mosquitoes to better understand their metabolism, development, and responses to microorganisms. Depending on the study, we found that miRNAs were dysregulated in a species-, sex-, stage-, and tissue/organ-specific manner. Aberrant miRNA expressions were observed in development, metabolism, host-pathogen interactions, and insecticide resistance. Of note, many miRNAs were down-regulated upon pathogen infection. The experimental studies have expanded the identification of miRNA target from the 3' untranslated regions (UTRs) of mRNAs of mosquitoes to the 5' UTRs of mRNAs of the virus. In addition, we discuss current trends in mosquito miRNA research and offer suggestions for future studies.
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17
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MicroRNA-Based Attenuation of Influenza Virus across Susceptible Hosts. J Virol 2018; 92:JVI.01741-17. [PMID: 29093096 DOI: 10.1128/jvi.01741-17] [Citation(s) in RCA: 38] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2017] [Accepted: 10/25/2017] [Indexed: 12/25/2022] Open
Abstract
Influenza A virus drives significant morbidity and mortality in humans and livestock. Annual circulation of the virus in livestock and waterfowl contributes to severe economic disruption and increases the risk of zoonotic transmission of novel strains into the human population, where there is no preexisting immunity. Seasonal vaccinations in humans help prevent infection and can reduce symptoms when infection does occur. However, current vaccination regimens available for livestock are limited in part due to safety concerns regarding reassortment/recombination with circulating strains. Therefore, inactivated vaccines are used instead of the more immunostimulatory live attenuated vaccines. MicroRNAs (miRNAs) have been used previously to generate attenuated influenza A viruses for use as a vaccine. Here, we systematically targeted individual influenza gene mRNAs using the same miRNA to determine the segment(s) that yields maximal attenuation potential. This analysis demonstrated that targeting of NP mRNA most efficiently ablates replication. We further increased the plasticity of miRNA-mediated attenuation of influenza A virus by exploiting a miRNA, miR-21, that is ubiquitously expressed across influenza-susceptible hosts. In order to construct this targeted virus, we used CRISPR/Cas9 to eliminate the universally expressed miR-21 from MDCK cells. miR-21-targeted viruses were attenuated in human, mouse, canine, and avian cells and drove protective immunity in mice. This strategy has the potential to enhance the safety of live attenuated vaccines in humans and zoonotic reservoirs.IMPORTANCE Influenza A virus circulates annually in both avian and human populations, causing significant morbidity, mortality, and economic burden. High incidence of zoonotic infections greatly increases the potential for transmission to humans, where no preexisting immunity or vaccine exists. There is a critical need for new vaccine strategies to combat emerging influenza outbreaks. MicroRNAs were used previously to attenuate influenza A viruses. We propose the development of a novel platform to produce live attenuated vaccines that are highly customizable, efficacious across a broad species range, and exhibit enhanced safety over traditional vaccination methods. This strategy exploits a microRNA that is expressed abundantly in influenza virus-susceptible hosts. By eliminating this ubiquitous microRNA from a cell line, targeted viruses that are attenuated across susceptible strains can be generated. This approach greatly increases the plasticity of the microRNA targeting approach and enhances vaccine safety.
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18
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Grabowski JM, Tsetsarkin KA, Long D, Scott DP, Rosenke R, Schwan TG, Mlera L, Offerdahl DK, Pletnev AG, Bloom ME. Flavivirus Infection of Ixodes scapularis (Black-Legged Tick) Ex Vivo Organotypic Cultures and Applications for Disease Control. mBio 2017; 8:e01255-17. [PMID: 28830948 PMCID: PMC5565970 DOI: 10.1128/mbio.01255-17] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2017] [Accepted: 07/25/2017] [Indexed: 12/21/2022] Open
Abstract
Ixodes scapularis ticks transmit many infectious agents that cause disease, including tick-borne flaviviruses (TBFVs). TBFV infections cause thousands of human encephalitis cases worldwide annually. In the United States, human TBFV infections with Powassan virus (POWV) are increasing and have a fatality rate of 10 to 30%. Additionally, Langat virus (LGTV) is a TBFV of low neurovirulence and is used as a model TBFV. TBFV replication and dissemination within I. scapularis organs are poorly characterized, and a deeper understanding of virus biology in this vector may inform effective countermeasures to reduce TBFV transmission. Here, we describe short-term, I. scapularis organ culture models of TBFV infection. Ex vivo organs were metabolically active for 9 to 10 days and were permissive to LGTV and POWV replication. Imaging and videography demonstrated replication and spread of green fluorescent protein-expressing LGTV in the organs. Immunohistochemical staining confirmed LGTV envelope and POWV protein synthesis within the infected organs. LGTV- and POWV-infected organs produced infectious LGTV and POWV; thus, the ex vivo cultures were suitable for study of virus replication in individual organs. LGTV- and POWV-infected midgut and salivary glands were subjected to double-stranded RNA (dsRNA) transfection with dsRNA to the LGTV 3' untranslated region (UTR), which reduced infectious LGTV and POWV replication, providing a proof-of-concept use of RNA interference in I. scapularis organ cultures to study the effects on TBFV replication. The results contribute important information on TBFV localization within ex vivo I. scapularis organs and provide a significant translational tool for evaluating recombinant, live vaccine candidates and potential tick transcripts and proteins for possible therapeutic use and vaccine development to reduce TBFV transmission.IMPORTANCE Tick-borne flavivirus (TBFV) infections cause neurological and/or hemorrhagic disease in humans worldwide. There are currently no licensed therapeutics or vaccines against Powassan virus (POWV), the only TBFV known to circulate in North America. Evaluating tick vector targets for antitick vaccines directed at reducing TBFV infection within the arthropod vector is a critical step in identifying efficient approaches to controlling TBFV transmission. This study characterized infection of female Ixodes scapularis tick organ cultures of midgut, salivary glands, and synganglion with the low-neurovirulence Langat virus (LGTV) and the more pathogenic POWV. Cell types of specific organs were susceptible to TBFV infection, and a difference in LGTV and POWV replication was noted in TBFV-infected organs. This tick organ culture model of TBFV infection will be useful for various applications, such as screening of tick endogenous dsRNA corresponding to potential control targets within midgut and salivary glands to confirm restriction of TBFV infection.
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Affiliation(s)
- Jeffrey M Grabowski
- Biology of Vector-Borne Viruses Section, Laboratory of Virology, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana, USA
| | - Konstantin A Tsetsarkin
- Neurotropic Flaviviruses Section, Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, Maryland, USA
| | - Dan Long
- Rocky Mountain Veterinary Branch, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana, USA
| | - Dana P Scott
- Rocky Mountain Veterinary Branch, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana, USA
| | - Rebecca Rosenke
- Rocky Mountain Veterinary Branch, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana, USA
| | - Tom G Schwan
- Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana, USA
| | - Luwanika Mlera
- Biology of Vector-Borne Viruses Section, Laboratory of Virology, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana, USA
| | - Danielle K Offerdahl
- Biology of Vector-Borne Viruses Section, Laboratory of Virology, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana, USA
| | - Alexander G Pletnev
- Neurotropic Flaviviruses Section, Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, Maryland, USA
| | - Marshall E Bloom
- Biology of Vector-Borne Viruses Section, Laboratory of Virology, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, Montana, USA
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19
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Synergistic Internal Ribosome Entry Site/MicroRNA-Based Approach for Flavivirus Attenuation and Live Vaccine Development. mBio 2017; 8:mBio.02326-16. [PMID: 28420742 PMCID: PMC5395672 DOI: 10.1128/mbio.02326-16] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
The recent emergence of Zika virus underscores the need for new strategies for a rapid development of safe flavivirus vaccines. Using another flavivirus (Langat virus [LGTV]) that belongs to the group of tick-borne flaviviruses as a model, we describe a dual strategy for virus attenuation which synergistically accesses the specificity of microRNA (miRNA) genome targeting and the effectiveness of internal ribosome entry site (IRES) insertion. To increase the stability and immunogenicity of bicistronic LGTVs, we developed a novel approach in which the capsid (C) protein gene was relocated into the 3′ noncoding region (NCR) and expressed under translational control from an IRES. Engineered bicistronic LGTVs carrying multiple target sequences for brain-specific miRNAs were stable in Vero cells and induced adaptive immunity in mice. Importantly, miRNA-targeted bicistronic LGTVs were not pathogenic for either newborn mice after intracranial inoculation or adult immunocompromised mice (SCID or type I interferon receptor knockout) after intraperitoneal injection. Moreover, bicistronic LGTVs were restricted for replication in tick-derived cells, suggesting an interruption of viral transmission in nature by arthropod vectors. This approach is suitable for reliable attenuation of many flaviviruses and may enable development of live attenuated flavivirus vaccines. The recent emergence of Zika virus underscores the need for new strategies for a rapid development of safe flavivirus vaccines. Allied separately attenuating approaches based on (i) microRNA genome targeting and (ii) internal ribosome entry site insertion are not sufficient for relievable attenuation of neurotropic flavivirus pathogenesis. Here, we describe a novel dual strategy that combines the specificity of miRNA-based and the effectiveness of IRES-based attenuating approaches, allowing us to overcome these critical limitations. This developed approach provides a robust platform for reliable attenuation of many flaviviruses and may enable development of live flavivirus vaccines.
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20
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Tsetsarkin KA, Liu G, Kenney H, Hermance M, Thangamani S, Pletnev AG. Concurrent micro-RNA mediated silencing of tick-borne flavivirus replication in tick vector and in the brain of vertebrate host. Sci Rep 2016; 6:33088. [PMID: 27620807 PMCID: PMC5020608 DOI: 10.1038/srep33088] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2016] [Accepted: 08/19/2016] [Indexed: 01/01/2023] Open
Abstract
Tick-borne viruses include medically important zoonotic pathogens that can cause life-threatening diseases. Unlike mosquito-borne viruses, whose impact can be restrained via mosquito population control programs, for tick-borne viruses only vaccination remains the reliable means of disease prevention. For live vaccine viruses a concern exists, that spillovers from viremic vaccinees could result in introduction of genetically modified viruses into sustainable tick-vertebrate host transmission cycle in nature. To restrict tick-borne flavivirus (Langat virus, LGTV) vector tropism, we inserted target sequences for tick-specific microRNAs (mir-1, mir-275 and mir-279) individually or in combination into several distant regions of LGTV genome. This caused selective attenuation of viral replication in tick-derived cells. LGTV expressing combinations of target sequences for tick- and vertebrate CNS-specific miRNAs were developed. The resulting viruses replicated efficiently and remained stable in simian Vero cells, which do not express these miRNAs, however were severely restricted to replicate in tick-derived cells. In addition, simultaneous dual miRNA targeting led to silencing of virus replication in live Ixodes ricinus ticks and abolished virus neurotropism in highly permissive newborn mice. The concurrent restriction of adverse replication events in vertebrate and invertebrate hosts will, therefore, ensure the environmental safety of live tick-borne virus vaccine candidates.
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Affiliation(s)
- Konstantin A. Tsetsarkin
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Guangping Liu
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Heather Kenney
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
| | - Meghan Hermance
- Department of Pathology, University of Texas Medical Branch (UTMB), Galveston, Texas, USA
| | - Saravanan Thangamani
- Department of Pathology, Galveston National Laboratory, UTMB, Galveston, Texas, USA
| | - Alexander G. Pletnev
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
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21
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A Full-Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus-Host Interactions and Vaccine Development. mBio 2016; 7:mBio.01114-16. [PMID: 27555311 PMCID: PMC4999549 DOI: 10.1128/mbio.01114-16] [Citation(s) in RCA: 109] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
UNLABELLED An arthropod-borne virus, Zika virus (ZIKV), has recently emerged as a major human pathogen. Associated with complications during perinatal development and Guillain-Barré syndrome in adults, ZIKV raises new challenges for understanding the molecular determinants of flavivirus pathogenesis. This underscores the necessity for the development of a reverse genetic system based on an epidemic ZIKV strain. Here, we describe the generation and characterization in cell cultures of an infectious cDNA clone of ZIKV isolated from the 2015 epidemic in Brazil. The cDNA-derived ZIKV replicated efficiently in a variety of cell lines, including those of both neuronal and placental origin. We observed that the growth of cDNA-derived virus was attenuated compared to the growth of the parental isolate in most cell lines, which correlates with substantial differences in sequence heterogeneity between these viruses that were determined by deep-sequencing analysis. Our findings support the role of genetic diversity in maintaining the replicative fitness of viral populations under changing conditions. Moreover, these results indicate that caution should be exercised when interpreting the results of reverse-genetics experiments in attempts to accurately predict the biology of natural viruses. Finally, a Vero cell-adapted cDNA clone of ZIKV was generated that can be used as a convenient platform for studies aimed at the development of ZIKV vaccines and therapeutics. IMPORTANCE The availability of genetic tools and laboratory models determines the progress in understanding mechanisms of virus emergence and pathogenesis. Recent large-scale outbreaks of Zika virus (ZIKV) that were linked to complications during perinatal development and Guillain-Barré syndrome in adults emphasize the urgency for the development of a reverse-genetics system based on an epidemic ZIKV strain. Here, we report a stable infectious cDNA clone for ZIKV isolated during the 2015 epidemic in Brazil, as well as a Vero cell-adapted version of it, which will be used for virus-host interaction studies and vaccine development.
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22
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Kim GW, Lee SH, Cho H, Kim M, Shin EC, Oh JW. Hepatitis C Virus Core Protein Promotes miR-122 Destabilization by Inhibiting GLD-2. PLoS Pathog 2016; 12:e1005714. [PMID: 27366906 PMCID: PMC4930175 DOI: 10.1371/journal.ppat.1005714] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2016] [Accepted: 06/01/2016] [Indexed: 12/12/2022] Open
Abstract
The liver-specific microRNA miR-122, which has essential roles in liver development and metabolism, is a key proviral factor for hepatitis C virus (HCV). Despite its crucial role in the liver and HCV life cycle, little is known about the molecular mechanism of miR-122 expression regulation by HCV infection. Here, we show that the HCV core protein downregulates the abundance of miR-122 by promoting its destabilization via the inhibition of GLD-2, a non-canonical cytoplasmic poly(A) polymerase. The decrease in miR-122 expression resulted in the dysregulation of the known functions of miR-122, including its proviral activity for HCV. By high-throughput sequencing of small RNAs from human liver biopsies, we found that the 22-nucleotide (nt) prototype miR-122 is modified at its 3' end by 3'-terminal non-templated and templated nucleotide additions. Remarkably, the proportion of miR-122 isomers bearing a single nucleotide tail of any ribonucleotide decreased in liver specimens from patients with HCV. We found that these single-nucleotide-tailed miR-122 isomers display increased miRNA activity and stability over the 22-nt prototype miR-122 and that the 3'-terminal extension is catalyzed by the unique terminal nucleotidyl transferase activity of GLD-2, which is capable of adding any single ribonucleotide without preference of adenylate to the miR-122 3' end. The HCV core protein specifically inhibited GLD-2, and its interaction with GLD-2 in the cytoplasm was found to be responsible for miR-122 downregulation. Collectively, our results provide new insights into the regulatory role of the HCV core protein in controlling viral RNA abundance and miR-122 functions through miR-122 stability modulation.
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Affiliation(s)
- Geon-Woo Kim
- Department of Biotechnology, Yonsei University, Seoul, Korea
| | - Seung-Hoon Lee
- Department of Biotechnology, Yonsei University, Seoul, Korea
| | - Hee Cho
- Department of Biotechnology, Yonsei University, Seoul, Korea
| | - Minwoo Kim
- Department of Biotechnology, Yonsei University, Seoul, Korea
| | - Eui-Cheol Shin
- Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon, Korea
| | - Jong-Won Oh
- Department of Biotechnology, Yonsei University, Seoul, Korea
- * E-mail:
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23
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Geisler A, Fechner H. MicroRNA-regulated viral vectors for gene therapy. World J Exp Med 2016; 6:37-54. [PMID: 27226955 PMCID: PMC4873559 DOI: 10.5493/wjem.v6.i2.37] [Citation(s) in RCA: 60] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/27/2015] [Revised: 03/02/2016] [Accepted: 03/17/2016] [Indexed: 02/06/2023] Open
Abstract
Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene. Besides traditional approaches, such as transcriptional and transductional targeting, microRNA-dependent post-transcriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. MicroRNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3' untranslated region (UTR) of the mRNA. To control exogenous transgene expression, tandem repeats of artificial microRNA target sites are usually incorporated into the 3' UTR of the transgene expression cassette, leading to subsequent degradation of transgene mRNA in cells expressing the corresponding microRNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying microRNA-regulation, highlights new developments in this field and gives an overview of applications of microRNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases.
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24
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Tsetsarkin KA, Liu G, Shen K, Pletnev AG. Kissing-loop interaction between 5' and 3' ends of tick-borne Langat virus genome 'bridges the gap' between mosquito- and tick-borne flaviviruses in mechanisms of viral RNA cyclization: applications for virus attenuation and vaccine development. Nucleic Acids Res 2016; 44:3330-50. [PMID: 26850640 PMCID: PMC4838367 DOI: 10.1093/nar/gkw061] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2015] [Accepted: 01/24/2016] [Indexed: 01/23/2023] Open
Abstract
Insertion of microRNA target sequences into the flavivirus genome results in selective tissue-specific attenuation and host-range restriction of live attenuated vaccine viruses. However, previous strategies for miRNA-targeting did not incorporate a mechanism to prevent target elimination under miRNA-mediated selective pressure, restricting their use in vaccine development. To overcome this limitation, we developed a new approach for miRNA-targeting of tick-borne flavivirus (Langat virus, LGTV) in the duplicated capsid gene region (DCGR). Genetic stability of viruses with DCGR was ensured by the presence of multiple cis-acting elements within the N-terminal capsid coding region, including the stem-loop structure (5′SL6) at the 3′ end of the promoter. We found that the 5′SL6 functions as a structural scaffold for the conserved hexanucleotide motif at its tip and engages in a complementary interaction with the region present in the 3′ NCR to enhance viral RNA replication. The resulting kissing-loop interaction, common in tick-borne flaviviruses, supports a single pair of cyclization elements (CYC) and functions as a homolog of the second pair of CYC that is present in the majority of mosquito-borne flaviviruses. Placing miRNA targets into the DCGR results in superior attenuation of LGTV in the CNS and does not interfere with development of protective immunity in immunized mice.
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Affiliation(s)
- Konstantin A Tsetsarkin
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, 20892-3203 USA
| | - Guangping Liu
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, 20892-3203 USA
| | - Kui Shen
- Bioinformatics and Computational Biosciences Branch, NIAID, NIH, Bethesda, MD, 20892, USA
| | - Alexander G Pletnev
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, 20892-3203 USA
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25
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Charley PA, Wilusz J. Standing your ground to exoribonucleases: Function of Flavivirus long non-coding RNAs. Virus Res 2016; 212:70-7. [PMID: 26368052 PMCID: PMC4744573 DOI: 10.1016/j.virusres.2015.09.009] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2015] [Revised: 09/04/2015] [Accepted: 09/10/2015] [Indexed: 01/18/2023]
Abstract
Members of the Flaviviridae (e.g., Dengue virus, West Nile virus, and Hepatitis C virus) contain a positive-sense RNA genome that encodes a large polyprotein. It is now also clear most if not all of these viruses also produce an abundant subgenomic long non-coding RNA. These non-coding RNAs, which are called subgenomic flavivirus RNAs (sfRNAs) or Xrn1-resistant RNAs (xrRNAs), are stable decay intermediates generated from the viral genomic RNA through the stalling of the cellular exoribonuclease Xrn1 at highly structured regions. Several functions of these flavivirus long non-coding RNAs have been revealed in recent years. The generation of these sfRNAs/xrRNAs from viral transcripts results in the repression of Xrn1 and the dysregulation of cellular mRNA stability. The abundant sfRNAs also serve directly as a decoy for important cellular protein regulators of the interferon and RNA interference antiviral pathways. Thus the generation of long non-coding RNAs from flaviviruses, hepaciviruses and pestiviruses likely disrupts aspects of innate immunity and may directly contribute to viral replication, cytopathology and pathogenesis.
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Affiliation(s)
- Phillida A Charley
- Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA
| | - Jeffrey Wilusz
- Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
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