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Bahojb Mahdavi SZ, Jebelli A, Aghbash PS, Baradaran B, Amini M, Oroojalian F, Pouladi N, Baghi HB, de la Guardia M, Mokhtarzadeh AA. A comprehensive overview on the crosstalk between microRNAs and viral pathogenesis and infection. Med Res Rev 2025; 45:349-425. [PMID: 39185567 PMCID: PMC11796338 DOI: 10.1002/med.22073] [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: 11/06/2021] [Revised: 04/11/2023] [Accepted: 08/04/2024] [Indexed: 08/27/2024]
Abstract
Infections caused by viruses as the smallest infectious agents, pose a major threat to global public health. Viral infections utilize different host mechanisms to facilitate their own propagation and pathogenesis. MicroRNAs (miRNAs), as small noncoding RNA molecules, play important regulatory roles in different diseases, including viral infections. They can promote or inhibit viral infection and have a pro-viral or antiviral role. Also, viral infections can modulate the expression of host miRNAs. Furthermore, viruses from different families evade the host immune response by producing their own miRNAs called viral miRNAs (v-miRNAs). Understanding the replication cycle of viruses and their relation with host miRNAs and v-miRNAs can help to find new treatments against viral infections. In this review, we aim to outline the structure, genome, and replication cycle of various viruses including hepatitis B, hepatitis C, influenza A virus, coronavirus, human immunodeficiency virus, human papillomavirus, herpes simplex virus, Epstein-Barr virus, Dengue virus, Zika virus, and Ebola virus. We also discuss the role of different host miRNAs and v-miRNAs and their role in the pathogenesis of these viral infections.
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Affiliation(s)
- Seyedeh Zahra Bahojb Mahdavi
- Department of Biology, Faculty of Basic SciencesAzarbaijan Shahid Madani UniversityTabrizIran
- Immunology Research CenterTabriz University of Medical SciencesTabrizIran
| | - Asiyeh Jebelli
- Department of Biological Science, Faculty of Basic ScienceHigher Education Institute of Rab‐RashidTabrizIran
- Tuberculosis and Lung Diseases Research CenterTabriz University of Medical SciencesTabrizIran
| | | | - Behzad Baradaran
- Immunology Research CenterTabriz University of Medical SciencesTabrizIran
| | - Mohammad Amini
- Immunology Research CenterTabriz University of Medical SciencesTabrizIran
| | - Fatemeh Oroojalian
- Department of Advanced Sciences and Technologies in Medicine, School of MedicineNorth Khorasan University of Medical SciencesBojnurdIran
| | - Nasser Pouladi
- Department of Biology, Faculty of Basic SciencesAzarbaijan Shahid Madani UniversityTabrizIran
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Guo SY, Feng WH. Host microRNAs as regulators of porcine reproductive and respiratory syndrome virus infection. Virology 2025; 603:110361. [PMID: 39721195 DOI: 10.1016/j.virol.2024.110361] [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: 10/10/2024] [Revised: 12/01/2024] [Accepted: 12/16/2024] [Indexed: 12/28/2024]
Abstract
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is a significant pathogen in the swine industry. MicroRNAs (miRNAs), a class of small non-coding RNA molecules, have risen to prominence as key regulators of gene expression at the post-transcriptional level. Their significance in regulating virus-host interactions is now widely acknowledged. So far, more than 30 miRNAs have been found to play a role in PRRSV infection. They can regulate viral genome stability and protein synthesis by targeting PRRSV RNA, and modulate the host immune response, thus affecting PRRSV replication. Understanding the role of miRNAs in PRRSV infection can further elucidate the pathogenesis of PRRSV and pave the way for the development of new antiviral strategies through miRNA-based therapies. This review will focus on how host miRNAs alter PRRSV infection, underscoring their multifaceted involvement in the interplay between virus and host.
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Affiliation(s)
- Shu-Yuan Guo
- Frontiers Science Center for Molecular Design Breeding, College of Biological Sciences, China Agricultural University, Beijing, 100193, China; State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Beijing, 100193, China; Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China; Department of Microbiology and Immunology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China
| | - Wen-Hai Feng
- Frontiers Science Center for Molecular Design Breeding, College of Biological Sciences, China Agricultural University, Beijing, 100193, China; State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Beijing, 100193, China; Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China; Department of Microbiology and Immunology, College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
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Robles-Remacho A, Martos-Jamai I, Tabraue-Chávez M, Aguilar-González A, Laz-Ruiz JA, Cano-Cortés MV, López-Delgado FJ, Guardia-Monteagudo JJ, Pernagallo S, Diaz-Mochon JJ, Sanchez-Martin RM. Click chemistry-based dual nanosystem for microRNA-122 detection with single-base specificity from tumour cells. J Nanobiotechnology 2024; 22:791. [PMID: 39710710 DOI: 10.1186/s12951-024-03071-6] [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: 10/15/2024] [Accepted: 12/11/2024] [Indexed: 12/24/2024] Open
Abstract
MicroRNAs (miRNAs) have been recognised as potential biomarkers due to their specific expression patterns in different biological tissues and their changes in expression under pathological conditions. MicroRNA-122 (miR-122) is a vertebrate-specific miRNA that is predominantly expressed in the liver and plays an important role in liver metabolism and development. Dysregulation of miR-122 expression is associated with several liver-related diseases, including hepatocellular carcinoma and drug-induced liver injury (DILI). Given the potential of miR-122 as a biomarker, its effective detection is important for accurate diagnosis. However, miRNA detection methods still face challenges, particularly in terms of accurately identifying miRNA isoforms that may differ by only a single base. Here, with the aim of advancing accessible methods for the detection of miRNAs with single-base specificity, we have developed a robust dual nanosystem that leverages the simplicity of click chemistry reactions. Using the dual nanosystem, we successfully detected miR-122 at single-base resolution using flow cytometry and analysed its expression in various tumour cell lines with high specificity and strong correlation with TaqMan assay results. We also detected miR-122 in serum and identified four single nucleotide variations in its sequence. The chemistry employed in this dual nanosystem is highly versatile and offers a promising opportunity to develop nanoparticle-based strategies that incorporate click chemistry and bioorthogonal chemistry for the detection of miRNAs and their isoforms.
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Affiliation(s)
- Agustín Robles-Remacho
- GENYO, Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, Avenida de la Ilustración, 18016, Granada, Spain.
- Department of Medicinal and Organic Chemistry and Excellence Research Unit of Chemistry Applied to Biomedicine and the Environment, School of Pharmacy, University of Granada, Campus Cartuja s/n, 18071, Granada, Spain.
- Instituto de Investigación Biosanitaria Ibs.GRANADA, Granada, Spain.
- Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
| | - Ismael Martos-Jamai
- GENYO, Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, Avenida de la Ilustración, 18016, Granada, Spain
- Department of Medicinal and Organic Chemistry and Excellence Research Unit of Chemistry Applied to Biomedicine and the Environment, School of Pharmacy, University of Granada, Campus Cartuja s/n, 18071, Granada, Spain
- Instituto de Investigación Biosanitaria Ibs.GRANADA, Granada, Spain
| | - Mavys Tabraue-Chávez
- DESTINA Genomica S.L, PTS Granada, Avenida de la Innovación 1, Edificio BIC, 18016, Armilla, Spain
| | - Araceli Aguilar-González
- GENYO, Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, Avenida de la Ilustración, 18016, Granada, Spain
- Department of Medicinal and Organic Chemistry and Excellence Research Unit of Chemistry Applied to Biomedicine and the Environment, School of Pharmacy, University of Granada, Campus Cartuja s/n, 18071, Granada, Spain
- Instituto de Investigación Biosanitaria Ibs.GRANADA, Granada, Spain
| | - Jose A Laz-Ruiz
- GENYO, Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, Avenida de la Ilustración, 18016, Granada, Spain
- Department of Medicinal and Organic Chemistry and Excellence Research Unit of Chemistry Applied to Biomedicine and the Environment, School of Pharmacy, University of Granada, Campus Cartuja s/n, 18071, Granada, Spain
- Instituto de Investigación Biosanitaria Ibs.GRANADA, Granada, Spain
| | - M Victoria Cano-Cortés
- GENYO, Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, Avenida de la Ilustración, 18016, Granada, Spain
- Department of Medicinal and Organic Chemistry and Excellence Research Unit of Chemistry Applied to Biomedicine and the Environment, School of Pharmacy, University of Granada, Campus Cartuja s/n, 18071, Granada, Spain
- Instituto de Investigación Biosanitaria Ibs.GRANADA, Granada, Spain
| | - F Javier López-Delgado
- DESTINA Genomica S.L, PTS Granada, Avenida de la Innovación 1, Edificio BIC, 18016, Armilla, Spain
| | | | - Salvatore Pernagallo
- DESTINA Genomica S.L, PTS Granada, Avenida de la Innovación 1, Edificio BIC, 18016, Armilla, Spain
| | - Juan J Diaz-Mochon
- GENYO, Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, Avenida de la Ilustración, 18016, Granada, Spain.
- Department of Medicinal and Organic Chemistry and Excellence Research Unit of Chemistry Applied to Biomedicine and the Environment, School of Pharmacy, University of Granada, Campus Cartuja s/n, 18071, Granada, Spain.
- Instituto de Investigación Biosanitaria Ibs.GRANADA, Granada, Spain.
| | - Rosario M Sanchez-Martin
- GENYO, Centre for Genomics and Oncological Research, Pfizer/University of Granada/Andalusian Regional Government, PTS Granada, Avenida de la Ilustración, 18016, Granada, Spain
- Department of Medicinal and Organic Chemistry and Excellence Research Unit of Chemistry Applied to Biomedicine and the Environment, School of Pharmacy, University of Granada, Campus Cartuja s/n, 18071, Granada, Spain
- Instituto de Investigación Biosanitaria Ibs.GRANADA, Granada, Spain
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Cai Z, Zhao H, Xue Y. Protocol for profiling virus-to-host RNA-RNA interactions in infected cells by RIC-seq. STAR Protoc 2024; 5:103149. [PMID: 38907997 PMCID: PMC11245969 DOI: 10.1016/j.xpro.2024.103149] [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: 02/20/2024] [Revised: 05/22/2024] [Accepted: 06/03/2024] [Indexed: 06/24/2024] Open
Abstract
Virus-to-host RNA-RNA interactions directly regulate host mRNA stability and viral replication. However, globally profiling virus-to-host in situ RNA-RNA interactions remains challenging. Here, we present an RNA in situ conformation sequencing (RIC-seq)-based protocol for mapping high-confidence virus-to-host in situ RNA-RNA interactions in infected cells. We detail steps for formaldehyde crosslinking, pCp-biotin labeling, in situ proximity ligation, chimeric RNA enrichment, strand-specific library construction, and data analysis. This protocol allows unbiased identification of virus-to-host RNA-RNA interactions for various RNA viruses and is potentially applicable to DNA virus-derived transcripts. For complete details on the use and execution of this protocol, please refer to Zhao et al.1.
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Affiliation(s)
- Zhaokui Cai
- Key Laboratory of Epigenetic Regulation and Intervention, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
| | - Hailian Zhao
- Key Laboratory of Epigenetic Regulation and Intervention, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yuanchao Xue
- Key Laboratory of Epigenetic Regulation and Intervention, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
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Mirzaei R, Karampoor S, Korotkova NL. The emerging role of miRNA-122 in infectious diseases: Mechanisms and potential biomarkers. Pathol Res Pract 2023; 249:154725. [PMID: 37544130 DOI: 10.1016/j.prp.2023.154725] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/06/2023] [Revised: 07/25/2023] [Accepted: 07/26/2023] [Indexed: 08/08/2023]
Abstract
microRNAs (miRNAs) are small, non-coding RNA molecules that play crucial regulatory roles in numerous cellular processes. Recent investigations have highlighted the significant involvement of miRNA-122 (miR-122) in the pathogenesis of infectious diseases caused by diverse pathogens, encompassing viral, bacterial, and parasitic infections. In the context of viral infections, miR-122 exerts regulatory control over viral replication by binding to the viral genome and modulating the host's antiviral response. For instance, in hepatitis B virus (HBV) infection, miR-122 restricts viral replication, while HBV, in turn, suppresses miR-122 expression. Conversely, miR-122 interacts with the hepatitis C virus (HCV) genome, facilitating viral replication. Regarding bacterial infections, miR-122 has been found to regulate host immune responses by influencing inflammatory cytokine production and phagocytosis. In Vibrio anguillarum infections, there is a significant reduction in miR-122 expression, contributing to the pathophysiology of bacterial infections. Toll-like receptor 14 (TLR14) has been identified as a novel target gene of miR-122, affecting inflammatory and immune responses. In the context of parasitic infections, miR-122 plays a crucial role in regulating host lipid metabolism and immune responses. For example, during Leishmania infection, miR-122-containing extracellular vesicles from liver cells are unable to enter infected macrophages, leading to a suppression of the inflammatory response. Furthermore, miR-122 exhibits promise as a potential biomarker for various infectious diseases. Its expression level in body fluids, particularly in serum and plasma, correlates with disease severity and treatment response in patients affected by HCV, HBV, and tuberculosis. This paper also discusses the potential of miR-122 as a biomarker in infectious diseases. In summary, this review provides a comprehensive and insightful overview of the emerging role of miR-122 in infectious diseases, detailing its mechanism of action and potential implications for the development of novel therapeutic strategies.
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Affiliation(s)
- Rasoul Mirzaei
- Venom and Biotherapeutics Molecules Lab, Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
| | - Sajad Karampoor
- Gastrointestinal and Liver Diseases Research Center, Iran University of Medical Sciences, Tehran, Iran.
| | - Nadezhda Lenoktovna Korotkova
- I.M. Sechenov First Moscow State Medical University (Sechenov University), Russia; Federal State Budgetary Educational Institution of Higher Education "Privolzhsky Research Medical University" of the Ministry of Health of the Russian Federation (FSBEI HE PRMU MOH Russia), Russia
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Toon K, Kalemera MD, Palor M, Rose NJ, Takeuchi Y, Grove J, Mattiuzzo G. GB Virus B and Hepatitis C Virus, Distantly Related Hepaciviruses, Share an Entry Factor, Claudin-1. J Virol 2023; 97:e0046923. [PMID: 37310242 PMCID: PMC10373534 DOI: 10.1128/jvi.00469-23] [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: 03/30/2023] [Accepted: 05/10/2023] [Indexed: 06/14/2023] Open
Abstract
Due to increased and broadened screening efforts, the last decade has seen a rapid expansion in the number of viral species classified into the Hepacivirus genus. Conserved genetic features of hepaciviruses suggest that they have undergone specific adaptation and have evolved to hijack similar host proteins for efficient propagation in the liver. Here, we developed pseudotyped viruses to elucidate the entry factors of GB virus B (GBV-B), the first hepacivirus described in an animal after hepatitis C virus (HCV). GBV-B-pseudotyped viral particles (GBVBpp) were shown to be uniquely sensitive to the sera of tamarins infected with GBV-B, validating their usefulness as a surrogate for GBV-B entry studies. We screened GBVBpp infection of human hepatoma cell lines that were CRISPR/Cas9 engineered to ablate the expression of individual HCV receptors/entry factors and found that claudin-1 is essential for GBV-B infection, indicating the GBV-B and HCV share an entry factor. Our data suggest that claudin-1 facilitates HCV and GBV-B entry through distinct mechanisms since the former requires the first extracellular loop and the latter is reliant on a C-terminal region containing the second extracellular loop. The observation that claudin-1 is an entry factor shared between these two hepaciviruses suggests that the tight junction protein is of fundamental mechanistic importance during cell entry. IMPORTANCE Hepatitis C virus (HCV) is a major public health burden; approximately 58 million individuals have chronic HCV infection and are at risk of developing cirrhosis and liver cancer. To achieve the World Health Organization's target of eliminating hepatitis by 2030, new therapeutics and vaccines are needed. Understanding how HCV enters cells can inform the design of new vaccines and treatments targeting the first stage of infection. However, the HCV cell entry mechanism is complex and has been sparsely described. Studying the entry of related hepaciviruses will increase the knowledge of the molecular mechanisms of the first stages of HCV infection, such as membrane fusion, and inform structure-guided HCV vaccine design; in this work, we have identified a protein, claudin-1, that facilitates the entry of an HCV-related hepacivirus but with a mechanism not described for HCV. Similar work on other hepaciviruses may unveil a commonality of entry factors and, possibly, new mechanisms.
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Affiliation(s)
- Kamilla Toon
- Science Research and Innovation, Medicines and Healthcare Products Regulatory Agency, South Mimms, United Kingdom
- Division of Infection and Immunity, University College London, London, United Kingdom
| | - Mphatso D. Kalemera
- Division of Infection and Immunity, University College London, London, United Kingdom
| | - Machaela Palor
- Division of Infection and Immunity, University College London, London, United Kingdom
| | - Nicola J. Rose
- Science Research and Innovation, Medicines and Healthcare Products Regulatory Agency, South Mimms, United Kingdom
| | - Yasuhiro Takeuchi
- Science Research and Innovation, Medicines and Healthcare Products Regulatory Agency, South Mimms, United Kingdom
- Division of Infection and Immunity, University College London, London, United Kingdom
| | - Joe Grove
- Division of Infection and Immunity, University College London, London, United Kingdom
- MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom
| | - Giada Mattiuzzo
- Science Research and Innovation, Medicines and Healthcare Products Regulatory Agency, South Mimms, United Kingdom
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Das O, Kundu J, Ghosh A, Gautam A, Ghosh S, Chakraborty M, Masid A, Gauri SS, Mitra D, Dutta M, Mukherjee B, Sinha S, Bhaumik M. AUF-1 knockdown in mice undermines gut microbial butyrate-driven hypocholesterolemia through AUF-1-Dicer-1-mir-122 hierarchy. Front Cell Infect Microbiol 2022; 12:1011386. [PMID: 36601302 PMCID: PMC9806232 DOI: 10.3389/fcimb.2022.1011386] [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: 08/04/2022] [Accepted: 11/28/2022] [Indexed: 12/23/2022] Open
Abstract
Introduction and objective Cholesterol homeostasis is a culmination of cellular synthesis, efflux, and catabolism to important physiological entities where short chain fatty acid, butyrate embodied as a key player. This discourse probes the mechanistic molecular details of butyrate action in maintaining host-cholesterol balance. Methods Hepatic mir-122 being the most indispensable regulator of cholesterol metabolic enzymes, we studied upstream players of mir-122 biogenesis in the presence and absence of butyrate in Huh7 cells and mice model. We synthesized unique self-transfecting GMO (guanidinium-morpholino-oligo) linked PMO (Phosphorodiamidate-Morpholino Oligo)-based antisense cell-penetrating reagent to selectively knock down the key player in butyrate mediated cholesterol regulation. Results We showed that butyrate treatment caused upregulation of RNA-binding protein, AUF1 resulting in RNase-III nuclease, Dicer1 instability, and significant diminution of mir-122. We proved the importance of AUF1 and sequential downstream players in AUF1-knock-down mice. Injection of GMO-PMO of AUF1 in mouse caused near absence of AUF1 coupled with increased Dicer1 and mir-122, and reduced serum cholesterol regardless of butyrate treatment indicating that butyrate acts through AUF1. Conclusion The roster of intracellular players was as follows: AUF1-Dicer1-mir-122 for triggering butyrate driven hypocholesterolemia. To our knowledge this is the first report linking AUF-1 with cholesterol biogenesis.
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Affiliation(s)
- Oishika Das
- Department of Immunology, Indian Council of Medical Research-National Institute of Cholera and Enteric Diseases, Kolkata, India
| | - Jayanta Kundu
- School of Applied and Interdisciplinary Sciences, Indian Associations for Cultivation of Science, Kolkata, India
| | - Atanu Ghosh
- School of Applied and Interdisciplinary Sciences, Indian Associations for Cultivation of Science, Kolkata, India
| | - Anupam Gautam
- Department of Algorithms in Bioinformatics, Institute for Bioinformatics and Medical Informatics, University of Tübingen, Tübingen, Germany,International Max Planck Research School “From Molecules to Organisms”, Max Planck Institute for Biology Tübingen, Tübingen, Germany,Cluster of Excellence: EXC 2124: Controlling Microbes to Fight Infection, University of Tübingen, Tübingen, Germany
| | - Souradeepa Ghosh
- School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, India
| | - Mainak Chakraborty
- Department of Immunology, Indian Council of Medical Research-National Institute of Cholera and Enteric Diseases, Kolkata, India
| | - Aaheli Masid
- Department of Immunology, Indian Council of Medical Research-National Institute of Cholera and Enteric Diseases, Kolkata, India
| | - Samiran Sona Gauri
- School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, India
| | - Debmalya Mitra
- Department of Immunology, Indian Council of Medical Research-National Institute of Cholera and Enteric Diseases, Kolkata, India
| | - Moumita Dutta
- Department of Immunology, Indian Council of Medical Research-National Institute of Cholera and Enteric Diseases, Kolkata, India
| | - Budhaditya Mukherjee
- School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, India
| | - Surajit Sinha
- School of Applied and Interdisciplinary Sciences, Indian Associations for Cultivation of Science, Kolkata, India
| | - Moumita Bhaumik
- Department of Immunology, Indian Council of Medical Research-National Institute of Cholera and Enteric Diseases, Kolkata, India,*Correspondence: Moumita Bhaumik,
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Joshi N, Chandane Tak M, Mukherjee A. The involvement of microRNAs in HCV and HIV infection. Ther Adv Vaccines Immunother 2022; 10:25151355221106104. [PMID: 35832725 PMCID: PMC9272158 DOI: 10.1177/25151355221106104] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2022] [Accepted: 05/24/2022] [Indexed: 11/17/2022] Open
Abstract
Approximately 2.3 million people are suffering from human immunodeficiency virus (HIV)/hepatitis C virus (HCV) co-infection worldwide. Faster disease progression and increased mortality rates during the HIV/HCV co-infection have become global health concerns. Effective therapeutics against co-infection and complete infection eradication has become a mandatory requirement. The study of small non-coding RNAs in cellular processes and viral infection has so far been beneficial in various terms. Currently, microRNAs are an influential candidate for disease diagnosis and treatment. Dysregulation in miRNA expression can lead to unfavorable outcomes; hence, this exact inevitable nature has made various studies a focal point. A considerable improvement in comprehending HIV and HCV mono-infection pathogenesis is seen using miRNAs. The prominent reason behind HIV/HCV co-infection is seen to be their standard route of transmission, while some pieces of evidence also suspect viral interplay between having a role in increased viral infection. This review highlights the involvement of microRNAs in HIV/HCV co-infection, along with their contribution in HIV mono- and HCV mono-infection. We also discuss miRNAs that carry the potentiality of becoming a biomarker for viral infection and early disease progression.
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Affiliation(s)
- Nicky Joshi
- Division of Virology, ICMR-National AIDS Research Institute, Pune, India
| | | | - Anupam Mukherjee
- Scientist D & RAMANUJAN Fellow, Division of Virology, ICMR-National AIDS Research Institute, Plot No. 73, 'G' Block, MIDC, Bhosari, Pune 411026, Maharashtra, India
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Salama II, Raslan HM, Abdel-Latif GA, Salama SI, Sami SM, Shaaban FA, Abdelmohsen AM, Fouad WA. Impact of direct-acting antiviral regimens on hepatic and extrahepatic manifestations of hepatitis C virus infection. World J Hepatol 2022; 14:1053-1073. [PMID: 35978668 PMCID: PMC9258264 DOI: 10.4254/wjh.v14.i6.1053] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/17/2022] [Revised: 04/01/2022] [Accepted: 05/22/2022] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) is a common cause of liver disease and is associated with various extrahepatic manifestations (EHMs). This mini-review outlines the currently available treatments for HCV infection and their prognostic effect on hepatic manifestations and EHMs. Direct-acting antiviral (DAA) regimens are considered pan-genotypic as they achieve a sustained virological response (SVR) > 85% after 12 wk through all the major HCV genotypes, with high percentages of SVR even in advanced fibrosis and cirrhosis. The risk factors for DAA failure include old males, cirrhosis, and the presence of resistance-associated substitutions (RAS) in the region targeted by the received DAAs. The effectiveness of DAA regimens is reduced in HCV genotype 3 with baseline RAS like A30K, Y93H, and P53del. Moreover, the European Association for the Study of the Liver recommended the identification of baseline RAS for HCV genotype 1a. The higher rate of hepatocellular carcinoma (HCC) after DAA therapy may be related to the fact that DAA regimens are offered to patients with advanced liver fibrosis and cirrhosis, where interferon was contraindicated to those patients. The change in the growth of pre-existing subclinical, undetectable HCC upon DAA treatment might be also a cause. Furthermore, after DAA therapy, the T cell-dependent immune response is much weaker upon HCV clearance, and the down-regulation of TNF-α or the elevated neutrophil to lymphocyte ratio might increase the risk of HCC. DAAs can result in reactivation of hepatitis B virus (HBV) in HCV co-infected patients. DAAs are effective in treating HCV-associated mixed cryoglobulinemia, with clinical and immunological responses, and have rapid and high effectiveness in thrombocytopenia. DAAs improve insulin resistance in 90% of patients, increase glomerular filtration rate, and decrease proteinuria, hematuria and articular manifestations. HCV clearance by DAAs allows a significant improvement in atherosclerosis and metabolic and immunological conditions, with a reduction of major cardiovascular events. They also improve physical function, fatigue, cognitive impairment, and quality of life. Early therapeutic approach with DAAs is recommended as it cure many of the EHMs that are still in a reversible stage and can prevent others that can develop due to delayed treatment.
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Affiliation(s)
- Iman Ibrahim Salama
- Department of Community Medicine Research, National Research Center, Giza 12622, Dokki, Egypt.
| | - Hala M Raslan
- Department of Internal Medicine, National Research Center, Giza 12622, Dokki, Egypt
| | - Ghada A Abdel-Latif
- Department of Community Medicine Research, National Research Center, Giza 12622, Dokki, Egypt
| | - Somaia I Salama
- Department of Community Medicine Research, National Research Center, Giza 12622, Dokki, Egypt
| | - Samia M Sami
- Department of Child Health, National Research Center, Giza 12622, Dokki, Egypt
| | - Fatma A Shaaban
- Department of Child Health, National Research Center, Giza 12622, Dokki, Egypt
| | - Aida M Abdelmohsen
- Department of Community Medicine Research, National Research Center, Giza 12622, Dokki, Egypt
| | - Walaa A Fouad
- Department of Community Medicine Research, National Research Center, Giza 12622, Dokki, Egypt
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10
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Alhetheel AF. Impact of Hepatitis C Virus Infection of Peripheral Blood Mononuclear Cells on the Immune System. FRONTIERS IN VIROLOGY 2022. [DOI: 10.3389/fviro.2021.810231] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Hepatitis C is a worldwide liver disease caused by hepatitis C virus (HCV) infection. The virus causes acute and chronic liver inflammation, and it is transmitted mainly by exposure to contaminated blood. HCV is capable of infecting hepatocytes and peripheral blood mononuclear cells, causing complications and disease progression. This mini review provides an overview of HCV infection, including details on the virological aspects, infection of the immune cells, and its impact on the immune system.
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11
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RNA Structures and Their Role in Selective Genome Packaging. Viruses 2021; 13:v13091788. [PMID: 34578369 PMCID: PMC8472981 DOI: 10.3390/v13091788] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2021] [Revised: 08/31/2021] [Accepted: 09/02/2021] [Indexed: 12/13/2022] Open
Abstract
To generate infectious viral particles, viruses must specifically select their genomic RNA from milieu that contains a complex mixture of cellular or non-genomic viral RNAs. In this review, we focus on the role of viral encoded RNA structures in genome packaging. We first discuss how packaging signals are constructed from local and long-range base pairings within viral genomes, as well as inter-molecular interactions between viral and host RNAs. Then, how genome packaging is regulated by the biophysical properties of RNA. Finally, we examine the impact of RNA packaging signals on viral evolution.
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12
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Gokhale NS, Smith JR, Van Gelder RD, Savan R. RNA regulatory mechanisms that control antiviral innate immunity. Immunol Rev 2021; 304:77-96. [PMID: 34405416 DOI: 10.1111/imr.13019] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Revised: 07/29/2021] [Accepted: 08/02/2021] [Indexed: 12/21/2022]
Abstract
From the initial sensing of viral nucleotides by pattern recognition receptors, through the induction of type I and III interferons (IFN), upregulation of antiviral effector proteins, and resolution of the inflammatory response, each step of innate immune signaling is under tight control. Though innate immunity is often associated with broad regulation at the level of gene transcription, RNA-centric post-transcriptional processes have emerged as critical mechanisms for ensuring a proper antiviral response. Here, we explore the diverse RNA regulatory mechanisms that modulate the innate antiviral immune response, with a focus on RNA sensing by RIG-I-like receptors (RLR), interferon (IFN) and IFN signaling pathways, viral pathogenesis, and host genetic variation that contributes to these processes. We address the post-transcriptional interactions with RNA-binding proteins, non-coding RNAs, transcript elements, and modifications that control mRNA stability, as well as alternative splicing events that modulate the innate immune antiviral response.
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Affiliation(s)
- Nandan S Gokhale
- Department of Immunology, School of Medicine, University of Washington, Seattle, Washington, USA
| | - Julian R Smith
- Department of Immunology, School of Medicine, University of Washington, Seattle, Washington, USA
| | - Rachel D Van Gelder
- Department of Immunology, School of Medicine, University of Washington, Seattle, Washington, USA
| | - Ram Savan
- Department of Immunology, School of Medicine, University of Washington, Seattle, Washington, USA
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13
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Yin L, Keeler GD, Zhang Y, Hoffman BE, Ling C, Qing K, Srivastava A. AAV3-miRNA vectors for growth suppression of human hepatocellular carcinoma cells in vitro and human liver tumors in a murine xenograft model in vivo. Gene Ther 2021; 28:422-434. [PMID: 32152434 PMCID: PMC7784898 DOI: 10.1038/s41434-020-0140-1] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2019] [Revised: 02/26/2020] [Accepted: 02/27/2020] [Indexed: 12/24/2022]
Abstract
We have previously reported that recombinant adeno-associated virus serotype 3 (AAV3) vectors transduce human liver tumors more efficiently in a mouse xenograft model following systemic administration. Others have utilized AAV8 vectors expressing miR-26a and miR-122 to achieve near total inhibition of growth of mouse liver tumors. Since AAV3 vectors transduce human hepatic cells more efficiently than AAV8 vectors, in the present studies, we wished to evaluate the efficacy of AAV3-miR-26a/122 vectors in suppressing the growth of human hepatocellular carcinoma (HCC) cells in vitro, and human liver tumors in a mouse model in vivo. To this end, a human HCC cell line, Huh7, was transduced with various multiplicities of infection (MOIs) of AAV3-miR-26a or scAAV3-miR-122 vectors, or both, which also co-expressed a Gaussia luciferase (GLuc) reporter gene. Only a modest level of dose-dependent growth inhibition of Huh7 cells (~12-13%) was observed at the highest MOI (1 × 105 vgs/cell) with each vector. When Huh7 cells were co-transduced with both vectors, the extent of growth inhibition was additive (~26%). However, AAV3-miR-26a and scAAV3-miR-122 vectors led to ~70% inhibition of growth of Huh-derived human liver tumors in a mouse xenograft model in vivo. Thus, the combined use of miR-26a and scAAV3-miR-122 delivered by AAV3 vectors offers a potentially useful approach to target human liver tumors.
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Affiliation(s)
- Ling Yin
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai, China
- Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL, USA
- Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, USA
| | - Geoffrey D Keeler
- Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL, USA
- Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, USA
| | - Yuanhui Zhang
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai, China
- Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL, USA
- Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, USA
- Department of Oncology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China
| | - Brad E Hoffman
- Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL, USA
- Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, USA
| | - Chen Ling
- State Key Laboratory of Genetic Engineering, School of Life Sciences, Zhongshan Hospital, Fudan University, Shanghai, China.
- Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL, USA.
- Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, USA.
- Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, FL, USA.
| | - Keyun Qing
- Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL, USA
- Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, USA
| | - Arun Srivastava
- Division of Cellular and Molecular Therapy, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL, USA.
- Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL, USA.
- Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, FL, USA.
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Otoguro T, Tanaka T, Kasai H, Kobayashi N, Yamashita A, Fukuhara T, Ryo A, Fukai M, Taketomi A, Matsuura Y, Moriishi K. Establishment of a Cell Culture Model Permissive for Infection by Hepatitis B and C Viruses. Hepatol Commun 2021; 5:634-649. [PMID: 33860122 PMCID: PMC8034569 DOI: 10.1002/hep4.1653] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/15/2020] [Revised: 11/19/2020] [Accepted: 11/22/2020] [Indexed: 12/18/2022] Open
Abstract
Compared with each monoinfection, coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) is well known to increase the risks of developing liver cirrhosis and hepatocellular carcinoma. However, the mechanism by which HBV/HCV coinfection is established in hepatocytes is not well understood. Common cell culture models for coinfection are required to examine viral propagation. In this study, we aimed to establish a cell line permissive for both HBV and HCV infection. We first prepared a HepG2 cell line expressing sodium taurocholate cotransporting polypeptide, an HBV receptor, and then selected a cell line highly permissive for HBV infection, G2/NT18-B. After transduction with a lentivirus-encoding microRNA-122, the cell line harboring the highest level of replicon RNA was selected and then treated with anti-HCV compounds to eliminate the replicon RNA. The resulting cured cell line was transduced with a plasmid-encoding CD81. The cell line permissive for HCV infection was cloned and then designated the G2BC-C2 cell line, which exhibited permissiveness for HBV and HCV propagation. JAK inhibitor I potentiated the HCV superinfection of HBV-infected cells, and fluorescence-activated cell-sorting analysis indicated that HBV/HCV double-positive cells accounted for approximately 30% of the coinfected cells. Among several host genes tested, cyclooxygenase-2 showed synergistic induction by coinfection compared with each monoinfection. Conclusion: These data indicate that our in vitro HBV/HCV coinfection system provides an easy-to-use platform for the study of host and viral responses against coinfection and the development of antiviral agents targeting HBV and HCV.
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Affiliation(s)
- Teruhime Otoguro
- Department of MicrobiologyGraduate School of Medical ScienceUniversity of YamanashiYamanashiJapan
| | - Tomohisa Tanaka
- Department of MicrobiologyGraduate School of Medical ScienceUniversity of YamanashiYamanashiJapan
| | - Hirotake Kasai
- Department of MicrobiologyGraduate School of Medical ScienceUniversity of YamanashiYamanashiJapan
| | - Nobuhiro Kobayashi
- Department of Gastroenterological Surgery IGraduate School of MedicineHokkaido UniversityHokkaidoJapan
| | - Atsuya Yamashita
- Department of MicrobiologyGraduate School of Medical ScienceUniversity of YamanashiYamanashiJapan
| | - Takasuke Fukuhara
- Department of Molecular VirologyResearch Institute for Microbial DiseasesOsaka UniversityOsakaJapan.,Department of Microbiology and ImmunologyGraduate School of MedicineHokkaido UniversityHokkaidoJapan
| | - Akihide Ryo
- Department of MicrobiologyYokohama City University Graduate School of MedicineKanagawaJapan
| | - Moto Fukai
- Department of Gastroenterological Surgery IGraduate School of MedicineHokkaido UniversityHokkaidoJapan
| | - Akinobu Taketomi
- Department of Gastroenterological Surgery IGraduate School of MedicineHokkaido UniversityHokkaidoJapan
| | - Yoshiharu Matsuura
- Department of Molecular VirologyResearch Institute for Microbial DiseasesOsaka UniversityOsakaJapan
| | - Kohji Moriishi
- Department of MicrobiologyGraduate School of Medical ScienceUniversity of YamanashiYamanashiJapan
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15
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Shafaati M, Jamalidoust M, Kargar M, Arefian E, Kafilzadeh F. Downregulation of hepatitis C virus replication by miR-196a using lentiviral vectors. Microbiol Immunol 2021; 65:161-170. [PMID: 33470443 DOI: 10.1111/1348-0421.12875] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2020] [Revised: 11/25/2020] [Accepted: 01/08/2021] [Indexed: 11/29/2022]
Abstract
Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus that causes chronic hepatitis and hepatocellular carcinoma. Cellular microRNAs (miRNAs) directly modulate the viral infectivity and indirectly through targeting virus-related host factors. They play an essential role in the progression of different stages of HCV infection. The roles of miR-196 family in HCV infection and hepatocellular carcinoma progression remain poorly understood. Using ViTa databases, miR-196a as a high-score miRNA targeting the NS5 A region of HCV genome was selected. Using dual luciferase assay and an established cell-cultured HCV (HCVcc) system, the effect of miR-196a on HCV genome was assessed. In silico analysis demonstrated the significant role of miR-196a in the downregulation of HCV replication. Using dual luciferase assay, the liver-specific miR-196a and NS5 A gene binding was confirmed. To assess the experimental role of miR-196a, an HCVcc system was established in the Huh 7.5 cell lines. The HCV-RNA 1b derived from an infected patient was transfected into Huh 7.5 cells containing miR-196a lentiviral vectors (Huh 7.5/miR-196a), mocks (Huh 7.5/mock vector), and naïve Huh 7.5 cells. The rate of reduction of the HCV genome replication was assessed using relative real-time PCR assay. These results represent miR-196a overexpression and its roles in regulating HCV genome replication. However, miR-196a may inhibit HCV replication and accelerate the early stages of apoptosis. Overexpression of miR-196a in Huh 7.5 replicon cell is a potential new strategy to prevent hepatitis C infection. The results of this study suggest that miR-196a directly downregulates HCV replication and may serve as a new antiviral therapy.
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Affiliation(s)
- Maryam Shafaati
- Department of Microbiology, Faculty of Sciences, Jahrom Branch, Islamic Azad University, Jahrom, Iran
| | - Marzieh Jamalidoust
- Department of Virology, Professor Alborzi Clinical Microbiology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Mohammad Kargar
- Department of Microbiology, Faculty of Sciences, Jahrom Branch, Islamic Azad University, Jahrom, Iran
| | - Ehsan Arefian
- Department of Microbiology, School of Biology, College of Science, University of Tehran, Tehran, Iran
| | - Farshid Kafilzadeh
- Department of Microbiology, Faculty of Sciences, Jahrom Branch, Islamic Azad University, Jahrom, Iran
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Moghoofei M, Najafipour S, Mostafaei S, Tavakoli A, Bokharaei-Salim F, Ghorbani S, Javanmard D, Ghaffari H, Monavari SH. MicroRNAs Profiling in HIV, HCV, and HIV/HCV Co-Infected Patients. Curr HIV Res 2021; 19:27-34. [PMID: 32900348 DOI: 10.2174/1570162x18666200908112113] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2020] [Revised: 08/11/2020] [Accepted: 08/18/2020] [Indexed: 12/15/2022]
Abstract
BACKGROUND Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections are important public health issues. OBJECTIVE This study aimed to assess the association between microRNAs expression leveland immunological and viral markers in HIV, HCV, and HIV/HCV co-infected patients. METHODS The expression level of miR-29, miR-149, miR-199, miR-let7, miR-223, miR-155, miR-122, and miR-150 was evaluated in 20 HIV, 20 HCV, 20 co-infected patients, and 20 healthy controls using real-time PCR assay. HIV and HCVviral loads were measuredby real-time PCR, and also, CD4+ T-lymphocyte count was measuredby the PIMA CD4 analyzer. RESULTS The miRNA expression pattern in each mentioned group showed significantly different expression profiles, but some miRNA species were shared between the groups. MiR-122 and miR-155 were upregulated, while miR-29 and miR-223 were downregulated in three patients groups compared to healthy controls. A significant positive correlation was observed between the expression of miR-122 and HIV/HCV loads. But, miR-29 and let-7 were negatively correlated with HIV load, and miR-149 and let-7 were negatively correlated with HCV load. Also, miR-155 was positively correlated with HCV load. MiR-122 and miR-199 were negative while others were positively correlated with CD4+ T cell count. CONCLUSION These miRNAs are probably involved in the clinical progression and pathogenesis of HIV and HCV infections. Therefore, determining and manipulating these miRNAs can lead to opening a new gate to control these important infections.
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Affiliation(s)
- Mohsen Moghoofei
- Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | - Sohrab Najafipour
- Department of Microbiology, Faculty of Medicine, Fasa University of Medical Sciences, Fasa, Iran
| | - Shayan Mostafaei
- Medical Biology Research Center, Health Technology Institute, Kermanshah University of Medical Sciences, Kermanshah, Iran
| | - Ahmad Tavakoli
- Department of Virology, Faculty of Medicine, Iran University of Medical Science, Tehran, Iran
| | - Farah Bokharaei-Salim
- Department of Virology, Faculty of Medicine, Iran University of Medical Science, Tehran, Iran
| | - Saied Ghorbani
- Department of Virology, Faculty of Medicine, Iran University of Medical Science, Tehran, Iran
| | - Davod Javanmard
- Infectious Diseases Research Center, Birjand University of Medical Sciences, Birjand, Iran
| | - Hadi Ghaffari
- Department of Bacteriology and Virology, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran
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17
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Pham KM, Suter SR, Lu SS, Beal PA. Ester modification at the 3' end of anti-microRNA oligonucleotides increases potency of microRNA inhibition. Bioorg Med Chem 2021; 29:115894. [PMID: 33290908 PMCID: PMC8610567 DOI: 10.1016/j.bmc.2020.115894] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2020] [Revised: 11/17/2020] [Accepted: 11/19/2020] [Indexed: 11/17/2022]
Abstract
MicroRNAs (miRNAs) are short noncoding RNAs that play a fundamental role in gene regulation. Deregulation of miRNA expression has a strong correlation with disease and antisense oligonucleotides that bind and inhibit miRNAs associated with disease have therapeutic potential. Current research on the chemical modification of anti-miRNA oligonucleotides (anti-miRs) is focused on alterations of the phosphodiester-ribose backbone to improve nuclease resistance and binding affinity to miRNA strands. Here we describe a structure-guided approach for modification of the 3'-end of anti-miRs by screening for modifications compatible with a nucleotide-binding pocket present on human Argonaute2 (hAgo2). We computationally screened a library of 190 triazole-modified nucleoside analogs for complementarity to the t1A-binding pocket of hAgo2. Seventeen top scoring triazoles were then incorporated into the 3' end of anti-miR21 and potency was evaluated for each in a cell-based assay for anti-miR activity. Four triazole-modified anti-miRs showed higher potency than anti-miR21 bearing a 3' adenosine. In particular, a triazole-modified nucleoside bearing an ester substituent imparted a nine-fold and five-fold increase in activity for both anti-miR21 and anti-miR122 at 300 and 5 nM, respectively. The ester group was shown to be critical as a similar carboxylic acid and amide were inactive. Furthermore, anti-miR 3' end modification with triazole-modified nucleoside analogs improved resistance to snake venom phosphodiesterase, a 3'-exonuclease. Thus, the modifications described here are good candidates for improvement of anti-miR activity.
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Affiliation(s)
- Kevin M Pham
- Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, United States
| | - Scott R Suter
- Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, United States
| | - Shannon S Lu
- Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, United States
| | - Peter A Beal
- Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, United States.
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Lu C, Feng Y, Sun X, Li N, Kuang D, Wang W, Tong P, Han Y, Xia X, Dai J. Tree shrew bone marrow-derived mesenchymal stem cells express CD81, OCLN, and miR-122, facilitating the entire hepatitis C virus life cycle. J Med Virol 2020; 92:3465-3474. [PMID: 32056224 DOI: 10.1002/jmv.25710] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2019] [Accepted: 02/10/2020] [Indexed: 01/12/2023]
Abstract
Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and associated cirrhosis, and hepatocellular carcinoma worldwide. At present, there is no prophylactic vaccine against HCV due to the lack of in vivo and in vitro model systems. Although most recombinants of all major HCV genotypes replicate in Huh-7 cell line and derivatives, these cells are human hepatoma-derived cell line. Therefore, the development of un-tumor-derived cell systems facilitating the entire HCV life cycle is urgently needed. In this study, we aimed to establish a novel tree shrew-derived bone marrow-derived mesenchymal stem cell (BM-MSC) system to reconstruct the HCV life cycle. We transduction cluster of differentiation 81 (CD81), occludin (OCLN), and microRNA-122 (miR-122) into BM-MSCs, then used a well-established HCV, produced from the J6/JFH1-Huh7.5.1 culture system, to infect the cells. We observed that BM-MSCs transduction with CD81/OCLN or CD81/OCLN/miR-122 support HCV RNA replication and infectious virus production. We also found that the addition of exogenous vascular endothelial growth factor (VEGF) can enhance HCV infectivity in BM-MSCs, with HCV virus load up to 105 copies/mL. In conclusion, we identified the minimum essential factors required for HCV replication in tree shrew-derived nonhuman nonhepatic BM-MSCs. Further, we identified that exogenous addition of VEGF, and exogenous expression of CD81, OCLN, and miR-122, facilitates efficient viral replication and production of infectious particles. Our results describe a novel cell system capable of supporting the entire HCV life cycle, which may provide an essential tool for anti-HCV drug discovery, vaccine development, and study of pathogenesis.
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Affiliation(s)
- Caixia Lu
- Center of Tree Shrew Germplasm Resources, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming, China
- Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Kunming, China
- Yunnan Innovation Team of Standardization and Application Research in Tree Shrew, Kunming, China
| | - Yue Feng
- Yunnan Provincial Center for Molecular Medicine, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China
| | - Xiaomei Sun
- Center of Tree Shrew Germplasm Resources, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming, China
- Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Kunming, China
- Yunnan Innovation Team of Standardization and Application Research in Tree Shrew, Kunming, China
| | - Na Li
- Center of Tree Shrew Germplasm Resources, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming, China
- Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Kunming, China
| | - Dexuan Kuang
- Center of Tree Shrew Germplasm Resources, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming, China
| | - Wenguang Wang
- Center of Tree Shrew Germplasm Resources, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming, China
- Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Kunming, China
| | - Pinfen Tong
- Center of Tree Shrew Germplasm Resources, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming, China
- Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Kunming, China
| | - Yuanyuan Han
- Center of Tree Shrew Germplasm Resources, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming, China
- Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Kunming, China
| | - Xueshan Xia
- Yunnan Provincial Center for Molecular Medicine, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China
| | - Jiejie Dai
- Center of Tree Shrew Germplasm Resources, Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College, Kunming, China
- Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases, Kunming, China
- Yunnan Innovation Team of Standardization and Application Research in Tree Shrew, Kunming, China
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19
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Pierce JB, Simion V, Icli B, Pérez-Cremades D, Cheng HS, Feinberg MW. Computational Analysis of Targeting SARS-CoV-2, Viral Entry Proteins ACE2 and TMPRSS2, and Interferon Genes by Host MicroRNAs. Genes (Basel) 2020; 11:E1354. [PMID: 33207533 PMCID: PMC7696723 DOI: 10.3390/genes11111354] [Citation(s) in RCA: 51] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2020] [Revised: 10/16/2020] [Accepted: 10/16/2020] [Indexed: 01/18/2023] Open
Abstract
Rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), has led to a global pandemic, failures of local health care systems, and global economic recession. MicroRNAs (miRNAs) have recently emerged as important regulators of viral pathogenesis, particularly among RNA viruses, but the impact of host miRNAs on SARS-CoV-2 infectivity remains unknown. In this study, we utilize the combination of powerful bioinformatic prediction algorithms and miRNA profiling to predict endogenous host miRNAs that may play important roles in regulating SARS-CoV-2 infectivity. We provide a collection of high-probability miRNA binding sites within the SARS-CoV-2 genome as well as within mRNA transcripts of critical viral entry proteins ACE2 and TMPRSS2 and their upstream modulators, the interferons (IFN). By utilizing miRNA profiling datasets of SARS-CoV-2-resistant and -susceptible cell lines, we verify the biological plausibility of the predicted miRNA-target RNA interactions. Finally, we utilize miRNA profiling of SARS-CoV-2-infected cells to identify predicted miRNAs that are differentially regulated in infected cells. In particular, we identify predicted miRNA binders to SARS-CoV-2 ORFs (miR-23a (1ab), miR-29a, -29c (1ab, N), miR-151a, -151b (S), miR-4707-3p (S), miR-298 (5'-UTR), miR-7851-3p (5'-UTR), miR-8075 (5'-UTR)), ACE2 3'-UTR (miR-9-5p, miR-218-5p), TMPRSS2 3'-UTR (let-7d-5p, -7e-5p, miR-494-3p, miR-382-3p, miR-181c-5p), and IFN-α 3'-UTR (miR-361-5p, miR-410-3p). Overall, this study provides insight into potential novel regulatory mechanisms of SARS-CoV-2 by host miRNAs and lays the foundation for future investigation of these miRNAs as potential therapeutic targets or biomarkers.
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Affiliation(s)
- Jacob B. Pierce
- Department of Medicine, Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (J.B.P.); (V.S.); (B.I.); (D.P.-C.); (H.S.C.)
- Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
| | - Viorel Simion
- Department of Medicine, Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (J.B.P.); (V.S.); (B.I.); (D.P.-C.); (H.S.C.)
| | - Basak Icli
- Department of Medicine, Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (J.B.P.); (V.S.); (B.I.); (D.P.-C.); (H.S.C.)
| | - Daniel Pérez-Cremades
- Department of Medicine, Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (J.B.P.); (V.S.); (B.I.); (D.P.-C.); (H.S.C.)
| | - Henry S. Cheng
- Department of Medicine, Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (J.B.P.); (V.S.); (B.I.); (D.P.-C.); (H.S.C.)
| | - Mark W. Feinberg
- Department of Medicine, Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA; (J.B.P.); (V.S.); (B.I.); (D.P.-C.); (H.S.C.)
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20
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Kunden RD, Ghezelbash S, Khan JQ, Wilson JA. Location specific annealing of miR-122 and other small RNAs defines an Hepatitis C Virus 5' UTR regulatory element with distinct impacts on virus translation and genome stability. Nucleic Acids Res 2020; 48:9235-9249. [PMID: 32810257 PMCID: PMC7498337 DOI: 10.1093/nar/gkaa664] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2020] [Revised: 07/21/2020] [Accepted: 07/28/2020] [Indexed: 12/24/2022] Open
Abstract
Hepatitis C virus (HCV) replication requires annealing of a liver specific small-RNA, miR-122 to 2 sites on 5′ untranslated region (UTR). Annealing has been reported to (a) stabilize the genome, (b) stimulate translation and (c) promote the formation of translationally active Internal Ribosome Entry Site (IRES) RNA structure. In this report, we map the RNA element to which small RNA annealing promotes HCV to nucleotides 1–44 and identify the relative impact of small RNA annealing on virus translation promotion and genome stabilization. We mapped the optimal region on the HCV genome to which small RNA annealing promotes virus replication to nucleotides 19–37 and found the efficiency of viral RNA accumulation decreased as annealing moved away from this region. Then, by using a panel of small RNAs that promote replication with varying efficiencies we link the efficiency of lifecycle promotion with translation stimulation. By contrast small RNA annealing stabilized the viral genome even if they did not promote virus replication. Thus, we propose that miR-122 annealing promotes HCV replication by annealing to an RNA element that activates the HCV IRES and stimulates translation, and that miR-122 induced HCV genome stabilization is insufficient alone but enhances virus replication.
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Affiliation(s)
- Rasika D Kunden
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
| | - Sarah Ghezelbash
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
| | - Juveriya Q Khan
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
| | - Joyce A Wilson
- Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
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21
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Ono C, Fukuhara T, Li S, Wang J, Sato A, Izumi T, Fauzyah Y, Yamamoto T, Morioka Y, Dokholyan NV, Standley DM, Matsuura Y. Various miRNAs compensate the role of miR-122 on HCV replication. PLoS Pathog 2020; 16:e1008308. [PMID: 32574204 PMCID: PMC7337399 DOI: 10.1371/journal.ppat.1008308] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2019] [Revised: 07/06/2020] [Accepted: 05/18/2020] [Indexed: 02/07/2023] Open
Abstract
One of the determinants for tissue tropism of hepatitis C virus (HCV) is miR-122, a liver-specific microRNA. Recently, it has been reported that interaction of miR-122 to HCV RNA induces a conformational change of the 5'UTR internal ribosome entry site (IRES) structure to form stem-loop II structure (SLII) and hijack of translating 80S ribosome through the binding of SLIII to 40S subunit, which leads to efficient translation. On the other hand, low levels of HCV-RNA replication have also been detected in some non-hepatic cells; however, the details of extrahepatic replication remain unknown. These observations suggest the possibility that miRNAs other than miR-122 can support efficient replication of HCV-RNA in non-hepatic cells. Here, we identified a number of such miRNAs and show that they could be divided into two groups: those that bind HCV-RNA at two locations (miR-122 binding sites I and II), in a manner similar to miR-122 (miR-122-like), and those that target a single site that bridges sites I and II and masking both G28 and C29 in the 5'UTR (non-miR-122-like). Although the enhancing activity of these non-hepatic miRNAs were lower than those of miR-122, substantial expression was detected in various normal tissues. Furthermore, structural modeling indicated that both miR-122-like and non-miR-122-like miRNAs not only can facilitate the formation of an HCV IRES SLII but also can stabilize IRES 3D structure in order to facilitate binding of SLIII to the ribosome. Together, these results suggest that HCV facilitates miR-122-independent replication in non-hepatic cells through recruitment of miRNAs other than miR-122. And our findings can provide a more detailed mechanism of miR-122-dependent enhancement of HCV-RNA translation by focusing on IRES tertiary structure.
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Affiliation(s)
- Chikako Ono
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takasuke Fukuhara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Songling Li
- Department of Genome Informatics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Jian Wang
- Department of Pharmacology, Penn State University College of Medicine, Hershey, Pennsylvania, United States of America
| | - Asuka Sato
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takuma Izumi
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Yuzy Fauzyah
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takuya Yamamoto
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Yuhei Morioka
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Nikolay V. Dokholyan
- Department of Pharmacology, Penn State University College of Medicine, Hershey, Pennsylvania, United States of America
- Department of Biochemistry & Molecular Biology, Penn State University College of Medicine, Hershey, Pennsylvania, United States of America
| | - Daron M. Standley
- Department of Genome Informatics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Yoshiharu Matsuura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
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22
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Miyayama Y, Lee H, Song H, Abe-Chayama H, Miki D, Imamura M, Chayama K, Hijikata M. Comparative study on the replication of HCV1b genome between wild-type and cell culture-adaptive mutant in regard to sensitivities against anti-HCV drugs. Microbiol Immunol 2019; 64:296-303. [PMID: 31854467 DOI: 10.1111/1348-0421.12768] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2019] [Revised: 12/12/2019] [Accepted: 12/17/2019] [Indexed: 11/28/2022]
Abstract
The replicon system, which mimics viral genome replication in culture cells, has been widely used to analyze the genome replication of the hepatitis C virus (HCV). However, most HCV genomes used in the system include adaptive mutations (AMs) that are vital for replication in culture cells despite the nonexistence of such mutations in the genome of wild-type (WT) HCV in patients. In order to study the genome replications of WT HCV, new HCV subgenomic replicon (SGR) systems were established using Huh-7.5-derived cells producing Sec14-like protein 2 constitutively and SGR of KT9 (one of the HCV genotype 1b clones) with WT genome (SGR KT9WT) in this study. The replication efficiency and sensitivities of SGR KT9WT to anti-HCV drugs in the cloned cells permanently bearing replicon RNA, HS55-4 cells, were similar to those of reports using SGR, including AM. The SGR transient transfection system using SGR KT9WT and SGR KT9AM encoding secreted Nano-luciferase and HS55-4C cells established by the elimination of SGR KT9 RNA from HS55-4 cells, however, showed that the replication efficiency of SGR KT9WT was much lower than that of SGR KT9AM under a same condition. Furthermore, the sensitivities of SGR KT9WT to almost all tested anti-HCV reagents, except the inhibitor of miR-122, a cellular factor important for HCV replication, were quite low compared with SGR KT9AM. These results suggested that the new replicon systems might not only provide information about precise responses against new anti-HCV drugs but also reveal novel molecular mechanisms supporting negligent proliferation of HCV.
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Affiliation(s)
- Yohei Miyayama
- Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.,Graduate School of Biostudies, Kyoto University, Kyoto, Japan
| | - Heini Lee
- Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.,Graduate School of Biostudies, Kyoto University, Kyoto, Japan
| | - HoJoong Song
- Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.,Graduate School of Biostudies, Kyoto University, Kyoto, Japan
| | - Hiromi Abe-Chayama
- Department of Gastroenterology and Metabolism, Hiroshima University Hospital, Hiroshima, Japan
| | - Daiki Miki
- Department of Gastroenterology and Metabolism, Hiroshima University Hospital, Hiroshima, Japan
| | - Michio Imamura
- Department of Gastroenterology and Metabolism, Hiroshima University Hospital, Hiroshima, Japan
| | - Kazuaki Chayama
- Department of Gastroenterology and Metabolism, Hiroshima University Hospital, Hiroshima, Japan
| | - Makoto Hijikata
- Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.,Graduate School of Biostudies, Kyoto University, Kyoto, Japan
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23
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Lee J, Mou H, Ibraheim R, Liang SQ, Liu P, Xue W, Sontheimer EJ. Tissue-restricted genome editing in vivo specified by microRNA-repressible anti-CRISPR proteins. RNA (NEW YORK, N.Y.) 2019; 25:1421-1431. [PMID: 31439808 PMCID: PMC6795140 DOI: 10.1261/rna.071704.119] [Citation(s) in RCA: 59] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/01/2019] [Accepted: 08/09/2019] [Indexed: 05/20/2023]
Abstract
CRISPR-Cas systems are bacterial adaptive immune pathways that have revolutionized biotechnology and biomedical applications. Despite the potential for human therapeutic development, there are many hurdles that must be overcome before its use in clinical settings. Some clinical safety concerns arise from editing activity in unintended cell types or tissues upon in vivo delivery (e.g., by adeno-associated virus (AAV) vectors). Although tissue-specific promoters and serotypes with tissue tropisms can be used, suitably compact promoters are not always available for desired cell types, and AAV tissue tropism specificities are not absolute. To reinforce tissue-specific editing, we exploited anti-CRISPR proteins (Acrs) that have evolved as natural countermeasures against CRISPR immunity. To inhibit Cas9 in all ancillary tissues without compromising editing in the target tissue, we established a flexible platform in which an Acr transgene is repressed by endogenous, tissue-specific microRNAs (miRNAs). We demonstrate that miRNAs regulate the expression of an Acr transgene bearing miRNA-binding sites in its 3'-UTR and control subsequent genome editing outcomes in a cell-type specific manner. We also show that the strategy is applicable to multiple Cas9 orthologs and their respective anti-CRISPRs. Furthermore, we validate this approach in vivo by demonstrating that AAV9 delivery of Nme2Cas9, along with an AcrIIC3 Nme construct that is targeted for repression by liver-specific miR-122, allows editing in the liver while repressing editing in an unintended tissue (heart muscle) in adult mice. This strategy provides safeguards against off-tissue genome editing by confining Cas9 activity to selected cell types.
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Affiliation(s)
- Jooyoung Lee
- RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
| | - Haiwei Mou
- RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
| | - Raed Ibraheim
- RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
| | - Shun-Qing Liang
- RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
| | - Pengpeng Liu
- Program in Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
| | - Wen Xue
- RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
- Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
| | - Erik J Sontheimer
- RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
- Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA
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24
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Zhang X, Feng Y, Yan Y, Zheng Z, Wang W, Zhang Y, Zhou EM, Xiao S. Cellular microRNA miR-c89 inhibits replication of porcine reproductive and respiratory syndrome virus by targeting the host factor porcine retinoid X receptor β. J Gen Virol 2019; 100:1407-1416. [PMID: 31478827 DOI: 10.1099/jgv.0.001320] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
MicroRNAs (miRNAs) play critical roles in the complex networks of virus-host interactions. Our previous research showed that porcine reproductive and respiratory syndrome virus (PRRSV) infection markedly upregulates miR-c89 expression, suggesting that miR-c89 may play an important role in PRRSV infection. The present study sought to determine the function of miR-c89 and its molecular mechanism during PRRSV infection. Using quantitative reverse transcription PCR (RT-qPCR) verification, we demonstrated that both highly pathogenic PRRSV and low-pathogenic PRRSV infection induced miR-c89 expression. The overexpression of miR-c89 significantly suppressed the replication of a variety of PRRSV strains, regardless of the timing of infection. Further, miR-c89 can directly target the 3'UTR of porcine retinoid X receptor β (RXRB) mRNA in a sequence-specific manner. Knockdown affected RXRB expression, as siRNA can suppress the replication of a variety of PRRSV strains. This work not only provides new insights into PRRSV-cell interactions, but also highlights the potential for the use of miR-c89 in the development of new antiviral strategies to combat PRRSV infection.
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Affiliation(s)
- Xiaobin Zhang
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, PR China
| | - Yingtong Feng
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, PR China
| | - Yunhuan Yan
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, PR China
| | - Zifang Zheng
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, PR China
| | - Wenjing Wang
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, PR China
| | - Yichi Zhang
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, PR China
| | - En-Min Zhou
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, PR China
| | - Shuqi Xiao
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, 712100, PR China
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25
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Fukuhara T, Matsuura Y. Roles of secretory glycoproteins in particle formation of Flaviviridae viruses. Microbiol Immunol 2019; 63:401-406. [PMID: 31342548 DOI: 10.1111/1348-0421.12733] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2019] [Revised: 07/17/2019] [Accepted: 07/18/2019] [Indexed: 12/12/2022]
Abstract
The family Flaviviridae comprises four genera, namely, Flavivirus, Pestivirus, Pegivirus, and Hepacivirus. These viruses have similar genome structures, but the genomes of Pestivirus and Flavivirus encode the secretory glycoproteins Erns and NS1, respectively. Erns plays an important role in virus particle formation and cell entry, whereas NS1 participates in the formation of replication complexes and virus particles. Conversely, apolipoproteins are known to participate in the formation of infectious particles of hepatitis C virus (HCV) and various secretory glycoproteins play a similar role in HCV particles formation, suggesting that there is no strong specificity for the function of secretory glycoproteins in infectious-particle formation. In addition, recent studies have shown that host-derived apolipoproteins and virus-derived Erns and NS1 play comparable roles in infectious-particle formation of both HCV and pestiviruses. In this review, we summarize the roles of secretory glycoproteins in the formation of Flaviviridae virus particles.
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Affiliation(s)
- Takasuke Fukuhara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Yoshiharu Matsuura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
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26
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Comparison of three human liver cell lines for in vitro drug-induced liver injury assessment: Huh7, HepaRG, and stem cell-derived hepatocytes. Mol Cell Toxicol 2019. [DOI: 10.1007/s13273-019-0031-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
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27
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[Hepatic tropism of hepatitis C virus infection]. Uirusu 2019; 68:63-70. [PMID: 31105136 DOI: 10.2222/jsv.68.63] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
Hepatitis C virus (HCV) infects over 170 million people worldwide and is a major cause of life-threatening liver diseases such as liver cirrhosis and hepatocellular carcinoma. In current research, we aimed to clarify the mechanism of hepatic tropism of HCV infection. Although non-hepatic cells could not permit replication of HCV RNA, exogenous expression of liver specific miRNA, miR-122 facilitated efficient replication of viral RNA through direct interaction with 5'UTR of viral genome, indicating that miR-122 is one of the key determinants for hepatic tropism of HCV infection. In spite of efficient replication of viral RNA, formation of infectious particles was not observed in non-hepatic cells exogenously expressing miR-122. We found that expression of apolipoprotein E (ApoE) facilitated the formation of infectious HCV particles in non-hepatic cells, indicating that not only miR-122 but also ApoE participate in tissue tropism of HCV infection. To understand the exact roles of miR-122 and apolipoproteins in hepatic tropism of HCV, we established miR-122 and ApoB/ApoE knockout (KO) Huh7 cells, respectively. Although slight increase of intracellular HCV RNA and infectious titers in the culture supernatants was observed, propagation of HCV was impaired in miR-122 KO Huh7 cells. After serial passages of HCV in miR-122 KO cells, we obtained an adaptive mutant that possessed G28A substitutions in the 5'UTR of the HCV genome and exhibited efficient translation and replication in both miR-122 KO Huh7 and non-hepatic cells without exogenous expression of miR-122. These results suggest that HCV mutants replicating in non-hepatic cells in an miR-122-independent manner participate in the induction of extrahepatic manifestations in chronic hepatitis C patients. Deficiency of both ApoB and ApoE strongly inhibited the formation of infectious HCV particles. Interestingly, expression not only of ApoE but also of ApoA or ApoC could rescue the production of infectious HCV particles in ApoB/ApoE KO cells, suggesting that exchangeable apolipoproteins redundantly participate in the formation of infectious HCV particles.
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28
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Establishment and Characterization of a New Cell Line Permissive for Hepatitis C Virus Infection. Sci Rep 2019; 9:7943. [PMID: 31138826 PMCID: PMC6538753 DOI: 10.1038/s41598-019-44257-5] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2018] [Accepted: 03/12/2019] [Indexed: 01/02/2023] Open
Abstract
Hepatitis C virus (HCV) cell culture systems have facilitated the development of efficient direct-acting antivirals against HCV. Huh-7.5, a subline of the human hepatoma cell line Huh-7, has been used widely to amplify HCV because HCV can efficiently replicate in these cells due to a defect in innate antiviral signalling. Recently, we established a novel cell line, KH, derived from human hepatocellular carcinoma, which showed atypical uptake of gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) in a Gd-EOB-DTPA-enhanced magnetic resonance imaging study. KH cells expressed hepatocyte markers including microRNA-122 (miR-122) at a lower level than Huh-7.5 cells. We demonstrated that KH cells could support the entire life cycle of HCV; however, HCV replicated at a lower rate in KH cells compared to Huh-7.5 cells, and virus particles produced from KH cells seemed to have some disadvantages in viral assembly compared with those produced from Huh-7.5 cells. KH cells had more robust interferon-stimulated gene expression and induction upon HCV RNA transfection, interferon-α2b addition, and HCV infection than Huh-7.5 cells. Interestingly, both miR-122 supplementation and IRF3 knockout in KH cells boosted HCV replication to a similar level as in Huh-7.5 cells, suggesting that intact innate antiviral signalling and lower miR-122 expression limit HCV replication in KH cells. KH cells will enable a deeper understanding of the role of the innate immune response in persistent HCV infection.
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29
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Zhao Y, Lin Q, Li N, Babu VS, Fu X, Liu L, Liang H, Liu X, Lin L. MicroRNAs profiles of Chinese Perch Brain (CPB) cells infected with Siniperca chuatsi rhabdovirus (SCRV). FISH & SHELLFISH IMMUNOLOGY 2019; 84:1075-1082. [PMID: 30423456 DOI: 10.1016/j.fsi.2018.11.020] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/27/2018] [Revised: 11/03/2018] [Accepted: 11/06/2018] [Indexed: 06/09/2023]
Abstract
MicroRNAs are non-coding RNAs, which widely participate in biological processes. In recent years, Siniperca chuatsi rhabdovirus (SCRV) has caused mass mortality in Chinese perch (Siniperca chuatsi). To identify specific miRNAs involved in SCRV infection, deep sequencing of microRNA on Chinese perch brain cell line (CPB) with or without SCRV infection were performed at 6 and 12 h post of infection (hpi). Totally 382 miRNAs were identified, including 217 known miRNA aligned with zebrafish miRNAs and 165 novel miRNAs by MiRDeep2 program. Of which 15 and 35 differentially-expressed miRNAs were determined respectively to 6 and 12 hpi. Nine miRNAs were selected randomly from the differentially-expressed miRNAs and validated by quantitative real-time PCR (qRT-PCR). These results were consistent with the microRNA sequencing results. Besides, target genes of 98 differentially-expressed miRNAs were predicted. Three of miRNAs (miR-122, miR-214, miR-135a) were selected, and its effects were analyzed in CPC cells transfected with appropriate miRNA mimics/inhibitors to evaluate its regulation effects by qRT-PCR and western blot. The results demonstrated that miR-214 inhibited the replication of SCRV, while miR-122 promoted the replication of SCRV and there was no correlation between the miR-135a and SCRV replication. These results will pave a new way for the development of effective strategies against the SCRV infection.
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Affiliation(s)
- Yongliang Zhao
- Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei, 430070, China; Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou, Guangdong, 510380, China; Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Guangdong Provincial Key Laboratory of Waterfowl Healthy Breeding, College of Animal Sciences and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou, Guangdong, 510225, China
| | - Qiang Lin
- Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou, Guangdong, 510380, China.
| | - Ningqiu Li
- Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou, Guangdong, 510380, China
| | - V Sarath Babu
- Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Guangdong Provincial Key Laboratory of Waterfowl Healthy Breeding, College of Animal Sciences and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou, Guangdong, 510225, China
| | - Xiaozhe Fu
- Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou, Guangdong, 510380, China
| | - Lihui Liu
- Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou, Guangdong, 510380, China
| | - Hongru Liang
- Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Key Laboratory of Aquatic Animal Immune Technology, Guangzhou, Guangdong, 510380, China
| | - Xiaoling Liu
- Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan, Hubei, 430070, China
| | - Li Lin
- Guangzhou Key Laboratory of Aquatic Animal Diseases and Waterfowl Breeding, Guangdong Provincial Key Laboratory of Waterfowl Healthy Breeding, College of Animal Sciences and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou, Guangdong, 510225, China.
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30
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Mori H, Fukuhara T, Ono C, Tamura T, Sato A, Fauzyah Y, Wada M, Okamoto T, Noda T, Yoshimori T, Matsuura Y. Induction of selective autophagy in cells replicating hepatitis C virus genome. J Gen Virol 2018; 99:1643-1657. [DOI: 10.1099/jgv.0.001161] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Affiliation(s)
- Hiroyuki Mori
- 1Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
| | - Takasuke Fukuhara
- 1Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
| | - Chikako Ono
- 1Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
| | - Tomokazu Tamura
- 1Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
| | - Asuka Sato
- 1Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
| | - Yuzy Fauzyah
- 1Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
| | - Masami Wada
- 1Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
- †Present address: Division of Virology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
| | - Toru Okamoto
- 1Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
| | - Takeshi Noda
- 2Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, 1-8 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
| | - Tamotsu Yoshimori
- 3Department of Genetics, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
| | - Yoshiharu Matsuura
- 1Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan
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31
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Sequential S232/S235/S238 Phosphorylation of the Hepatitis C Virus Nonstructural Protein 5A. J Virol 2018; 92:JVI.01295-18. [PMID: 30089697 DOI: 10.1128/jvi.01295-18] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2018] [Accepted: 07/30/2018] [Indexed: 02/06/2023] Open
Abstract
The hepatitis C virus (HCV) protein NS5A is a phosphorylated protein with crucial roles in viral replication and assembly. NS5A was thought to undergo sequential phosphorylation on a series of conserved serine residues; however, the phosphorylation cascade remained obscure. Using three phosphorylation-specific antibodies, we found that phosphorylation at S232, S235, and S238 occurred in parallel in HCV-infected Huh7.5.1 cells, suggestive of intramolecular sequential NS5A phosphorylation from S232 through S235 to S238 by casein kinase Iα (CKIα). In line with this, alanine mutation at S225, S229, or S232 reduced, whereas aspartate mutation at the same sites rescued, NS5A phosphorylation at S232, S235, and S238. In contrast, alanine or aspartate mutation at S235 or S238 had little or no effect on S232 or S235 phosphorylation. Consistent with an intramolecular sequential phosphorylation cascade, S232, S235, and S238 phosphorylation coexisted on one single NS5A molecule. Phosphorylation of NH2-terminal serine residues in one NS5A molecule did not rescue phosphorylation of COOH-terminal serine residues in another NS5A molecule. CKIα inhibition reduced NS5A phosphorylation at S232, S235, and S238. In summary, our results are indicative of a CKIα-mediated intramolecular, sequential phosphorylation cascade from S232 through S235 to S238 of the HCV NS5A protein. S225 and S229 also contribute substantially to the above sequential phosphorylation cascade of NS5A.IMPORTANCE The nonstructural protein 5A (NS5A) of the hepatitis C virus was thought to undergo sequential intramolecular phosphorylation on a series of serine residues; however, direct evidence was missing. We offer the first direct evidence of a CKIα-mediated intramolecular sequential NS5A phosphorylation cascade from serine 232 through 235 to 238. This sequential phosphorylation cascade occurs in the disordered low-complexity sequence I region, which together with the domain I region forms an RNA-binding groove in an NS5A dimer. Sequential phosphorylation in the disordered region adds charge-charge repulsion to the RNA-binding groove and probably thereby regulates NS5A's RNA-binding ability and functions in viral RNA replication and assembly.
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Suda T, Tatsumi T, Nishio A, Kegasawa T, Yoshioka T, Yamada R, Furuta K, Kodama T, Shigekawa M, Hikita H, Sakamori R, Fukuhara T, Matsuura Y, Takehara T. CEACAM1 Is Associated With the Suppression of Natural Killer Cell Function in Patients With Chronic Hepatitis C. Hepatol Commun 2018; 2:1247-1258. [PMID: 30288478 PMCID: PMC6167072 DOI: 10.1002/hep4.1240] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/15/2017] [Accepted: 05/20/2018] [Indexed: 12/15/2022] Open
Abstract
Natural killer cells (NK cells) play an essential role in the immunological mechanism underlying chronic hepatitis C (CHC). Impairment of NK cell function facilitates persistent infection with hepatitis C virus (HCV) and hepatocellular carcinogenesis. However, the mechanism by which NK cell activity is suppressed in CHC is not completely understood. In this study, we focused on carcinoembryonic antigen–related cell‐adhesion molecule 1 (CEACAM1). CEACAM1 is thought to suppress NK cell function. We examined the effect of CEACAM1 on NK cell function in CHC. We investigated the function of CEACAM1 in vitro using Huh7.5.1 cells and the HCV‐Japanese fulminant hepatitis (JFH)‐1 strain. We analyzed serum CEACAM1 level, NK cell function, and CEACAM1 messenger RNA (mRNA) level in human liver samples. Levels of CEACAM1 on the cell surface, CEACAM1 mRNA levels, and soluble CEACAM1 levels in supernatants were significantly higher in Huh7.5.1 cells infected with JFH‐1 (Huh7.5.1/JFH‐1 cells) than in Huh7.5.1 cells. Significantly higher NK cell cytotoxicity was observed toward K562 cells after coculture with CEACAM1 knockout Huh7.5.1/JFH‐1 cells than after coculture with Huh7.5.1/JFH‐1 cells. CEACAM1 expression was induced by the HCV E2 glycoprotein in HCV infection. Significantly higher serum CEACAM1 levels were detected in patients with CHC compared with healthy subjects and patients who achieved sustained virological responses. The expression of CD107a on NK cells from patients with CHC was negatively correlated with serum CEACAM1 levels. Significantly higher levels of CEACAM1 mRNA were detected in HCV‐infected livers compared with uninfected livers. Conclusion: CEACAM1 expression was induced in hepatocytes following HCV infection and decreased NK cell cytotoxicity. These results demonstrate a possible role for CEACAM1 in the pathogenesis of CHC and hepatocellular carcinoma progression.
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Affiliation(s)
- Takahiro Suda
- Department of Gastroenterology and Hepatology Osaka University Graduate School of Medicine Suita Japan
| | - Tomohide Tatsumi
- Department of Gastroenterology and Hepatology Osaka University Graduate School of Medicine Suita Japan
| | - Akira Nishio
- Department of Gastroenterology and Hepatology Osaka University Graduate School of Medicine Suita Japan
| | - Tadashi Kegasawa
- Department of Gastroenterology and Hepatology Osaka University Graduate School of Medicine Suita Japan
| | - Teppei Yoshioka
- Department of Gastroenterology and Hepatology Osaka University Graduate School of Medicine Suita Japan
| | - Ryoko Yamada
- Department of Gastroenterology and Hepatology Osaka University Graduate School of Medicine Suita Japan
| | - Kunimaro Furuta
- Department of Gastroenterology and Hepatology Osaka University Graduate School of Medicine Suita Japan
| | - Takahiro Kodama
- Department of Gastroenterology and Hepatology Osaka University Graduate School of Medicine Suita Japan
| | - Minoru Shigekawa
- Department of Gastroenterology and Hepatology Osaka University Graduate School of Medicine Suita Japan
| | - Hayato Hikita
- Department of Gastroenterology and Hepatology Osaka University Graduate School of Medicine Suita Japan
| | - Ryotaro Sakamori
- Department of Gastroenterology and Hepatology Osaka University Graduate School of Medicine Suita Japan
| | - Takasuke Fukuhara
- Department of Molecular Virology Research Institute for Microbial Diseases, Osaka University Suita Japan
| | - Yoshiharu Matsuura
- Department of Molecular Virology Research Institute for Microbial Diseases, Osaka University Suita Japan
| | - Tetsuo Takehara
- Department of Gastroenterology and Hepatology Osaka University Graduate School of Medicine Suita Japan
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Ramirez S, Bukh J. Current status and future development of infectious cell-culture models for the major genotypes of hepatitis C virus: Essential tools in testing of antivirals and emerging vaccine strategies. Antiviral Res 2018; 158:264-287. [PMID: 30059723 DOI: 10.1016/j.antiviral.2018.07.014] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2018] [Revised: 07/17/2018] [Accepted: 07/20/2018] [Indexed: 02/08/2023]
Abstract
In this review, we summarize the relevant scientific advances that led to the development of infectious cell culture systems for hepatitis C virus (HCV) with the corresponding challenges and successes. We also provide an overview of how these systems have contributed to the study of antiviral compounds and their relevance for the development of a much-needed vaccine against this major human pathogen. An efficient infectious system to study HCV in vitro, using human hepatoma derived cells, has only been available since 2005, and was limited to a single isolate, named JFH1, until 2012. Successive developments have been slow and cumbersome, as each available system has been the result of a systematic effort for discovering adaptive mutations conferring culture replication and propagation to patient consensus clones that are inherently non-viable in vitro. High genetic heterogeneity is a paramount characteristic of this virus, and as such, it should preferably be reflected in basic, translational, and clinical studies. The limited number of efficient viral culture systems, in the context of the vast genetic diversity of HCV, continues to represent a major hindrance for the study of this virus, posing a significant barrier towards studies of antivirals (particularly of resistance) and for advancing vaccine development. Intensive research efforts, driven by isolate-specific culture adaptation, have only led to efficient full-length infectious culture systems for a few strains of HCV genotypes 1, 2, 3, and 6. Hence research aimed at identifying novel strategies that will permit universal culture of HCV will be needed to further our understanding of this unique virus causing 400 thousand deaths annually.
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Affiliation(s)
- Santseharay Ramirez
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
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Roles of the 5' Untranslated Region of Nonprimate Hepacivirus in Translation Initiation and Viral Replication. J Virol 2018; 92:JVI.01997-17. [PMID: 29343570 DOI: 10.1128/jvi.01997-17] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2017] [Accepted: 01/09/2018] [Indexed: 12/26/2022] Open
Abstract
The 5' untranslated region (UTR) of hepatitis C virus (HCV), which is composed of four domains (I, II, III, and IV) and a pseudoknot, is essential for translation and viral replication. Equine nonprimate hepacivirus (EHcV) harbors a 5' UTR consisting of a large 5'-terminal domain (I); three additional domains (I', II, and III), which are homologous to domains I, II, and III, respectively, of HCV; and a pseudoknot, in the order listed. In this study, we investigated the roles of the EHcV 5' UTR in translation and viral replication. The internal ribosome entry site (IRES) activity of the EHcV 5' UTR was lower than that of the HCV 5' UTR in several cell lines due to structural differences in domain III. Domains I and III of EHcV were functional in the HCV 5' UTR in terms of IRES activity and the replication of the subgenomic replicon (SGR), although domain II was not exchangeable between EHcV and HCV for SGR replication. Furthermore, the region spanning domains I and I' of EHcV (the 5'-proximal EHcV-specific region) improved RNA stability and provided the HCV SGR with microRNA 122 (miR-122)-independent replication capability, while EHcV domain I alone improved SGR replication and RNA stability irrespective of miR-122. These data suggest that the region spanning EHcV domains I and I' improves RNA stability and viral replication regardless of miR-122 expression. The 5'-proximal EHcV-specific region may represent an inherent mechanism to facilitate viral replication in nonhepatic tissues.IMPORTANCE EHcV is the closest viral homolog to HCV among other hepaciviruses. HCV exhibits a narrow host range and liver-specific tropism, while epidemiological reports suggest that EHcV infects the liver and respiratory organs in horses, donkeys, and dogs. However, the mechanism explaining the differences in host or organ tropism between HCV and EHcV is unknown. In this study, our data suggest that the 5' untranslated region (UTR) of EHcV is composed of an internal ribosome entry site (IRES) element that is functionally exchangeable with HCV IRES elements. Furthermore, the 5'-proximal EHcV-specific region enhances viral replication and RNA stability in a miR-122-independent manner. Our data suggest that the region upstream of domain II in the EHcV 5' UTR contributes to the differences in tissue tropism observed between these hepaciviruses.
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Fernández-Carrillo C, Pérez-Vilaró G, Díez J, Pérez-Del-Pulgar S. Hepatitis C virus plays with fire and yet avoids getting burned. A review for clinicians on processing bodies and stress granules. Liver Int 2018; 38:388-398. [PMID: 28782251 DOI: 10.1111/liv.13541] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/14/2016] [Accepted: 08/02/2017] [Indexed: 02/13/2023]
Abstract
Over the last few years, many reports have defined several types of RNA cell granules composed of proteins and messenger RNA (mRNA) that regulate gene expression on a post-transcriptional level. Processing bodies (P-bodies) and stress granules (SGs) are among the best-known RNA granules, only detectable when they accumulate into very dynamic cytosolic foci. Recently, a tight association has been found between positive-stranded RNA viruses, including hepatitis C virus (HCV), and these granules. The present article offers a comprehensive review on the complex and paradoxical relationship between HCV, P-bodies and SGs from a translational perspective. Despite the fact that components of P-bodies and SGs have assiduously controlled mRNA expression, either by sequestration or degradation, for thousands of years, HCV has learned how to dangerously exploit certain of them for its own benefit in an endless biological war. Thus, HCV has gained the ability to hack ancient host machineries inherited from prokaryotic times. While P-bodies and SGs are crucial to the HCV cycle, in the interferon-free era we still lack detailed knowledge of the mechanisms involved, processes that may underlie the long-term complications of HCV infection.
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Affiliation(s)
| | - Gemma Pérez-Vilaró
- Department of Experimental and Health Sciences, Molecular Virology, Universitat Pompeu Fabra, Barcelona, Spain
| | - Juana Díez
- Department of Experimental and Health Sciences, Molecular Virology, Universitat Pompeu Fabra, Barcelona, Spain
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Fournier C, Hoffmann TW, Morel V, Descamps V, Dubuisson J, Brochot E, Francois C, Duverlie G, Castelain S, Helle F. Claudin-1, miR-122 and apolipoprotein E transductions improve the permissivity of SNU-182, SNU-398 and SNU-449 hepatoma cells to hepatitis C virus. J Viral Hepat 2018; 25:63-71. [PMID: 28772350 DOI: 10.1111/jvh.12767] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/18/2017] [Accepted: 07/11/2017] [Indexed: 12/09/2022]
Abstract
Hepatitis C virus (HCV) is a human hepatotropic virus, but many hepatoma cell lines are not permissive to this virus. In a previous study, we observed that SNU-182, SNU-398 and SNU-449 hepatoma cell lines were nonpermissive to HCV. To understand the nonpermissivity, we evaluated the ability of each cell line to support the different steps of HCV life cycle (entry, replication and production of infectious particles). Using retroviral pseudoparticles pseudotyped with HCV envelope proteins and recombinant HCV produced in cell culture, we observed that low level or absence of claudin-1 (CLDN1) expression limited the viral entry process in SNU-182 and SNU-398 cells, respectively. Our results also showed that supplementation of the three cell lines with miR-122 partly restored the replication of a JFH1 HCV replicon. Finally, we observed that expression of apolipoprotein E (ApoE) was very low or undetectable in the three cell lines and that its ectopic expression permits the production of infectious viral particles in SNU-182 and SNU-398 cells but not in SNU-449 cells. Nevertheless, the supplementation of SNU-182, SNU-398 and SNU-449 cells with CLDN1, miR-122 and ApoE was not sufficient to render these cells as permissive as HuH-7 cells. Thus, these cell lines could serve as cell culture models for functional studies on the role of CLDN1, miR-122 and ApoE in HCV life cycle but also for the identification of new restriction and/or dependency host factors essential for HCV infection.
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Affiliation(s)
- C Fournier
- EA4294, Laboratoire de Virologie, Centre Universitaire de Recherche en Santé, Centre Hospitalier Universitaire et Université de Picardie Jules Verne, Amiens, France
| | - T W Hoffmann
- EA4294, Laboratoire de Virologie, Centre Universitaire de Recherche en Santé, Centre Hospitalier Universitaire et Université de Picardie Jules Verne, Amiens, France
| | - V Morel
- EA4294, Laboratoire de Virologie, Centre Universitaire de Recherche en Santé, Centre Hospitalier Universitaire et Université de Picardie Jules Verne, Amiens, France
| | - V Descamps
- EA4294, Laboratoire de Virologie, Centre Universitaire de Recherche en Santé, Centre Hospitalier Universitaire et Université de Picardie Jules Verne, Amiens, France
| | - J Dubuisson
- U1019 - UMR 8204, CIIL - Centre d'Infection et d'Immunité de Lille, CNRS, Institut Pasteur de Lille, Inserm, CHU Lille, Université Lille, Lille, France
| | - E Brochot
- EA4294, Laboratoire de Virologie, Centre Universitaire de Recherche en Santé, Centre Hospitalier Universitaire et Université de Picardie Jules Verne, Amiens, France
| | - C Francois
- EA4294, Laboratoire de Virologie, Centre Universitaire de Recherche en Santé, Centre Hospitalier Universitaire et Université de Picardie Jules Verne, Amiens, France
| | - G Duverlie
- EA4294, Laboratoire de Virologie, Centre Universitaire de Recherche en Santé, Centre Hospitalier Universitaire et Université de Picardie Jules Verne, Amiens, France
| | - S Castelain
- EA4294, Laboratoire de Virologie, Centre Universitaire de Recherche en Santé, Centre Hospitalier Universitaire et Université de Picardie Jules Verne, Amiens, France
| | - F Helle
- EA4294, Laboratoire de Virologie, Centre Universitaire de Recherche en Santé, Centre Hospitalier Universitaire et Université de Picardie Jules Verne, Amiens, France
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Huang K, Doyle F, Wurz ZE, Tenenbaum SA, Hammond RK, Caplan JL, Meyers BC. FASTmiR: an RNA-based sensor for in vitro quantification and live-cell localization of small RNAs. Nucleic Acids Res 2017; 45:e130. [PMID: 28586459 PMCID: PMC5737440 DOI: 10.1093/nar/gkx504] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2016] [Accepted: 05/27/2017] [Indexed: 01/19/2023] Open
Abstract
Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a variety of important regulatory roles in many eukaryotes. Their small size has made it challenging to study them directly in live cells. Here we describe an RNA-based fluorescent sensor for small RNA detection both in vitro and in vivo, adaptable for any small RNA. It utilizes an sxRNA switch for detection of miRNA–mRNA interactions combined with a fluorophore-binding sequence ‘Spinach’, a GFP-like RNA aptamer for which the RNA–fluorophore complex exhibits strong and consistent fluorescence under an excitation wavelength. Two example sensors, FASTmiR171 and FASTmiR122, can rapidly detect and quantify the levels of miR171 and miR122 in vitro. The sensors can determine relative levels of miRNAs in total RNA extracts with sensitivity similar to small RNA sequencing and northern blots. FASTmiR sensors were also used to estimate the copy number range of miRNAs in total RNA extracts. To localize and analyze the spatial distribution of small RNAs in live, single cells, tandem copies of FASTmiR122 were expressed in different cell lines. FASTmiR122 was able to quantitatively detect the differences in miR122 levels in Huh7 and HEK293T cells demonstrating its potential for tracking miRNA expression and localization in vivo.
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Affiliation(s)
- Kun Huang
- Bio-Imaging Center, Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA.,Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716, USA
| | - Francis Doyle
- Nanobioscience Constellation, State University of New York- Polytechnic Institute, College of Nanoscale Science and Engineering, Albany, NY 12203, USA
| | - Zachary E Wurz
- Nanobioscience Constellation, State University of New York- Polytechnic Institute, College of Nanoscale Science and Engineering, Albany, NY 12203, USA
| | - Scott A Tenenbaum
- Nanobioscience Constellation, State University of New York- Polytechnic Institute, College of Nanoscale Science and Engineering, Albany, NY 12203, USA
| | - Reza K Hammond
- Bio-Imaging Center, Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA.,Center for Bioinformatics and Computational Biology, University of Delaware, Newark, DE 19711, USA
| | - Jeffrey L Caplan
- Bio-Imaging Center, Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA.,Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716, USA
| | - Blake C Meyers
- Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716, USA.,Donald Danforth Plant Science Center, 975 North Warson Road, St Louis, MO 63132, USA.,University of Missouri-Columbia, Division of Plant Sciences, 52 Agriculture Lab, Columbia, MO 65211, USA
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Yu Y, Scheel TKH, Luna JM, Chung H, Nishiuchi E, Scull MA, Echeverría N, Ricardo-Lax I, Kapoor A, Lipkin IW, Divers TJ, Antczak DF, Tennant BC, Rice CM. miRNA independent hepacivirus variants suggest a strong evolutionary pressure to maintain miR-122 dependence. PLoS Pathog 2017; 13:e1006694. [PMID: 29084265 PMCID: PMC5679655 DOI: 10.1371/journal.ppat.1006694] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2017] [Revised: 11/09/2017] [Accepted: 10/14/2017] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV) requires the liver specific micro-RNA (miRNA), miR-122, to replicate. This was considered unique among RNA viruses until recent discoveries of HCV-related hepaciviruses prompting the question of a more general miR-122 dependence. Among hepaciviruses, the closest known HCV relative is the equine non-primate hepacivirus (NPHV). Here, we used Argonaute cross-linking immunoprecipitation (AGO-CLIP) to confirm AGO binding to the single predicted miR-122 site in the NPHV 5'UTR in vivo. To study miR-122 requirements in the absence of NPHV-permissive cell culture systems, we generated infectious NPHV/HCV chimeric viruses with the 5' end of NPHV replacing orthologous HCV sequences. These chimeras were viable even in cells lacking miR-122, although miR-122 presence enhanced virus production. No other miRNAs bound this region. By random mutagenesis, we isolated HCV variants partially dependent on miR-122 as well as robustly replicating NPHV/HCV variants completely independent of any miRNAs. These miRNA independent variants even replicate and produce infectious particles in non-hepatic cells after exogenous delivery of apolipoprotein E (ApoE). Our findings suggest that miR-122 independent HCV and NPHV variants have arisen and been sampled during evolution, yet miR-122 dependence has prevailed. We propose that hepaciviruses may use this mechanism to guarantee liver tropism and exploit the tolerogenic liver environment to avoid clearance and promote chronicity.
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Affiliation(s)
- Yingpu Yu
- Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY, United States of America
| | - Troels K. H. Scheel
- Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY, United States of America
- Copenhagen Hepatitis C Program, Department of Infectious Diseases, Hvidovre Hospital, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Joseph M. Luna
- Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY, United States of America
| | - Hachung Chung
- Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY, United States of America
| | - Eiko Nishiuchi
- Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY, United States of America
| | - Margaret A. Scull
- Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY, United States of America
| | - Natalia Echeverría
- Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY, United States of America
- Laboratorio de Virología Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
| | - Inna Ricardo-Lax
- Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY, United States of America
| | - Amit Kapoor
- Department of Pediatrics, College of Medicine, The Ohio State University, Columbus, OH, United States of America
- Center for Vaccines and Immunity, The Research Institute at Nationwide Children’s Hospital, Columbus, OH, United States of America
| | - Ian W. Lipkin
- Center for Infection and Immunity, Mailman School of Public Health and College of Physicians & Surgeons, Columbia University, New York, NY, United States of America
| | - Thomas J. Divers
- Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, United States of America
| | - Douglas F. Antczak
- Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY, United States of America
| | - Bud C. Tennant
- Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, United States of America
| | - Charles M. Rice
- Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, New York, NY, United States of America
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Liu F, Du Y, Feng WH. New perspective of host microRNAs in the control of PRRSV infection. Vet Microbiol 2017; 209:48-56. [DOI: 10.1016/j.vetmic.2017.01.004] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2016] [Revised: 12/22/2016] [Accepted: 01/03/2017] [Indexed: 02/09/2023]
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40
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Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation. Oncotarget 2017; 8:53899-53915. [PMID: 28903311 PMCID: PMC5589550 DOI: 10.18632/oncotarget.19108] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2017] [Accepted: 06/28/2017] [Indexed: 12/14/2022] Open
Abstract
Directed differentiation of human stem cells including induced pluripotent stem cells into hepatic cells potentially leads to acquired susceptibility to hepatitis C virus (HCV) infection. However, cellular determinants that change their expression during cell reprogramming or hepatic differentiation and are pivotal for supporting the HCV life cycle remain unclear. In this study, by introducing a set of reprogramming factors, we established HuH-7-derived oval-like cell lines, Hdo-17 and -23, which possess features of bipotential liver precursors. Upon induction of hepatocyte differentiation, expression of mature hepatocyte markers and hepatoblast markers in cells increased and decreased, respectively. In contrast, in response to cholangiocytic differentiation induction, gene expression of epithelium markers increased and cells formed round cysts with a central luminal space. Hdo cells lost their susceptibility to HCV infection and viral RNA replication. Hepatic differentiation of Hdo cells potentially led to recovery of permissiveness to HCV RNA replication. Gene expression profiling showed that most host-cell factors known to be involved in the HCV life cycle, except CD81, are expressed in Hdo cells comparable to HuH-7 cells. HCV pseudoparticle infectivity was significantly but partially recovered by ectopic expression of CD81, suggesting possible involvement of additional unidentified factors in HCV entry. In addition, we identified miR200a-3p, which is highly expressed in Hdo cells and stem cells but poorly expressed in differentiated cells and mature hepatocytes, as a novel negative regulator of HCV replication. In conclusion, our results showed that epigenetic reprogramming of human hepatoma cells potentially changes their permissivity to HCV.
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Fukuhara T, Tamura T, Ono C, Shiokawa M, Mori H, Uemura K, Yamamoto S, Kurihara T, Okamoto T, Suzuki R, Yoshii K, Kurosu T, Igarashi M, Aoki H, Sakoda Y, Matsuura Y. Host-derived apolipoproteins play comparable roles with viral secretory proteins Erns and NS1 in the infectious particle formation of Flaviviridae. PLoS Pathog 2017. [PMID: 28644867 PMCID: PMC5500379 DOI: 10.1371/journal.ppat.1006475] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Amphipathic α-helices of exchangeable apolipoproteins have shown to play crucial roles in the formation of infectious hepatitis C virus (HCV) particles through the interaction with viral particles. Among the Flaviviridae members, pestivirus and flavivirus possess a viral structural protein Erns or a non-structural protein 1 (NS1) as secretory glycoproteins, respectively, while Hepacivirus including HCV has no secretory glycoprotein. In case of pestivirus replication, the C-terminal long amphipathic α-helices of Erns are important for anchoring to viral membrane. Here we show that host-derived apolipoproteins play functional roles similar to those of virally encoded Erns and NS1 in the formation of infectious particles. We examined whether Erns and NS1 could compensate for the role of apolipoproteins in particle formation of HCV in apolipoprotein B (ApoB) and ApoE double-knockout Huh7 (BE-KO), and non-hepatic 293T cells. We found that exogenous expression of either Erns or NS1 rescued infectious particle formation of HCV in the BE-KO and 293T cells. In addition, expression of apolipoproteins or NS1 partially rescued the production of infectious pestivirus particles in cells upon electroporation with an Erns-deleted non-infectious RNA. As with exchangeable apolipoproteins, the C-terminal amphipathic α-helices of Erns play the functional roles in the formation of infectious HCV or pestivirus particles. These results strongly suggest that the host- and virus-derived secretory glycoproteins have overlapping roles in the viral life cycle of Flaviviridae, especially in the maturation of infectious particles, while Erns and NS1 also participate in replication complex formation and viral entry, respectively. Considering the abundant hepatic expression and liver-specific propagation of these apolipoproteins, HCV might have evolved to utilize them in the formation of infectious particles through deletion of a secretory viral glycoprotein gene. The family Flaviviridae consists of 4 genera, namely Flavivirus, Pestivirus, Pegivirus, and Hepacivirus. Flaviviruses and pestiviruses can infect various species and tissues; however, infection of pegivirus and hepacivirus is observed in a strikingly restricted range of tissue and hosts. Although all the Flaviviridae viruses possess a similar genome structure, hepatitis C virus (HCV) from Hepacivirus encodes no secretory glycoprotein, such as Erns of pestivirus and NS1 of flavivirus. The apolipoproteins, one of the host secretory glycoproteins, play important roles in the formation of infectious HCV particles through the interaction with viral particles. The data presented here show that the host-derived apolipoproteins and viral-derived Erns and NS1 have overlapping roles in the maturation of infectious particles of Flaviviridae. Considering an abundant expression of apolipoproteins in the liver and their liver-specific propagation, HCV might have evolved to utilize the apolipoproteins in the formation of infectious particles through deletion of a gene encoding a secretory viral glycoprotein. The data of this manuscript also suggest that utilization of host factors in the viral life cycle is closely associated with the tissue- and species-specificities and evolution among Flaviviridae viruses.
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Affiliation(s)
- Takasuke Fukuhara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Tomokazu Tamura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
- Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan
| | - Chikako Ono
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Mai Shiokawa
- School of Veterinary Nursing and Technology, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, Tokyo, Japan
| | - Hiroyuki Mori
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Kentaro Uemura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Satomi Yamamoto
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takeshi Kurihara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Toru Okamoto
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Ryosuke Suzuki
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Kentaro Yoshii
- Laboratory of Public Health, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan
| | - Takeshi Kurosu
- Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan
| | - Manabu Igarashi
- Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, Hokkaido, Japan
- Division of Global Epidemiology, Research Center for Zoonosis Control, Hokkaido University, Hokkaido, Japan
| | - Hiroshi Aoki
- School of Veterinary Nursing and Technology, Faculty of Veterinary Science, Nippon Veterinary and Life Science University, Tokyo, Japan
| | - Yoshihiro Sakoda
- Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Hokkaido, Japan
- Global Institution for Collaborative Research and Education (GI-CoRE), Hokkaido University, Hokkaido, Japan
| | - Yoshiharu Matsuura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
- * E-mail:
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Ono C, Fukuhara T, Motooka D, Nakamura S, Okuzaki D, Yamamoto S, Tamura T, Mori H, Sato A, Uemura K, Fauzyah Y, Kurihara T, Suda T, Nishio A, Hmwe SS, Okamoto T, Tatsumi T, Takehara T, Chayama K, Wakita T, Koike K, Matsuura Y. Characterization of miR-122-independent propagation of HCV. PLoS Pathog 2017; 13:e1006374. [PMID: 28494029 PMCID: PMC5441651 DOI: 10.1371/journal.ppat.1006374] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2017] [Revised: 05/23/2017] [Accepted: 04/24/2017] [Indexed: 12/12/2022] Open
Abstract
miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV122KO, possessing G28A substitution in the 5’UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocyte-derived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients. A liver-specific microRNA, miR-122, is one of the key determinants of hepatitis C virus (HCV) hepatotropism and is required for efficient propagation of HCV. On the other hand, chronic infection with HCV is often associated with extrahepatic manifestations (EHMs), and a low level of HCV-RNA replication has been detected in some non-hepatic cells. Nonetheless, the detailed mechanisms underlying these phenomena remain unknown. Here, we show that miR-122 is dispensable for low-level replication or infectious particle formation, and a mutant virus adapted to miR-122-knockout cells exhibited efficient but miR-122-independent propagation. The adaptive virus of HCV genotype 2a possessed a G28A substitution in the 5’UTR and facilitated efficient replication complex formation under an miR-122-deficient condition, while it propagated at a level comparable to the wild type HCV in the presence of miR-122. Moreover, various adaptive mutations including C30U were introduced into genotype 1b, which originally had an adenine at the nucleotide position 29. These observations suggest that substitutions that yield miR-122-independent propagation are not induced during propagation in hepatocytes; however, treatment with an miR-122 inhibitor or persistent infection of HCV in non-hepatic cells may induce the emergence of mutant viruses, as evidenced by clinical samples.
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Affiliation(s)
- Chikako Ono
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takasuke Fukuhara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Daisuke Motooka
- Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Shota Nakamura
- Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Daisuke Okuzaki
- DNA-Chip Developmental Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Satomi Yamamoto
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Tomokazu Tamura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Hiroyuki Mori
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Asuka Sato
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Kentaro Uemura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Yuzy Fauzyah
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takeshi Kurihara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takahiro Suda
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Osaka University, Osaka, Japan
| | - Akira Nishio
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Osaka University, Osaka, Japan
| | - Su Su Hmwe
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Toru Okamoto
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Tomohide Tatsumi
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Osaka University, Osaka, Japan
| | - Tetsuo Takehara
- Department of Gastroenterology and Hepatology, Graduate School of Medicine, Osaka University, Osaka, Japan
| | - Kazuaki Chayama
- Department of Medicine and Molecular Science, Hiroshima University School of Medicine, Hiroshima, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Kazuhiko Koike
- Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
| | - Yoshiharu Matsuura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
- * E-mail:
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Regulation of hepatitis C virus genome replication by microRNA-122. Uirusu 2017; 65:277-286. [PMID: 27760927 DOI: 10.2222/jsv.65.277] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
microRNA-122 (miR-122) is an abundant, liver-specific miRNA that regulates gene expression post-transcriptionally, typically by binding to the 3' untranslated region (UTR) of mRNAs, repressing their translation and mediating their degradation. Hepatitis C virus (HCV) is uniquely dependent on miR-122. Similar to conventional miRNA action, miR-122 recruits Argonaute-2 (AGO2) protein to the 5' UTR of the viral genome. However, in contrast to typical miRNA function, this stabilizes HCV RNA and slows its decay in infected cells. We found that HCV RNA is degraded primarily by the cytoplasmic 5' exonuclease XRN1 and that miR-122 acts to protect the viral RNA from XRN1-mediated 5' exonucleolytic decay. However, HCV replication still requires miR-122 in XRN1-depleted cells, suggesting additional functions. We also showed that miR-122 enhances HCV RNA synthesis by reducing viral genomes engaged in translation while increasing the fraction available for RNA synthesis. In this review, we summarize the recent progress on the regulatory mechanisms of HCV genome replication by miR-122.
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Fukuhara T, Yamamoto S, Ono C, Nakamura S, Motooka D, Mori H, Kurihara T, Sato A, Tamura T, Motomura T, Okamoto T, Imamura M, Ikegami T, Yoshizumi T, Soejima Y, Maehara Y, Chayama K, Matsuura Y. Quasispecies of Hepatitis C Virus Participate in Cell-Specific Infectivity. Sci Rep 2017; 7:45228. [PMID: 28327559 PMCID: PMC5361118 DOI: 10.1038/srep45228] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2016] [Accepted: 02/21/2017] [Indexed: 02/08/2023] Open
Abstract
It is well documented that a variety of viral quasispecies are found in the patients with chronic infection of hepatitis C virus (HCV). However, the significance of quasispecies in the specific infectivity to individual cell types remains unknown. In the present study, we analyzed the role of quasispecies of the genotype 2a clone, JFH1 (HCVcc), in specific infectivity to the hepatic cell lines, Huh7.5.1 and Hep3B. HCV RNA was electroporated into Huh7.5.1 cells and Hep3B/miR-122 cells expressing miR-122 at a high level. Then, we adapted the viruses to Huh7 and Hep3B/miR-122 cells by serial passages and termed the resulting viruses HCVcc/Huh7 and HCVcc/Hep3B, respectively. Interestingly, a higher viral load was obtained in the homologous combination of HCVcc/Huh7 in Huh7.5.1 cells or HCVcc/Hep3B in Hep3B/miR-122 cells compared with the heterologous combination. By using a reverse genetics system and deep sequence analysis, we identified several adaptive mutations involved in the high affinity for each cell line, suggesting that quasispecies of HCV participate in cell-specific infectivity.
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Affiliation(s)
- Takasuke Fukuhara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Satomi Yamamoto
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.,Laboratory of Veterinary Microbiology, School of Veterinary Medicine, Kitasato University, Aomori, Japan
| | - Chikako Ono
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Shota Nakamura
- Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Daisuke Motooka
- Department of Infection Metagenomics, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Hiroyuki Mori
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takeshi Kurihara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Asuka Sato
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Tomokazu Tamura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takashi Motomura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.,Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Toru Okamoto
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Michio Imamura
- Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical &Health Sciences, Hiroshima University, Hiroshima 734-8551, Japan
| | - Toru Ikegami
- Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Tomoharu Yoshizumi
- Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Yuji Soejima
- Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Yoshihiko Maehara
- Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
| | - Kazuaki Chayama
- Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical &Health Sciences, Hiroshima University, Hiroshima 734-8551, Japan
| | - Yoshiharu Matsuura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
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Tsai WL, Yeh PH, Tsai CY, Ting CT, Chiu YH, Tao MH, Li WC, Hung SC. Efficient programming of human mesenchymal stem cell-derived hepatocytes by epigenetic regulations. J Gastroenterol Hepatol 2017; 32:261-269. [PMID: 27218433 DOI: 10.1111/jgh.13451] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Accepted: 05/16/2016] [Indexed: 12/15/2022]
Abstract
BACKGROUND AND AIM In view of its unique properties of detoxification and involvement of metabolic and biochemical functions, in vitro hepatocyte culture serves as a valuable material for drug screening and mechanistic analysis for pathology of liver diseases. The restriction of rapid de-differentiation and inaccessibility of human hepatocytes from routine clinical procedure, however, limits its use. METHODS To address this issue, the effort to direct human mesenchymal stem cells (hMSCs) into hepatocytes using a modified protocol was proposed. With the additional treatment of histone deacetylase inhibitor (HDACi) and DNA methyltransferase inhibitor (DNMTi), in vitro hMSC-derived hepatocytes were cultivated and their hepatic characteristics were examined. RESULTS By using a modified protocol, it was shown that Trichostatin A and 5-aza-2-deoxycitidine protected differentiating cells from death and could sufficiently trigger a wide range of liver-specific markers as well as liver functions including albumin production, glycogen storage, and urea cycle in hMSC-derived hepatocytes. The increased mRNA expression for hepatitis C virus (HCV) entry including CD81, Occludin, LDL receptor, and scavenger receptor class B type I in hMSC-derived hepatocytes was also detected, implying its potential to be utilized as an in vitro model to analyze dynamic HCV infection. CONCLUSIONS The present study successfully established a protocol to direct hMSCs into hepatocyte-like cells suggesting the beneficial impact to apply HDACi and DNMTi as potent modulators for hMSCs to liver differentiation.
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Affiliation(s)
- Wei-Lun Tsai
- Division of Gastroenterology, Department of Internal Medicine, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.,Medical School, National Yang-Ming University, Taipei, Taiwan
| | - Po-Hung Yeh
- Stem Cell Laboratory, Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.,Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, Taipei, Taiwan
| | - Chia-Yun Tsai
- Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan.,Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei, Taiwan
| | - Chin-Tsung Ting
- Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan.,Division of Gastrointestinal Surgery, Department of Surgery, Ren-Ai Branch, Taipei City Hospital, Taipei, Taiwan
| | - Yen-Hui Chiu
- Department of Education and Research, Taipei City Hospital, Taipei, Taiwan
| | - Mi-Hua Tao
- Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
| | - Wan-Chun Li
- Institute of Oral Biology, School of Dentistry, National Yang-Ming University, Taipei, Taiwan.,Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei, Taiwan.,Department of Education and Research, Taipei City Hospital, Taipei, Taiwan
| | - Shih-Chieh Hung
- Stem Cell Laboratory, Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.,Department of Orthopaedics and Traumatology, Taipei Veterans General Hospital, Taipei, Taiwan.,Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.,Institute of Clinical Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan.,Integrative Stem Cell Center, Department of Orthopedics, China Medical University Hospital, Taichung, Taiwan.,Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan
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46
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Nishio A, Tatsumi T, Nawa T, Suda T, Yoshioka T, Onishi Y, Aono S, Shigekawa M, Hikita H, Sakamori R, Okuzaki D, Fukuhara T, Matsuura Y, Hiramatsu N, Takehara T. CD14 + monocyte-derived galectin-9 induces natural killer cell cytotoxicity in chronic hepatitis C. Hepatology 2017; 65:18-31. [PMID: 27640362 DOI: 10.1002/hep.28847] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/31/2016] [Accepted: 08/23/2016] [Indexed: 12/18/2022]
Abstract
UNLABELLED Natural killer (NK) cell activation is associated with both liver injury and persistent infection in chronic hepatitis C (CHC); however, the detailed mechanism of this activation has not yet been fully elucidated. Because galectin-9 (Gal-9) has been reported to be increased in the serum and liver tissue of CHC patients, we investigated the function of Gal-9 in NK cell activation in CHC. First, we evaluated the function of Gal-9 on NK cytotoxicity in vitro. Gal-9 treatment resulted in increased cytotoxicity of naïve NK cells, and the Gal-9-activated NK cells demonstrated cytotoxicity toward hepatoma cells and T cells. Additionally, coculturing peripheral blood mononuclear cells (PBMCs) with JFH-1/Huh7.5.1 cells increased both Gal-9 production and NK cell cytotoxicity. Next, we investigated the source of Gal-9 and the mechanism of Gal-9 production. Deletion of CD14+ monocytes from PBMCs resulted in reduced Gal-9 production in the coculture with JFH-1/Huh7.5.1 cells. Gal-9 production was driven by coculturing of PBMCs with apoptotic hepatocytes. Blocking integrin αv β3 , a receptor for phosphatidylserine expressed on apoptotic cells, also resulted in decreased Gal-9 production. Finally, we found that serum Gal-9 levels were significantly higher in CHC patients than in healthy donors and patients who achieved sustained virologic response. Among CHC patients, serum Gal-9 levels were significantly higher in patients with elevated alanine aminotransferase (ALT) than in those with normal ALT. CONCLUSION These results demonstrate that CD14+ monocyte-derived Gal-9 increases NK cell cytotoxicity in HCV infection, which might be associated with liver injury and persistent infection. (Hepatology 2017;65:18-31).
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Affiliation(s)
- Akira Nishio
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Tomohide Tatsumi
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Takatoshi Nawa
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Takahiro Suda
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Teppei Yoshioka
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Yoshiki Onishi
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Satoshi Aono
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Minoru Shigekawa
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Hayato Hikita
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Ryotaro Sakamori
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Daisuke Okuzaki
- Department of DNA-Chip Developmental Center for Infectious Diseases, Osaka University, Suita, Japan
| | - Takasuke Fukuhara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Japan
| | - Yoshiharu Matsuura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Japan
| | - Naoki Hiramatsu
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
| | - Tetsuo Takehara
- Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Japan
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Lee KY, Chen YH, Hsu SC, Yu MJ. Phosphorylation of Serine 235 of the Hepatitis C Virus Non-Structural Protein NS5A by Multiple Kinases. PLoS One 2016; 11:e0166763. [PMID: 27875595 PMCID: PMC5119781 DOI: 10.1371/journal.pone.0166763] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2016] [Accepted: 11/03/2016] [Indexed: 12/14/2022] Open
Abstract
Phosphorylation at serine 235 (S235) of the hepatitis C virus (HCV) non-structural protein 5A (NS5A) plays a critical role in the viral life cycle. For medical and virological interests, we exploited the HEK293T kidney cells to test 3 candidate protein kinases on NS5A S235 phosphorylation. Inhibitors that inhibit casein kinase I α (CKIα), polo-like kinase I (PlKI) or calmodulin-dependent kinase II (CaMKII) all reduced NS5A S235 phosphorylation. CKIα was studied previously and PlKI had severe cytotoxicity, thus CaMKII was selected for validation in the Huh7.5.1 liver cells. In the HCV (J6/JFH1)-infected Huh7.5.1 cells, CaMKII inhibitor reduced NS5A S235 phosphorylation and HCV RNA levels without apparent cytotoxicity. RT-PCR analysis showed expression of CaMKII γ and δ isoforms in the Huh7.5.1 cells. Both CaMKII γ and δ directly phosphorylated NS5A S235 in vitro. CaMKII γ or δ single knockdown did not affect NS5A S235 phosphorylation but elevated the HCV RNA levels in the infected cells. CKIα plus CaMKII (γ or δ) double knockdown reduced NS5A S235 phosphorylation and reduced HCV RNA levels; however, the HCV RNA levels were higher than those in the infected cells with CKIα single knockdown. We conclude that CKIα-mediated NS5A S235 phosphorylation is critical for HCV replication. CaMKII γ and δ may have negative roles in the HCV life cycle.
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Affiliation(s)
- Kuan-Ying Lee
- Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, 10051, Taiwan
| | - Yi-Hung Chen
- Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, 10051, Taiwan
| | - Shih-Chin Hsu
- Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, 10051, Taiwan
| | - Ming-Jiun Yu
- Institute of Biochemistry and Molecular Biology, National Taiwan University College of Medicine, Taipei, 10051, Taiwan
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Human Cathelicidin Compensates for the Role of Apolipoproteins in Hepatitis C Virus Infectious Particle Formation. J Virol 2016; 90:8464-77. [PMID: 27440892 DOI: 10.1128/jvi.00471-16] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2016] [Accepted: 07/05/2016] [Indexed: 12/12/2022] Open
Abstract
UNLABELLED Exchangeable apolipoproteins (ApoA, -C, and -E) have been shown to redundantly participate in the formation of infectious hepatitis C virus (HCV) particles during the assembly process, although their precise role in the viral life cycle is not well understood. Recently, it was shown that the exogenous expression of only short sequences containing amphipathic α-helices from various apolipoproteins is sufficient to restore the formation of infectious HCV particles in ApoB and ApoE double-gene-knockout Huh7 (BE-KO) cells. In this study, through the expression of a small library of human secretory proteins containing amphipathic α-helix structures, we identified the human cathelicidin antimicrobial peptide (CAMP), the only known member of the cathelicidin family of antimicrobial peptides (AMPs) in humans and expressed mainly in bone marrow and leukocytes. We showed that CAMP is able to rescue HCV infectious particle formation in BE-KO cells. In addition, we revealed that the LL-37 domain in CAMP containing amphipathic α-helices is crucial for the compensation of infectivity in BE-KO cells, and the expression of CAMP in nonhepatic 293T cells expressing claudin 1 and microRNA miR-122 confers complete propagation of HCV. These results suggest the possibility of extrahepatic propagation of HCV in cells with low-level or no expression of apolipoproteins but expressing secretory proteins containing amphipathic α-helices such as CAMP. IMPORTANCE Various exchangeable apolipoproteins play a pivotal role in the formation of infectious HCV during the assembly of viral particles, and amphipathic α-helix motifs in the apolipoproteins have been shown to be a key factor. To the best of our knowledge, we have identified for the first time the human cathelicidin CAMP as a cellular protein that can compensate for the role of apolipoproteins in the life cycle of HCV. We have also identified the domain in CAMP that contains amphipathic α-helices crucial for compensation and show that the expression of CAMP in nonhepatic cells expressing claudin 1 and miR-122 confers complete propagation of HCV. We speculate that low levels of HCV propagation might be possible in extrahepatic tissues expressing secretory proteins containing amphipathic α-helices without the expression of apolipoproteins.
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Abstract
A hepatitis C virus (HCV) cell culture system incorporating the JFH-1 strain and the human hepatoma cell line HuH-7 enabled the production of infectious HCV particles. Several host factors were identified as essential for HCV replication. Supplementation of these factors in nonhepatic human cell lines enabled HCV replication and particle production. Vero cells established from monkey kidney are commonly used for the production of vaccines against a variety of viruses. In this study, we aimed to establish a novel Vero cell line to reconstruct the HCV life cycle. Unmodified Vero cells did not allow HCV infection or replication. The expression of microRNA 122 (miR-122), an essential factor for HCV replication, is notably low in Vero cells. Therefore, we supplemented Vero cells with miR-122 and found that HCV replication was enhanced. However, Vero cells that expressed miR-122 still did not allow HCV infection. We supplemented HCV receptor molecules and found that scavenger receptor class B type I (SRBI) was essential for HCV infection in Vero cells. The supplementation of apolipoprotein E (ApoE), a host factor important for virus production, enabled the production of infectious virus in Vero cells. Finally, we created a Vero cell line that expressed the essential factors miR-122, SRBI, and ApoE; the entire HCV life cycle, including infection, replication, and infectious virus production, was completed in these cells. In conclusion, we demonstrated that miR-122, SRBI, and ApoE were necessary and sufficient for the completion of the entire HCV life cycle in nonhuman, nonhepatic Vero cells. HCV is a major cause of chronic liver diseases worldwide, and an effective prophylactic HCV vaccine is needed. For safety reasons, the current HCV cell culture system using HuH-7 cells, which was established from a hepatocellular carcinoma, is not suitable for the production of a vaccine against HCV. A robust HCV production system using non-cancer-derived cells is indispensable for this purpose. In this study, we wanted to establish a novel HCV cell culture system using Vero cells, which are widely used in the production of vaccines against different viruses. We identified the minimum essential host factors for the completion of the entire HCV life cycle in Vero cells to develop a novel HCV cell culture system. A cell culture system that uses Vero cells will be useful not only for HCV vaccine production but also for the further elucidation of the mechanisms of various HCV-host interactions.
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Yamamoto S, Fukuhara T, Ono C, Uemura K, Kawachi Y, Shiokawa M, Mori H, Wada M, Shima R, Okamoto T, Hiraga N, Suzuki R, Chayama K, Wakita T, Matsuura Y. Lipoprotein Receptors Redundantly Participate in Entry of Hepatitis C Virus. PLoS Pathog 2016; 12:e1005610. [PMID: 27152966 PMCID: PMC4859476 DOI: 10.1371/journal.ppat.1005610] [Citation(s) in RCA: 68] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2015] [Accepted: 04/12/2016] [Indexed: 02/07/2023] Open
Abstract
Scavenger receptor class B type 1 (SR-B1) and low-density lipoprotein receptor (LDLR) are known to be involved in entry of hepatitis C virus (HCV), but their precise roles and their interplay are not fully understood. In this study, deficiency of both SR-B1 and LDLR in Huh7 cells was shown to impair the entry of HCV more strongly than deficiency of either SR-B1 or LDLR alone. In addition, exogenous expression of not only SR-B1 and LDLR but also very low-density lipoprotein receptor (VLDLR) rescued HCV entry in the SR-B1 and LDLR double-knockout cells, suggesting that VLDLR has similar roles in HCV entry. VLDLR is a lipoprotein receptor, but the level of its hepatic expression was lower than those of SR-B1 and LDLR. Moreover, expression of mutant lipoprotein receptors incapable of binding to or uptake of lipid resulted in no or slight enhancement of HCV entry in the double-knockout cells, suggesting that binding and/or uptake activities of lipid by lipoprotein receptors are essential for HCV entry. In addition, rescue of infectivity in the double-knockout cells by the expression of the lipoprotein receptors was not observed following infection with pseudotype particles bearing HCV envelope proteins produced in non-hepatic cells, suggesting that lipoproteins associated with HCV particles participate in the entry through their interaction with lipoprotein receptors. Buoyant density gradient analysis revealed that HCV utilizes these lipoprotein receptors in a manner dependent on the lipoproteins associated with HCV particles. Collectively, these results suggest that lipoprotein receptors redundantly participate in the entry of HCV. Hepatitis C virus (HCV) utilizes several receptors to enter hepatocytes, including scavenger receptor class B type 1 (SR-B1) receptor and low-density lipoprotein receptor (LDLR). HCV particles interact with lipoprotein and apolipoproteins to form complexes termed lipoviroparticles. Several reports have shown that SR-B1 and LDLR participate in the entry of lipoviroparticles through interaction with lipoproteins. However, the precise roles of SR-B1 and LDLR in HCV entry have not been fully clarified. In this study, we showed that SR-B1 and LDLR have a redundant role in HCV entry. In addition, we showed that very low-density lipoprotein receptor (VLDLR) played a role in HCV entry similar to the roles of SR-B1 and LDLR. Interestingly, VLDLR expression was low in the liver in contrast to the abundant expressions of SR-B1 and LDLR, but high in several extrahepatic tissues. Our data suggest that lipoprotein receptors participate in the entry of HCV particles associated with various lipoproteins.
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Affiliation(s)
- Satomi Yamamoto
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Takasuke Fukuhara
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Chikako Ono
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Kentaro Uemura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Yukako Kawachi
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Mai Shiokawa
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Hiroyuki Mori
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Masami Wada
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Ryoichi Shima
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Toru Okamoto
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
| | - Nobuhiko Hiraga
- Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Ryosuke Suzuki
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Kazuaki Chayama
- Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Yoshiharu Matsuura
- Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
- * E-mail:
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