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Zhang Y, Pan C, Wang S, Zhou Y, Chen J, Yu X, Peng R, Zhang N, Yang H. Distinctive function of Tetraspanins: Implication in viral infections. Virulence 2025; 16:2474188. [PMID: 40053412 PMCID: PMC11901453 DOI: 10.1080/21505594.2025.2474188] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2024] [Revised: 02/03/2025] [Accepted: 02/25/2025] [Indexed: 03/09/2025] Open
Abstract
Harboring four transmembrane domains in their structural hallmark, Tetraspanins (Tspans) are a family of glycoproteins with pivotal functions in a variety of biological and cellular processes. Through interacting laterally with each other or specific membrane proteins, Tspans organize tetraspanin-enriched microdomains (TEMs), modulating cellular signaling, adhesion, fusion, and proliferation. An abundance of evidence has identified the multiple functions in the progression of cancer as well as the underlying molecular mechanisms. Recently, plenty of studies have focused on the utilities of Tspans by pathogens for infection, especially the infection of viruses. The expression of Tspans correlates with the phase of viral infection, the type of virus, and targeted therapies. In particular, perturbations of Tspans in host cells can affect viral attachment, intracellular trafficking, translation, virus assembly, and release. In this review, we summarize and provide a historical overview of the discovery and characterization of various kinds of virus infection and highlight their diversity and complexity, along with the virus life cycle. Furthermore, we examined the current understanding of how various Tspans are involved in the regulatory mechanisms underlying viral infection. This review aims to offer a comprehensive understanding of the targeting of Tspans for therapeutic intervention in infections caused by diverse pathogens.
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Affiliation(s)
- Yuzhi Zhang
- School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China
- Engineering Research Center of Chinese Ministry of Education for Biological Diagnosis, Treatment and Protection Technology and Equipment, Xi’an, China
- Research Center of Special Environmental Biomechanics & Medical Engineering, Northwestern Polytechnical University, Xi’an, Shaanxi, China
| | - Chengwei Pan
- Department of Mechanical Engineering, University of Victoria, Victoria, BC, Canada
| | - Sijie Wang
- School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China
- Engineering Research Center of Chinese Ministry of Education for Biological Diagnosis, Treatment and Protection Technology and Equipment, Xi’an, China
- Research Center of Special Environmental Biomechanics & Medical Engineering, Northwestern Polytechnical University, Xi’an, Shaanxi, China
| | - Yidan Zhou
- School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China
- Engineering Research Center of Chinese Ministry of Education for Biological Diagnosis, Treatment and Protection Technology and Equipment, Xi’an, China
- Research Center of Special Environmental Biomechanics & Medical Engineering, Northwestern Polytechnical University, Xi’an, Shaanxi, China
| | - Jiawei Chen
- School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China
- Engineering Research Center of Chinese Ministry of Education for Biological Diagnosis, Treatment and Protection Technology and Equipment, Xi’an, China
- Research Center of Special Environmental Biomechanics & Medical Engineering, Northwestern Polytechnical University, Xi’an, Shaanxi, China
| | - Xiaoyu Yu
- School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China
- Engineering Research Center of Chinese Ministry of Education for Biological Diagnosis, Treatment and Protection Technology and Equipment, Xi’an, China
- Research Center of Special Environmental Biomechanics & Medical Engineering, Northwestern Polytechnical University, Xi’an, Shaanxi, China
| | - Ruining Peng
- School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China
- Engineering Research Center of Chinese Ministry of Education for Biological Diagnosis, Treatment and Protection Technology and Equipment, Xi’an, China
- Research Center of Special Environmental Biomechanics & Medical Engineering, Northwestern Polytechnical University, Xi’an, Shaanxi, China
| | - Nu Zhang
- School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China
- Engineering Research Center of Chinese Ministry of Education for Biological Diagnosis, Treatment and Protection Technology and Equipment, Xi’an, China
- Research Center of Special Environmental Biomechanics & Medical Engineering, Northwestern Polytechnical University, Xi’an, Shaanxi, China
| | - Hui Yang
- School of Life Sciences, Northwestern Polytechnical University, Xi’an, Shaanxi, China
- Engineering Research Center of Chinese Ministry of Education for Biological Diagnosis, Treatment and Protection Technology and Equipment, Xi’an, China
- Research Center of Special Environmental Biomechanics & Medical Engineering, Northwestern Polytechnical University, Xi’an, Shaanxi, China
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2
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Tok D. Analysis of articles on hepatitis C by scientific mapping: 1989-2022. World J Clin Cases 2024; 12:4301-4316. [PMID: 39015889 PMCID: PMC11235547 DOI: 10.12998/wjcc.v12.i20.4301] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/22/2024] [Revised: 05/01/2024] [Accepted: 05/24/2024] [Indexed: 06/30/2024] Open
Abstract
BACKGROUND Hepatitis C virus (HCV) poses a significant quandary about public health. It is challenging to study the literature in a particular discipline comprehensively today. One solution is bibliometric analysis, which is often used to track the attributes and evolutionary trajectories of scientific outputs. AIM To examine the 35-year scientific evolution of articles focused on HCV. METHODS This study examined the 35-year scientific evolution of articles focused on HCV. Our study utilized the Web of Science database. The study encompassed a total of 11930 articles. RESULTS Regarding the cumulative count of articles, the leading countries are the United States, Japan, and Italy. Rice CM is the author with the highest recorded H-index and G-index values. The journal with the highest recorded H-index and G-index values is the Journal of Virology. The Journal of Viral Hepatitis contributed 10.94% of the articles, whereas the Journal of Virology published 9.68%. According to the strategic diagram, the keywords most frequently used in 2020-2022 are HCV, epidemiology, and sofosbuvir. CONCLUSION This study provides valuable information about 40 years of academic knowledge on HCV.
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Affiliation(s)
- Duran Tok
- Department of Infectious Diseases, Liv Hospital, Ankara 06100, Türkiye
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Reiter S, Sun T, Gärtner S, Pöhlmann S, Winkler M. Development of rhesus macaque astrocyte cell lines supporting infection with a panel of viruses. PLoS One 2024; 19:e0303059. [PMID: 38743751 PMCID: PMC11093292 DOI: 10.1371/journal.pone.0303059] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2023] [Accepted: 04/19/2024] [Indexed: 05/16/2024] Open
Abstract
Non-human primate (NHP)-based model systems are highly relevant for biomedical research. However, only few NHP cell lines are available and the generation of additional cell lines is an urgent need to help in the refinement and replacement of these models. Using lentiviral transduction of c-Fos, we established cell lines from the brain of rhesus macaques (Macaca mulatta). Transcriptome analysis revealed that these cell lines are closely related to astrocytes, which was confirmed by immunoblot and immunofluorescence microscopy detecting expression of the astrocyte marker glial fibrillary acidic protein (GFAP). Quantitative real-time PCR (qRT-PCR) demonstrated that major pathways of the interferon (IFN) system are intact. Using retroviral pseudotypes we found that the cell lines are susceptible to entry driven by the glycoproteins of vesicular stomatitis virus (VSV), lymphocytic choriomeningitis virus (LCMV) and to a lesser extent influenza A virus (IAV). Finally, these cells supported growth of Zika virus (ZIKV) and Papiine alphaherpesvirus 2 (PaHV2). In summary, we developed IFN-responsive cell lines from the rhesus macaque brain that allowed entry driven by several viral glycoproteins and were permissive to infection with ZIKV and a primate simplexvirus. These cell lines will be useful for efforts to analyze neurotropic viral infections in rhesus macaque models.
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Affiliation(s)
- Stefanie Reiter
- German Primate Center—Leibniz Institute for Primate Research, Infection Biology Unit, Göttingen, Germany
| | - Ting Sun
- Department of Neurogenetics, Max Planck Institute for Multidisciplinary Sciences (City Campus), Göttingen, Germany
| | - Sabine Gärtner
- German Primate Center—Leibniz Institute for Primate Research, Infection Biology Unit, Göttingen, Germany
| | - Stefan Pöhlmann
- German Primate Center—Leibniz Institute for Primate Research, Infection Biology Unit, Göttingen, Germany
- Faculty of Biology and Psychology, Georg-August-University Göttingen, Göttingen, Germany
| | - Michael Winkler
- German Primate Center—Leibniz Institute for Primate Research, Infection Biology Unit, Göttingen, Germany
- Faculty of Biology and Psychology, Georg-August-University Göttingen, Göttingen, Germany
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4
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Trischitta P, Tamburello MP, Venuti A, Pennisi R. Pseudovirus-Based Systems for Screening Natural Antiviral Agents: A Comprehensive Review. Int J Mol Sci 2024; 25:5188. [PMID: 38791226 PMCID: PMC11121416 DOI: 10.3390/ijms25105188] [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: 03/27/2024] [Revised: 05/03/2024] [Accepted: 05/08/2024] [Indexed: 05/26/2024] Open
Abstract
Since the outbreak of COVID-19, researchers have been working tirelessly to discover effective ways to combat coronavirus infection. The use of computational drug repurposing methods and molecular docking has been instrumental in identifying compounds that have the potential to disrupt the binding between the spike glycoprotein of SARS-CoV-2 and human ACE2 (hACE2). Moreover, the pseudovirus approach has emerged as a robust technique for investigating the mechanism of virus attachment to cellular receptors and for screening targeted small molecule drugs. Pseudoviruses are viral particles containing envelope proteins, which mediate the virus's entry with the same efficiency as that of live viruses but lacking pathogenic genes. Therefore, they represent a safe alternative to screen potential drugs inhibiting viral entry, especially for highly pathogenic enveloped viruses. In this review, we have compiled a list of antiviral plant extracts and natural products that have been extensively studied against enveloped emerging and re-emerging viruses by pseudovirus technology. The review is organized into three parts: (1) construction of pseudoviruses based on different packaging systems and applications; (2) knowledge of emerging and re-emerging viruses; (3) natural products active against pseudovirus-mediated entry. One of the most crucial stages in the life cycle of a virus is its penetration into host cells. Therefore, the discovery of viral entry inhibitors represents a promising therapeutic option in fighting against emerging viruses.
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Affiliation(s)
- Paola Trischitta
- Department of Chemical, Biological, Pharmaceutical and Environmental Science, University of Messina, Viale Ferdinando Stagno d’Alcontres 31, 98166 Messina, Italy; (P.T.); (M.P.T.)
- Department of Chemistry, Biology, and Biotechnology, University of Perugia, Via Elce di Sotto 8, 06123 Perugia, Italy
| | - Maria Pia Tamburello
- Department of Chemical, Biological, Pharmaceutical and Environmental Science, University of Messina, Viale Ferdinando Stagno d’Alcontres 31, 98166 Messina, Italy; (P.T.); (M.P.T.)
| | - Assunta Venuti
- International Agency for Research on Cancer (IARC), World Health Organization, 69366 Lyon, CEDEX 07, France;
| | - Rosamaria Pennisi
- Department of Chemical, Biological, Pharmaceutical and Environmental Science, University of Messina, Viale Ferdinando Stagno d’Alcontres 31, 98166 Messina, Italy; (P.T.); (M.P.T.)
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Jäger N, Ayyub SA, Peske F, Liedtke D, Bohne J, Hoffmann M, Rodnina MV, Pöhlmann S. The Inhibition of Gag-Pol Expression by the Restriction Factor Shiftless Is Dispensable for the Restriction of HIV-1 Infection. Viruses 2024; 16:583. [PMID: 38675925 PMCID: PMC11055011 DOI: 10.3390/v16040583] [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: 03/01/2024] [Revised: 04/03/2024] [Accepted: 04/04/2024] [Indexed: 04/28/2024] Open
Abstract
The interferon-induced host cell protein Shiftless (SFL) inhibits -1 programmed ribosomal frameshifting (-1PRF) required for the expression of HIV-1 Gal-Pol and the formation of infectious HIV-1 particles. However, the specific regions in SFL required for antiviral activity and the mechanism by which SFL inhibits -1PRF remain unclear. Employing alanine scanning mutagenesis, we found that basic amino acids in the predicted zinc ribbon motif of SFL are essential for the suppression of Gag-Pol expression but dispensable for anti-HIV-1 activity. We have shown that SFL inhibits the expression of the murine leukemia virus (MLV) Gag-Pol polyprotein and the formation of infectious MLV particles, although Gag-Pol expression of MLV is independent of -1PRF but requires readthrough of a stop codon. These findings indicate that SFL might inhibit HIV-1 infection by more than one mechanism and that SFL might target programmed translational readthrough as well as -1PRF signals, both of which are regulated by mRNA secondary structure elements.
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Affiliation(s)
- Niklas Jäger
- Infection Biology Unit, German Primate Center–Leibniz Institute for Primate Research, 37077 Göttingen, Germany;
- Faculty of Biology and Psychology, University Göttingen, 37073 Göttingen, Germany
| | - Shreya Ahana Ayyub
- Max Planck Institute for Multidisciplinary Sciences, 37077 Göttingen, Germany; (S.A.A.); (F.P.); (D.L.); (M.V.R.)
| | - Frank Peske
- Max Planck Institute for Multidisciplinary Sciences, 37077 Göttingen, Germany; (S.A.A.); (F.P.); (D.L.); (M.V.R.)
| | - David Liedtke
- Max Planck Institute for Multidisciplinary Sciences, 37077 Göttingen, Germany; (S.A.A.); (F.P.); (D.L.); (M.V.R.)
| | - Jens Bohne
- Institute of Virology, Hannover Medical School, 30625 Hannover, Germany;
| | - Markus Hoffmann
- Infection Biology Unit, German Primate Center–Leibniz Institute for Primate Research, 37077 Göttingen, Germany;
- Faculty of Biology and Psychology, University Göttingen, 37073 Göttingen, Germany
| | - Marina V. Rodnina
- Max Planck Institute for Multidisciplinary Sciences, 37077 Göttingen, Germany; (S.A.A.); (F.P.); (D.L.); (M.V.R.)
| | - Stefan Pöhlmann
- Infection Biology Unit, German Primate Center–Leibniz Institute for Primate Research, 37077 Göttingen, Germany;
- Faculty of Biology and Psychology, University Göttingen, 37073 Göttingen, Germany
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Petrovskis I, Skrastina D, Jansons J, Dislers A, Bogans J, Spunde K, Neprjakhina A, Zakova J, Zajakina A, Sominskaya I. Toward a SARS-CoV-2 VLP Vaccine: HBc/G as a Carrier for SARS-CoV-2 Spike RBM and Nucleocapsid Protein-Derived Peptides. Vaccines (Basel) 2024; 12:267. [PMID: 38543900 PMCID: PMC10974900 DOI: 10.3390/vaccines12030267] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2023] [Revised: 02/22/2024] [Accepted: 02/29/2024] [Indexed: 11/12/2024] Open
Abstract
Virus-like particles (VLPs) offer an attractive possibility for the development of vaccines. Recombinant core antigen (HBc) of Hepatitis B virus (HBV) was expressed in different systems, and the E. coli expression system was shown to be effective for the production of HBc VLPs. Here, we used HBc of the HBV genotype G (HBc/G) as a technologically promising VLP carrier for the presentation of spike RBM and nucleocapsid protein-derived peptides of the SARS-CoV-2 Delta variant for subsequent immunological evaluations of obtained fusion proteins. The major immunodominant region (MIR) of the HBc/G protein was modified through the insertion of a receptor binding motif (RBM) from the S protein or B-cell epitope-containing peptide from the N protein. The C-terminus of the two truncated HBc/G proteins was used for the insertion of a group of five cytotoxic T lymphocyte (CTL) epitopes from the N protein. After expression in E. coli, the MIR-derived proteins were found to be insoluble and were recovered through step-wise solubilization with urea, followed by refolding. Despite the lack of correct VLPs, the chimeric proteins induced high levels of antibodies in BALB/c mice. These antibodies specifically recognized either eukaryotically expressed hRBD or bacterially expressed N protein (2-220) of SARS-CoV-2. CTL-epitope-containing proteins were purified as VLPs. The production of cytokines was analyzed through flow cytometry after stimulation of T-cells with target CTL peptides. Only a protein with a deleted polyarginine (PA) domain was able to induce the specific activation of T-cells. At the same time, the T-cell response against the carrier HBc/G protein was detected for both proteins. The neutralization of SARS-CoV-2 pseudotyped murine retrovirus with anti-HBc/G-RBM sera was found to be low.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | - Irina Sominskaya
- Latvian Biomedical Research and Study Centre, Ratsupites 1, LV-1067 Riga, Latvia; (I.P.); (D.S.); (J.J.); (A.D.); (J.B.); (K.S.); (A.N.); (J.Z.); (A.Z.)
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7
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Chumbe A, Grobben M, Capella-Pujol J, Koekkoek SM, Zon I, Slamanig S, Merat SJ, Beaumont T, Sliepen K, Schinkel J, van Gils MJ. A panel of hepatitis C virus glycoproteins for the characterization of antibody responses using antibodies with diverse recognition and neutralization patterns. Virus Res 2024; 341:199308. [PMID: 38171391 PMCID: PMC10821612 DOI: 10.1016/j.virusres.2024.199308] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2023] [Revised: 12/28/2023] [Accepted: 01/01/2024] [Indexed: 01/05/2024]
Abstract
A vaccine against Hepatitis C virus (HCV) is urgently needed to limit the spread of HCV. The large antigenic diversity of the HCV glycoprotein E1E2 makes it difficult to design a vaccine but also to fully understand the antibody response after infection or vaccination. Here we designed a panel of HCV pseudoparticles (HCVpps) that cover a wide range of genetically and antigenically diverse E1E2s. We validate our panel using neutralization and a binding antibody multiplex assay (BAMA). The panel of HCVpps includes E1E2 glycoproteins from acute and chronically infected cases in the Netherlands, as well as E1E2 glycoproteins from previously reported HCVs. Using eight monoclonal antibodies targeting multiple antigenic regions on E1E2, we could categorize four groups of neutralization sensitive viruses with viruses showing neutralization titers over a 100-fold range. One HCVpp (AMS0230) was extremely neutralization resistant and only neutralized by AR4-targeting antibodies. In addition, using binding antibody multiplex competition assay, we delineated mAb epitopes and their interactions. The binding and neutralization sensitivity of the HCVpps were confirmed using patient sera. At the end, eleven HCVpps with unique antibody binding and neutralization profiles were selected as the final panel for standardized HCV antibody assessments. In conclusion, this HCVpp panel can be used to evaluate antibody binding and neutralization breadth and potency as well as delineate the epitopes targeted in sera from patients or candidate vaccine trials. The HCVpp panel in combination with the established antibody competition assay present highly valuable tools for HCV vaccine development and evaluation.
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Affiliation(s)
- Ana Chumbe
- Amsterdam UMC, University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Amsterdam, the Netherlands; Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, the Netherlands
| | - Marloes Grobben
- Amsterdam UMC, University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Amsterdam, the Netherlands; Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, the Netherlands
| | - Joan Capella-Pujol
- Amsterdam UMC, University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Amsterdam, the Netherlands; Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, the Netherlands
| | - Sylvie M Koekkoek
- Amsterdam UMC, University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Amsterdam, the Netherlands; Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, the Netherlands
| | - Ian Zon
- Amsterdam UMC, University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Amsterdam, the Netherlands; Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, the Netherlands
| | - Stefan Slamanig
- Amsterdam UMC, University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Amsterdam, the Netherlands; Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, the Netherlands
| | | | - Tim Beaumont
- Amsterdam UMC, University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Amsterdam, the Netherlands; Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, the Netherlands; AIMM Therapeutics, Amsterdam, the Netherlands
| | - Kwinten Sliepen
- Amsterdam UMC, University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Amsterdam, the Netherlands; Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, the Netherlands
| | - Janke Schinkel
- Amsterdam UMC, University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Amsterdam, the Netherlands; Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, the Netherlands.
| | - Marit J van Gils
- Amsterdam UMC, University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Amsterdam, the Netherlands; Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, the Netherlands.
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8
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Bosquillon de Jarcy L, Akbil B, Mhlekude B, Leyens J, Postmus D, Harnisch G, Jansen J, Schmidt ML, Aigner A, Pott F, Chua RL, Krist L, Gentile R, Mühlemann B, Jones TC, Niemeyer D, Fricke J, Keil T, Pischon T, Janke J, Conrad C, Iacobelli S, Drosten C, Corman VM, Ralser M, Eils R, Kurth F, Sander L, Goffinet C. 90K/LGALS3BP expression is upregulated in COVID-19 but may not restrict SARS-CoV-2 infection. Clin Exp Med 2023; 23:3689-3700. [PMID: 37162650 PMCID: PMC10170455 DOI: 10.1007/s10238-023-01077-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2023] [Accepted: 04/12/2023] [Indexed: 05/11/2023]
Abstract
Glycoprotein 90K, encoded by the interferon-stimulated gene LGALS3BP, displays broad antiviral activity. It reduces HIV-1 infectivity by interfering with Env maturation and virion incorporation, and increases survival of Influenza A virus-infected mice via antiviral innate immune signaling. Its antiviral potential in SARS-CoV-2 infection remains largely unknown. Here, we analyzed the expression of 90K/LGALS3BP in 44 hospitalized COVID-19 patients at multiple levels. We quantified 90K protein concentrations in serum and PBMCs as well as LGALS3BP mRNA levels. Complementary, we analyzed two single cell RNA-sequencing datasets for expression of LGALS3BP in respiratory specimens and PBMCs from COVID-19 patients. Finally, we analyzed the potential of 90K to interfere with SARS-CoV-2 infection of HEK293T/ACE2, Calu-3 and Caco-2 cells using authentic virus. 90K protein serum concentrations were significantly elevated in COVID-19 patients compared to uninfected sex- and age-matched controls. Furthermore, PBMC-associated concentrations of 90K protein were overall reduced by SARS-CoV-2 infection in vivo, suggesting enhanced secretion into the extracellular space. Mining of published PBMC scRNA-seq datasets uncovered monocyte-specific induction of LGALS3BP mRNA expression in COVID-19 patients. In functional assays, neither 90K overexpression in susceptible cell lines nor exogenous addition of purified 90K consistently inhibited SARS-CoV-2 infection. Our data suggests that 90K/LGALS3BP contributes to the global type I IFN response during SARS-CoV-2 infection in vivo without displaying detectable antiviral properties in vitro.
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Affiliation(s)
- Laure Bosquillon de Jarcy
- Institute of Virology, Campus Charité Mitte, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
- Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 , Berlin, Germany
- Speciality Network: Infectious Diseases and Respiratory Medicine, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
| | - Bengisu Akbil
- Institute of Virology, Campus Charité Mitte, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
- Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 , Berlin, Germany
| | - Baxolele Mhlekude
- Institute of Virology, Campus Charité Mitte, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
- Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 , Berlin, Germany
| | - Johanna Leyens
- Institute of Virology, Campus Charité Mitte, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
| | - Dylan Postmus
- Institute of Virology, Campus Charité Mitte, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
- Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 , Berlin, Germany
| | - Greta Harnisch
- Institute of Virology, Campus Charité Mitte, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
| | - Jenny Jansen
- Institute of Virology, Campus Charité Mitte, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
| | - Marie L Schmidt
- Institute of Virology, Campus Charité Mitte, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
| | - Annette Aigner
- Institute of Biometry and Clinical Epidemiology, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
| | - Fabian Pott
- Institute of Virology, Campus Charité Mitte, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
- Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 , Berlin, Germany
| | - Robert Lorenz Chua
- Center for Digital Health, Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Berlin, Germany, Charitéplatz 1, 10117, Berlin, Germany
| | - Lilian Krist
- Institute of Social Medicine, Epidemiology and Health Economics, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
| | | | - Barbara Mühlemann
- Institute of Virology, Campus Charité Mitte, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
| | - Terence C Jones
- Institute of Virology, Campus Charité Mitte, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
- Department of Zoology, Centre for Pathogen Evolution, University of Cambridge, Downing St., Cambridge, CB2 3EJ, UK
| | - Daniela Niemeyer
- Institute of Virology, Campus Charité Mitte, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
- German Center for Infection Research, Associated Partner Charité, Berlin, Germany
| | - Julia Fricke
- Institute of Social Medicine, Epidemiology and Health Economics, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
| | - Thomas Keil
- Institute of Social Medicine, Epidemiology and Health Economics, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
- Institute of Clinical Epidemiology and Biometry, University of Würzburg, Josef-Schneiderstr. 2, 97080, Würzburg, Germany
- State Institute of Health, Bavarian Health and Food Safety Authority, Eggenreuther Weg 43, 91058, Erlangen, Germany
| | - Tobias Pischon
- Molecular Epidemiology Research Group, Max-Delbrueck-Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany
- Biobank Technology Platform, Max-Delbrueck-Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany
- Core Facility Biobank, Berlin Institute of Health at Charité - Universitätsmedizin Berlin, 10178, Berlin, Germany
- Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität Zu Berlin, 10117, Berlin, Germany
| | - Jürgen Janke
- Biobank Technology Platform, Max-Delbrueck-Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany
| | - Christian Conrad
- Center for Digital Health, Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Berlin, Germany, Charitéplatz 1, 10117, Berlin, Germany
| | | | - Christian Drosten
- Institute of Virology, Campus Charité Mitte, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
- German Center for Infection Research, Associated Partner Charité, Berlin, Germany
| | - Victor M Corman
- Institute of Virology, Campus Charité Mitte, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
- German Center for Infection Research, Associated Partner Charité, Berlin, Germany
| | - Markus Ralser
- Department of Biochemistry, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
- Molecular Biology of Metabolism Laboratory, The Francis Crick Institute, London, NW11AT, UK
| | - Roland Eils
- Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 , Berlin, Germany
- Center for Digital Health, Berlin Institute of Health (BIH) at Charité - Universitätsmedizin Berlin, Berlin, Germany, Charitéplatz 1, 10117, Berlin, Germany
- German Center for Lung Research (DZL), 35392, Gießen, Germany
- Health Data Science Unit, Heidelberg University Hospital and BioQuant, 69120, Heidelberg, Germany
| | - Florian Kurth
- Speciality Network: Infectious Diseases and Respiratory Medicine, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
- Department of Tropical Medicine, Bernhard Nocht Institute for Tropical Medicine, 20359, Hamburg, Germany
- Department of Medicine, University Medical Center, Hamburg-Eppendorf, 20251, Hamburg, Germany
| | - Leif Sander
- Speciality Network: Infectious Diseases and Respiratory Medicine, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany
- German Center for Lung Research (DZL), 35392, Gießen, Germany
| | - Christine Goffinet
- Institute of Virology, Campus Charité Mitte, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117, Berlin, Germany.
- Berlin Institute of Health at Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 , Berlin, Germany.
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9
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Granier C, Toesca J, Mialon C, Ritter M, Freitas N, Boson B, Pécheur EI, Cosset FL, Denolly S. Low-density hepatitis C virus infectious particles are protected from oxidation by secreted cellular proteins. mBio 2023; 14:e0154923. [PMID: 37671888 PMCID: PMC10653866 DOI: 10.1128/mbio.01549-23] [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: 06/17/2023] [Accepted: 07/04/2023] [Indexed: 09/07/2023] Open
Abstract
IMPORTANCE Assessments of viral stability on surfaces or in body fluids under different environmental conditions and/or temperatures are often performed, as they are key to understanding the routes and parameters of viral transmission and to providing clues on the epidemiology of infections. However, for most viruses, the mechanisms of inactivation vs stability of viral particles remain poorly defined. Although they are structurally diverse, with different compositions, sizes, and shapes, enveloped viruses are generally less stable than non-enveloped viruses, pointing out the role of envelopes themselves in virus lability. In this report, we investigated the properties of hepatitis C virus (HCV) particles with regards to their stability. We found that, compared to alternative enveloped viruses such as Dengue virus (DENV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), hepatitis delta virus (HDV), and Crimean-Congo hemorrhagic fever virus (CCHFV) that infect the liver, HCV particles are intrinsically labile. We determined the mechanisms that drastically alter their specific infectivity through oxidation of their lipids, and we highlighted that they are protected from lipid oxidation by secreted cellular proteins, which can protect their membrane fusion capacity and overall infectivity.
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Affiliation(s)
- Christelle Granier
- CIRI – Centre International de Recherche en Infectiologie, Univ. Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308 ENS de Lyon, Lyon, France
| | - Johan Toesca
- CIRI – Centre International de Recherche en Infectiologie, Univ. Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308 ENS de Lyon, Lyon, France
| | - Chloé Mialon
- CIRI – Centre International de Recherche en Infectiologie, Univ. Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308 ENS de Lyon, Lyon, France
| | - Maureen Ritter
- CIRI – Centre International de Recherche en Infectiologie, Univ. Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308 ENS de Lyon, Lyon, France
| | - Natalia Freitas
- CIRI – Centre International de Recherche en Infectiologie, Univ. Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308 ENS de Lyon, Lyon, France
| | - Bertrand Boson
- CIRI – Centre International de Recherche en Infectiologie, Univ. Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308 ENS de Lyon, Lyon, France
| | - Eve-Isabelle Pécheur
- Centre Léon Bérard, Centre de Recherche en Cancérologie de Lyon, CNRS 5286, Inserm U1052, Université Claude Bernard Lyon 1, Lyon, France
| | - François-Loïc Cosset
- CIRI – Centre International de Recherche en Infectiologie, Univ. Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308 ENS de Lyon, Lyon, France
| | - Solène Denolly
- CIRI – Centre International de Recherche en Infectiologie, Univ. Lyon, Université Claude Bernard Lyon 1, Inserm, U1111, CNRS, UMR5308 ENS de Lyon, Lyon, France
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
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10
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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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11
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Capella-Pujol J, de Gast M, Radić L, Zon I, Chumbe A, Koekkoek S, Olijhoek W, Schinkel J, van Gils MJ, Sanders RW, Sliepen K. Signatures of V H1-69-derived hepatitis C virus neutralizing antibody precursors defined by binding to envelope glycoproteins. Nat Commun 2023; 14:4036. [PMID: 37419906 PMCID: PMC10328973 DOI: 10.1038/s41467-023-39690-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2022] [Accepted: 06/23/2023] [Indexed: 07/09/2023] Open
Abstract
An effective preventive vaccine for hepatitis C virus (HCV) remains a major unmet need. Antigenic region 3 (AR3) on the E1E2 envelope glycoprotein complex overlaps with the CD81 receptor binding site and represents an important epitope for broadly neutralizing antibodies (bNAbs) and is therefore important for HCV vaccine design. Most AR3 bNAbs utilize the VH1-69 gene and share structural features that define the AR3C-class of HCV bNAbs. In this work, we identify recombinant HCV glycoproteins based on a permuted E2E1 trimer design that bind to the inferred VH1-69 germline precursors of AR3C-class bNAbs. When presented on nanoparticles, these recombinant E2E1 glycoproteins efficiently activate B cells expressing inferred germline AR3C-class bNAb precursors as B cell receptors. Furthermore, we identify critical signatures in three AR3C-class bNAbs that represent two subclasses of AR3C-class bNAbs that will allow refined protein design. These results provide a framework for germline-targeting vaccine design strategies against HCV.
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Affiliation(s)
- Joan Capella-Pujol
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105, AZ, Amsterdam, Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105, AZ, Amsterdam, Netherlands
| | - Marlon de Gast
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105, AZ, Amsterdam, Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105, AZ, Amsterdam, Netherlands
| | - Laura Radić
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105, AZ, Amsterdam, Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105, AZ, Amsterdam, Netherlands
| | - Ian Zon
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105, AZ, Amsterdam, Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105, AZ, Amsterdam, Netherlands
| | - Ana Chumbe
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105, AZ, Amsterdam, Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105, AZ, Amsterdam, Netherlands
| | - Sylvie Koekkoek
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105, AZ, Amsterdam, Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105, AZ, Amsterdam, Netherlands
| | - Wouter Olijhoek
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105, AZ, Amsterdam, Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105, AZ, Amsterdam, Netherlands
| | - Janke Schinkel
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105, AZ, Amsterdam, Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105, AZ, Amsterdam, Netherlands
| | - Marit J van Gils
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105, AZ, Amsterdam, Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105, AZ, Amsterdam, Netherlands
| | - Rogier W Sanders
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105, AZ, Amsterdam, Netherlands.
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105, AZ, Amsterdam, Netherlands.
- Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY, 10065, USA.
| | - Kwinten Sliepen
- Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam UMC, University of Amsterdam, 1105, AZ, Amsterdam, Netherlands.
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, 1105, AZ, Amsterdam, Netherlands.
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12
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Tsutsumi Y, Ito S, Shiratori S, Teshima T. Hepatitis C Virus (HCV)-Ribonucleic Acid (RNA) As a Biomarker for Lymphoid Malignancy with HCV Infection. Cancers (Basel) 2023; 15:2852. [PMID: 37345190 DOI: 10.3390/cancers15102852] [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: 04/25/2023] [Revised: 05/15/2023] [Accepted: 05/15/2023] [Indexed: 06/23/2023] Open
Abstract
The hepatitis C virus (HCV) is potentially associated with liver cancer, and advances in various drugs have led to progress in the treatment of hepatitis C and attempts to prevent its transition to liver cancer. Furthermore, reactivation of HCV has been observed in the treatment of lymphoma, during which the immortalization and proliferation of lymphocytes occur, which leads to the possibility of further stimulating cytokines and the like and possibly to the development of lymphoid malignancy. There are also cases in which the disappearance of lymphoid malignancy has been observed by treating HCV and suppressing HCV-Ribonucleic acid (RNA), as well as cases of recurrence with an increase in HCV-RNA. While HCV-associated lymphoma has a poor prognosis, improving the prognosis with Direct Acting Antivirals (DAA) has recently been reported. The reduction and eradication of HCV-RNA by means of DAA is thus important for the treatment of lymphoid malignancy associated with HCV infection, and HCV-RNA can presumably play a role as a biomarker. This review provides an overview of what is currently known about HCV-associated lymphoma, its epidemiology, the mechanisms underlying the progression to lymphoma, its treatment, the potential and limits of HCV-RNA as a therapeutic biomarker, and biomarkers that are expected now that DAA therapy has been developed.
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Affiliation(s)
- Yutaka Tsutsumi
- Department of Hematology, Hakodate Municipal Hospital, Hakodate, 1-10-1, Minato-cho, Hakodate 041-8680, Japan
| | - Shinichi Ito
- Department of Hematology, Hakodate Municipal Hospital, Hakodate, 1-10-1, Minato-cho, Hakodate 041-8680, Japan
| | - Souichi Shiratori
- Department of Hematology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan
| | - Takanori Teshima
- Department of Hematology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan
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13
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Wolfisberg R, Holmbeck K, Billerbeck E, Thorselius CE, Batista MN, Fahnøe U, Lundsgaard EA, Kennedy MJ, Nielsen L, Rice CM, Bukh J, Scheel TKH. Molecular Determinants of Mouse Adaptation of Rat Hepacivirus. J Virol 2023; 97:e0181222. [PMID: 36971565 PMCID: PMC10134885 DOI: 10.1128/jvi.01812-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2022] [Accepted: 02/28/2023] [Indexed: 03/29/2023] Open
Abstract
The lack of robust immunocompetent animal models for hepatitis C virus (HCV) impedes vaccine development and studies of immune responses. Norway rat hepacivirus (NrHV) infection in rats shares HCV-defining characteristics, including hepatotropism, chronicity, immune responses, and aspects of liver pathology. To exploit genetic variants and research tools, we previously adapted NrHV to prolonged infection in laboratory mice. Through intrahepatic RNA inoculation of molecular clones of the identified variants, we here characterized four mutations in the envelope proteins responsible for mouse adaptation, including one disrupting a glycosylation site. These mutations led to high-titer viremia, similar to that observed in rats. In 4-week-old mice, infection was cleared after around 5 weeks compared to 2 to 3 weeks for nonadapted virus. In contrast, the mutations led to persistent but attenuated infection in rats, and they partially reverted, accompanied by an increase in viremia. Attenuated infection in rat but not mouse hepatoma cells demonstrated that the characterized mutations were indeed mouse adaptive rather than generally adaptive across species and that species determinants and not immune interactions were responsible for attenuation in rats. Unlike persistent NrHV infection in rats, acute resolving infection in mice was not associated with the development of neutralizing antibodies. Finally, infection of scavenger receptor B-I (SR-BI) knockout mice suggested that adaptation to mouse SR-BI was not a primary function of the identified mutations. Rather, the virus may have adapted to lower dependency on SR-BI, thereby potentially surpassing species-specific differences. In conclusion, we identified specific determinants of NrHV mouse adaptation, suggesting species-specific interactions during entry. IMPORTANCE A prophylactic vaccine is required to achieve the World Health Organization's objective for hepatitis C virus elimination as a serious public health threat. However, the lack of robust immunocompetent animal models supporting hepatitis C virus infection impedes vaccine development as well as studies of immune responses and viral evasion. Hepatitis C virus-related hepaciviruses were discovered in a number of animal species and provide useful surrogate infection models. Norway rat hepacivirus is of particular interest, as it enables studies in rats, an immunocompetent and widely used small laboratory animal model. Its adaptation to robust infection also in laboratory mice provides access to a broader set of mouse genetic lines and comprehensive research tools. The presented mouse-adapted infectious clones will be of utility for reverse genetic studies, and the Norway rat hepacivirus mouse model will facilitate studies of hepacivirus infection for in-depth characterization of virus-host interactions, immune responses, and liver pathology.
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Affiliation(s)
- Raphael Wolfisberg
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- CO-HEP, Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
| | - Kenn Holmbeck
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- CO-HEP, Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
| | - Eva Billerbeck
- Department of Medicine, Division of Hepatology, Albert Einstein College of Medicine, New York, New York, USA
- Department of Microbiology and Immunology, Albert Einstein College of Medicine, New York, New York, USA
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, USA
| | - Caroline E. Thorselius
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- CO-HEP, Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
| | - Mariana N. Batista
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, USA
| | - Ulrik Fahnøe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- CO-HEP, Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
| | - Emma A. Lundsgaard
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- CO-HEP, Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
| | - Matthew J. Kennedy
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- CO-HEP, Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
| | - Louise Nielsen
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- CO-HEP, Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
| | - Charles M. Rice
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, USA
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- CO-HEP, Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
| | - Troels K. H. Scheel
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- CO-HEP, Department of Infectious Diseases, Copenhagen University Hospital, Hvidovre, Denmark
- Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, USA
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14
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Reiter S, Gärtner S, Decker K, Pöhlmann S, Winkler M. Development of immortalized rhesus macaque kidney cells supporting infection with a panel of viruses. PLoS One 2023; 18:e0284048. [PMID: 37146034 PMCID: PMC10162512 DOI: 10.1371/journal.pone.0284048] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2022] [Accepted: 03/22/2023] [Indexed: 05/07/2023] Open
Abstract
Non-human primate (NHP)-based model systems faithfully reproduce various viral diseases including Ebola, influenza, AIDS and Zika. However, only a small number of NHP cell lines are available and generation of additional cell lines could help to refine these models. We immortalized rhesus macaque kidney cells by lentiviral transduction with a vector encoding telomerase reverse transcriptase (TERT) and report the generation of three TERT-immortalized cell lines derived from rhesus macaque kidney. Expression of the kidney podocyte marker podoplanin on these cells was demonstrated by flow cytometry. Quantitative real-time PCR (qRT-PCR) was employed to demonstrate induction of MX1 expression upon stimulation with interferon (IFN) or viral infection, suggesting a functional IFN system. Further, the cell lines were susceptible to entry driven by the glycoproteins of vesicular stomatitis virus, influenza A virus, Ebola virus, Nipah virus and Lassa virus as assessed by infection with retroviral pseudotypes. Finally, these cells supported growth of Zika virus and the primate simplexviruses Cercopithecine alphaherpesvirus 2 and Papiine alphaherpesvirus 2. In summary, we developed IFN-responsive rhesus macaque kidney cell lines that allowed entry driven by diverse viral glycoproteins and were permissive to infection with Zika virus and primate simplexviruses. These cell lines will be useful for efforts to analyze viral infections of the kidney in macaque models.
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Affiliation(s)
- Stefanie Reiter
- Infection Biology Unit, German Primate Center, Leibniz Institute for Primate Research, Göttingen, Germany
| | - Sabine Gärtner
- Infection Biology Unit, German Primate Center, Leibniz Institute for Primate Research, Göttingen, Germany
| | - Katharina Decker
- Infection Biology Unit, German Primate Center, Leibniz Institute for Primate Research, Göttingen, Germany
| | - Stefan Pöhlmann
- Infection Biology Unit, German Primate Center, Leibniz Institute for Primate Research, Göttingen, Germany
- Faculty of Biology and Psychology, Georg-August-University Göttingen, Göttingen, Germany
| | - Michael Winkler
- Infection Biology Unit, German Primate Center, Leibniz Institute for Primate Research, Göttingen, Germany
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15
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Zhang L, Wang X, Ming A, Tan W. Pseudotyped Virus for Flaviviridae. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2023; 1407:313-327. [PMID: 36920705 DOI: 10.1007/978-981-99-0113-5_17] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/16/2023]
Abstract
Members of Flaviviridae are enveloped single positive-stranded RNA viruses including hepacivirus, pestivirus, pegivirus, and mosquito-transmitted flavivirus, which are important pathogens of infectious diseases and pose serious threats to human health. Pseudotyped virus is an artificially constructed virus-like particle, which could infect host cells similar to a live virus but cannot produce infectious progeny virus. Therefore, pseudotyped virus has the advantages of a wide host range, high transfection efficiency, low biosafety risk, and accurate and objective quantification. It has been widely used in biological characteristics, drug screening, detection methods, and vaccine evaluation of Flaviviridae viruses like hepatitis C virus, Japanese encephalitis virus, dengue virus, and Zika virus.
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Affiliation(s)
- Leiliang Zhang
- Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
- Department of Pathogen Biology, School of Clinical and Basic Medical Sciences, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong, China
| | - Xiao Wang
- Department of Pathogen Biology, School of Clinical and Basic Medical Sciences, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong, China
| | - Annan Ming
- Department of Pathogen Biology, School of Clinical and Basic Medical Sciences, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong, China
| | - Wenjie Tan
- NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
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16
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Angulo J, Cáceres CJ, Contreras N, Fernández-García L, Chamond N, Ameur M, Sargueil B, López-Lastra M. Polypyrimidine-Tract-Binding Protein Isoforms Differentially Regulate the Hepatitis C Virus Internal Ribosome Entry Site. Viruses 2022; 15:8. [PMID: 36680049 PMCID: PMC9864772 DOI: 10.3390/v15010008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Revised: 12/03/2022] [Accepted: 12/16/2022] [Indexed: 12/24/2022] Open
Abstract
Translation initiation of the hepatitis C virus (HCV) mRNA depends on an internal ribosome entry site (IRES) that encompasses most of the 5'UTR and includes nucleotides of the core coding region. This study shows that the polypyrimidine-tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the HCV 5'UTR, stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Our results show that PTB1 and PTB4, but not PTB2, stimulate HCV IRES activity in HuH-7 and HEK293T cells. In HuH-7 cells, PTB1 promotes HCV IRES-mediated initiation more strongly than PTB4. Mutations in PTB1, PTB4, RRM1/RRM2, or RRM3/RRM4, which disrupt the RRM's ability to bind RNA, abrogated the protein's capacity to stimulate HCV IRES activity in HuH-7 cells. In HEK293T cells, PTB1 and PTB4 stimulate HCV IRES activity to similar levels. In HEK293T cells, mutations in RRM1/RRM2 did not impact PTB1's ability to promote HCV IRES activity; and mutations in PTB1 RRM3/RRM4 domains reduced, but did not abolish, the protein's capacity to stimulate HCV IRES activity. In HEK293T cells, mutations in PTB4 RRM1/RRM2 abrogated the protein's ability to promote HCV IRES activity, and mutations in RRM3/RRM4 have no impact on PTB4 ability to enhance HCV IRES activity. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity in a cell type-specific manner. We conclude that PTB1 and PTB4, but not PTB2, act as IRES transacting factors of the HCV IRES.
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Affiliation(s)
- Jenniffer Angulo
- Laboratorio de Virología Molecular, Centro de Investigaciones Médicas, Instituto Milenio de Inmunología e Inmunoterapia, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile
- Facultad de Odontología, Universidad Finis Terrae, Santiago 7501015, Chile
| | - C. Joaquín Cáceres
- Laboratorio de Virología Molecular, Centro de Investigaciones Médicas, Instituto Milenio de Inmunología e Inmunoterapia, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile
- Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA
| | - Nataly Contreras
- Laboratorio de Virología Molecular, Centro de Investigaciones Médicas, Instituto Milenio de Inmunología e Inmunoterapia, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile
- Facultad de Medicina Veterinaria y Agronomía, Universidad de Las Américas, Santiago 7500975, Chile
| | - Leandro Fernández-García
- Laboratorio de Virología Molecular, Centro de Investigaciones Médicas, Instituto Milenio de Inmunología e Inmunoterapia, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile
| | - Nathalie Chamond
- Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8038, Laboratoire CiTCoM, Université Paris Cité, 75006 Paris, France
| | - Melissa Ameur
- Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8038, Laboratoire CiTCoM, Université Paris Cité, 75006 Paris, France
| | - Bruno Sargueil
- Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8038, Laboratoire CiTCoM, Université Paris Cité, 75006 Paris, France
| | - Marcelo López-Lastra
- Laboratorio de Virología Molecular, Centro de Investigaciones Médicas, Instituto Milenio de Inmunología e Inmunoterapia, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica de Chile, Santiago 8330024, Chile
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17
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Sliepen K, Radić L, Capella-Pujol J, Watanabe Y, Zon I, Chumbe A, Lee WH, de Gast M, Koopsen J, Koekkoek S, Del Moral-Sánchez I, Brouwer PJM, Ravichandran R, Ozorowski G, King NP, Ward AB, van Gils MJ, Crispin M, Schinkel J, Sanders RW. Induction of cross-neutralizing antibodies by a permuted hepatitis C virus glycoprotein nanoparticle vaccine candidate. Nat Commun 2022; 13:7271. [PMID: 36434005 PMCID: PMC9700739 DOI: 10.1038/s41467-022-34961-8] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2022] [Accepted: 11/14/2022] [Indexed: 11/27/2022] Open
Abstract
Hepatitis C virus (HCV) infection affects approximately 58 million people and causes ~300,000 deaths yearly. The only target for HCV neutralizing antibodies is the highly sequence diverse E1E2 glycoprotein. Eliciting broadly neutralizing antibodies that recognize conserved cross-neutralizing epitopes is important for an effective HCV vaccine. However, most recombinant HCV glycoprotein vaccines, which usually include only E2, induce only weak neutralizing antibody responses. Here, we describe recombinant soluble E1E2 immunogens that were generated by permutation of the E1 and E2 subunits. We displayed the E2E1 immunogens on two-component nanoparticles and these nanoparticles induce significantly more potent neutralizing antibody responses than E2. Next, we generated mosaic nanoparticles co-displaying six different E2E1 immunogens. These mosaic E2E1 nanoparticles elicit significantly improved neutralization compared to monovalent E2E1 nanoparticles. These results provide a roadmap for the generation of an HCV vaccine that induces potent and broad neutralization.
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Affiliation(s)
- Kwinten Sliepen
- Amsterdam UMC, location University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam, The Netherlands.
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, The Netherlands.
| | - Laura Radić
- Amsterdam UMC, location University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam, The Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, The Netherlands
| | - Joan Capella-Pujol
- Amsterdam UMC, location University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam, The Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, The Netherlands
| | - Yasunori Watanabe
- School of Biological Sciences, University of Southampton, Southampton, UK
| | - Ian Zon
- Amsterdam UMC, location University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam, The Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, The Netherlands
| | - Ana Chumbe
- Amsterdam UMC, location University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam, The Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, The Netherlands
| | - Wen-Hsin Lee
- Department of Structural Biology and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA
| | - Marlon de Gast
- Amsterdam UMC, location University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam, The Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, The Netherlands
| | - Jelle Koopsen
- Amsterdam UMC, location University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam, The Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, The Netherlands
| | - Sylvie Koekkoek
- Amsterdam UMC, location University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam, The Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, The Netherlands
| | - Iván Del Moral-Sánchez
- Amsterdam UMC, location University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam, The Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, The Netherlands
| | - Philip J M Brouwer
- Amsterdam UMC, location University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam, The Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, The Netherlands
| | - Rashmi Ravichandran
- Department of Biochemistry, University of Washington, Seattle, USA
- Institute for Protein Design, University of Washington, Seattle, USA
| | - Gabriel Ozorowski
- Department of Structural Biology and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA
| | - Neil P King
- Department of Biochemistry, University of Washington, Seattle, USA
- Institute for Protein Design, University of Washington, Seattle, USA
| | - Andrew B Ward
- Department of Structural Biology and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA
| | - Marit J van Gils
- Amsterdam UMC, location University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam, The Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, The Netherlands
| | - Max Crispin
- School of Biological Sciences, University of Southampton, Southampton, UK
| | - Janke Schinkel
- Amsterdam UMC, location University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam, The Netherlands
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, The Netherlands
| | - Rogier W Sanders
- Amsterdam UMC, location University of Amsterdam, Department of Medical Microbiology and Infection Prevention, Laboratory of Experimental Virology, Amsterdam, The Netherlands.
- Amsterdam Institute for Infection and Immunity, Infectious Diseases, Amsterdam, The Netherlands.
- Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, USA.
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18
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Chumbe A, Urbanowicz RA, Sliepen K, Koekkoek SM, Molenkamp R, Tarr AW, Ball JK, Schinkel J, van Gils MJ. Optimization of the pseudoparticle system for standardized assessments of neutralizing antibodies against hepatitis C virus. J Gen Virol 2022; 103. [PMID: 36399377 DOI: 10.1099/jgv.0.001801] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
A better understanding of the antibody response during natural infection and the effect on disease progression and reinfection is necessary for the development of a protective hepatitis C virus (HCV) vaccine. The HCV pseudoparticle (HCVpp) system enables the study of viral entry and inhibition by antibody neutralization. A robust and comparable neutralization assay is crucial for the development and evaluation of experimental vaccines.With the aim of optimizing the HCVpp-murine leukaemia virus (MLV) system, we tested the neutralization of HCVpp-harbouring E1E2 from 21 HCV isolates representing 6 different genotypes by several monoclonal antibodies (mAbs). HCVpps are generated by expressing functional envelope glycoproteins (E1E2) onto pseudoparticles derived from env-deleted MLV. Adjustments of E1E2, gag-pol and luciferase plasmid ratios resulted in increased yields for most HCVpps and recovery of one non-infectious HCVpp. We simplified and improved the protocol to achieve higher signal/noise ratios and minimized the amount of HCVpps and mAbs needed for the detection of neutralization. Using our optimized protocol, we demonstrated comparable results to previously reported data with both diluted and freeze-thawed HCVpps.In conclusion, we successfully established a simplified and reproducible HCVpp neutralization protocol for studying a wide range of HCV variants. This simplified protocol provides highly consistent results and could be easily adopted by others to evaluate precious biological material. This will contribute to a better understanding of the antibody response during natural infection and help evaluate experimental HCV vaccines.
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Affiliation(s)
- Ana Chumbe
- Department of Medical Microbiology and Infection Prevention, Amsterdam UMC, University of Amsterdam, Amsterdam Institute for Infection and Immunity, Amsterdam, Netherlands
| | - Richard A Urbanowicz
- School of Life Sciences, Faculty of Medicine and Health Sciences, The University of Nottingham, Nottingham, UK
- Wolfson Centre for Global Virus Research, The University of Nottingham, Nottingham, UK
- National Institute for Health Research Nottingham Biomedical Research Centre, Nottingham University Hospitals National Health Service Trust, Nottingham, UK
- Department of Infection Biology and Microbiomes, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L3 5RF, UK
| | - Kwinten Sliepen
- Department of Medical Microbiology and Infection Prevention, Amsterdam UMC, University of Amsterdam, Amsterdam Institute for Infection and Immunity, Amsterdam, Netherlands
| | - Sylvie M Koekkoek
- Department of Medical Microbiology and Infection Prevention, Amsterdam UMC, University of Amsterdam, Amsterdam Institute for Infection and Immunity, Amsterdam, Netherlands
| | | | - Alexander W Tarr
- School of Life Sciences, Faculty of Medicine and Health Sciences, The University of Nottingham, Nottingham, UK
- Wolfson Centre for Global Virus Research, The University of Nottingham, Nottingham, UK
- National Institute for Health Research Nottingham Biomedical Research Centre, Nottingham University Hospitals National Health Service Trust, Nottingham, UK
| | - Jonathan K Ball
- School of Life Sciences, Faculty of Medicine and Health Sciences, The University of Nottingham, Nottingham, UK
- Wolfson Centre for Global Virus Research, The University of Nottingham, Nottingham, UK
- National Institute for Health Research Nottingham Biomedical Research Centre, Nottingham University Hospitals National Health Service Trust, Nottingham, UK
| | - Janke Schinkel
- Department of Medical Microbiology and Infection Prevention, Amsterdam UMC, University of Amsterdam, Amsterdam Institute for Infection and Immunity, Amsterdam, Netherlands
| | - Marit J van Gils
- Department of Medical Microbiology and Infection Prevention, Amsterdam UMC, University of Amsterdam, Amsterdam Institute for Infection and Immunity, Amsterdam, Netherlands
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19
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Characterization of a multipurpose NS3 surface patch coordinating HCV replicase assembly and virion morphogenesis. PLoS Pathog 2022; 18:e1010895. [PMID: 36215335 PMCID: PMC9616216 DOI: 10.1371/journal.ppat.1010895] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Revised: 10/28/2022] [Accepted: 09/25/2022] [Indexed: 11/16/2022] Open
Abstract
The hepatitis C virus (HCV) life cycle is highly regulated and characterized by a step-wise succession of interactions between viral and host cell proteins resulting in the assembly of macromolecular complexes, which catalyse genome replication and/or virus production. Non-structural (NS) protein 3, comprising a protease and a helicase domain, is involved in orchestrating these processes by undergoing protein interactions in a temporal fashion. Recently, we identified a multifunctional NS3 protease surface patch promoting pivotal protein-protein interactions required for early steps of the HCV life cycle, including NS3-mediated NS2 protease activation and interactions required for replicase assembly. In this work, we extend this knowledge by identifying further NS3 surface determinants important for NS5A hyperphosphorylation, replicase assembly or virion morphogenesis, which map to protease and helicase domain and form a contiguous NS3 surface area. Functional interrogation led to the identification of phylogenetically conserved amino acid positions exerting a critical function in virion production without affecting RNA replication. These findings illustrate that NS3 uses a multipurpose protein surface to orchestrate the step-wise assembly of functionally distinct multiprotein complexes. Taken together, our data provide a basis to dissect the temporal formation of viral multiprotein complexes required for the individual steps of the HCV life cycle.
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20
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Yokokawa H, Shinohara M, Teraoka Y, Imamura M, Nakamura N, Watanabe N, Date T, Aizaki H, Iwamura T, Narumi H, Chayama K, Wakita T. Patient-derived monoclonal antibody neutralizes HCV infection in vitro and vivo without generating escape mutants. PLoS One 2022; 17:e0274283. [PMID: 36137152 PMCID: PMC9499215 DOI: 10.1371/journal.pone.0274283] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2022] [Accepted: 08/24/2022] [Indexed: 11/18/2022] Open
Abstract
In recent years, new direct-acting antivirals for hepatitis C virus (HCV) have been approved, but hepatitis C continues to pose a threat to human health. It is important to develop neutralizing anti-HCV antibodies to prevent medical and accidental infection, such as might occur via liver transplantation of chronic HCV patients and needle-stick accidents in the clinic. In this study, we sought to obtain anti-HCV antibodies using phage display screening. Phages displaying human hepatocellular carcinoma patient-derived antibodies were screened by 4 rounds of biopanning with genotype-1b and -2a HCV envelope E2 protein adsorbed to magnetic beads. The three antibodies obtained from this screen had reactivity against E2 proteins derived from both genotype-1b and -2a strains. However, in epitope analysis, these antibodies did not recognize linear peptides from an overlapping E2 epitope peptide library, and did not bind to denatured E2 protein. In addition, these antibodies showed cross-genotypic neutralizing activity against genotype-1a, -1b, -2a, and -3a cell culture-generated infectious HCV particles (HCVcc). Moreover, emergence of viral escape mutants was not observed after repeated rounds of passaging of HCV-infected cells in the presence of one such antibody, e2d066. Furthermore, injection of the e2d066 antibody into human hepatocyte-transplanted immunodeficient mice inhibited infection by J6/JFH-1 HCVcc. In conclusion, we identified conformational epitope-recognizing, cross-genotypic neutralizing antibodies using phage display screening. Notably, e2d066 antibody did not select for escape mutant emergence in vitro and demonstrated neutralizing activity in vivo. Our results suggested that these antibodies may serve as prophylactic and therapeutic agents.
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Affiliation(s)
- Hiroshi Yokokawa
- Pharmaceutical Research Laboratory, Toray Industries, Inc., Kanagawa, Japan
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- * E-mail: (HY); (TW)
| | | | - Yuji Teraoka
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan
- Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Michio Imamura
- Department of Gastroenterology and Metabolism, Graduate School of Biomedical and Health Science, Hiroshima University, Hiroshima, Japan
- Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
| | - Noriko Nakamura
- Pharmaceutical Research Laboratory, Toray Industries, Inc., Kanagawa, Japan
| | - Noriyuki Watanabe
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Tomoko Date
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Hideki Aizaki
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Tomokatsu Iwamura
- Pharmaceutical Research Laboratory, Toray Industries, Inc., Kanagawa, Japan
| | - Hideki Narumi
- Pharmaceutical Research Laboratory, Toray Industries, Inc., Kanagawa, Japan
| | - Kazuaki Chayama
- Research Center for Hepatology and Gastroenterology, Hiroshima University, Hiroshima, Japan
- Collaborative Research Laboratory of Medical Innovation, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan
- RIKEN Center for Integrative Medical Sciences, Yokohama, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- * E-mail: (HY); (TW)
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21
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Kazmierski J, Friedmann K, Postmus D, Emanuel J, Fischer C, Jansen J, Richter A, Bosquillon de Jarcy L, Schüler C, Sohn M, Sauer S, Drosten C, Saliba A, Sander LE, Müller MA, Niemeyer D, Goffinet C. Nonproductive exposure of PBMCs to SARS-CoV-2 induces cell-intrinsic innate immune responses. Mol Syst Biol 2022; 18:e10961. [PMID: 35975552 PMCID: PMC9382356 DOI: 10.15252/msb.202210961] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2022] [Revised: 07/28/2022] [Accepted: 07/28/2022] [Indexed: 12/15/2022] Open
Abstract
Cell-intrinsic responses mounted in PBMCs during mild and severe COVID-19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or result from physical interaction with virus particles remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS-CoV and SARS-CoV-2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. RT-PCR experiments and single-cell RNA sequencing revealed JAK/STAT-dependent induction of interferon-stimulated genes (ISGs) but not proinflammatory cytokines. This SARS-CoV-2-specific response was most pronounced in monocytes. SARS-CoV-2-RNA-positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG baseline profile or delivery of a SARS-CoV-2-specific sensing antagonist upon efficient particle internalization. Together, nonproductive physical interaction of PBMCs with SARS-CoV-2- and, to a much lesser extent, SARS-CoV particles stimulate JAK/STAT-dependent, monocyte-accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID-19.
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Affiliation(s)
- Julia Kazmierski
- Institute of Virology, Campus Charité Mitte, Charité – Universitätsmedizin BerlinBerlinGermany
- Berlin Institute of HealthBerlinGermany
| | - Kirstin Friedmann
- Institute of Virology, Campus Charité Mitte, Charité – Universitätsmedizin BerlinBerlinGermany
| | - Dylan Postmus
- Institute of Virology, Campus Charité Mitte, Charité – Universitätsmedizin BerlinBerlinGermany
- Berlin Institute of HealthBerlinGermany
| | - Jackson Emanuel
- Institute of Virology, Campus Charité Mitte, Charité – Universitätsmedizin BerlinBerlinGermany
| | - Cornelius Fischer
- Scientific Genomics Platforms, Laboratory of Functional Genomics, Nutrigenomics and Systems BiologyMax Delbrück Center for Molecular MedicineBerlinGermany
| | - Jenny Jansen
- Institute of Virology, Campus Charité Mitte, Charité – Universitätsmedizin BerlinBerlinGermany
- Berlin Institute of HealthBerlinGermany
| | - Anja Richter
- Institute of Virology, Campus Charité Mitte, Charité – Universitätsmedizin BerlinBerlinGermany
| | - Laure Bosquillon de Jarcy
- Institute of Virology, Campus Charité Mitte, Charité – Universitätsmedizin BerlinBerlinGermany
- Department of Infectious Diseases and Respiratory MedicineCharité ‐ Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt‐Universität zu Berlin, and Berlin Institute of Health (BIH)BerlinGermany
| | - Christiane Schüler
- Institute of Virology, Campus Charité Mitte, Charité – Universitätsmedizin BerlinBerlinGermany
- Berlin Institute of HealthBerlinGermany
| | - Madlen Sohn
- Scientific Genomics Platforms, Laboratory of Functional Genomics, Nutrigenomics and Systems BiologyMax Delbrück Center for Molecular MedicineBerlinGermany
| | - Sascha Sauer
- Scientific Genomics Platforms, Laboratory of Functional Genomics, Nutrigenomics and Systems BiologyMax Delbrück Center for Molecular MedicineBerlinGermany
| | - Christian Drosten
- Institute of Virology, Campus Charité Mitte, Charité – Universitätsmedizin BerlinBerlinGermany
- German Center for Infection Research, Associated Partner CharitéBerlinGermany
| | - Antoine‐Emmanuel Saliba
- Helmholtz Institute for RNA‐based Infection Research (HIRI)Helmholtz‐Center for Infection Research (HZI)WürzburgGermany
| | - Leif Erik Sander
- Department of Infectious Diseases and Respiratory MedicineCharité ‐ Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt‐Universität zu Berlin, and Berlin Institute of Health (BIH)BerlinGermany
| | - Marcel A Müller
- Institute of Virology, Campus Charité Mitte, Charité – Universitätsmedizin BerlinBerlinGermany
- German Center for Infection Research, Associated Partner CharitéBerlinGermany
| | - Daniela Niemeyer
- Institute of Virology, Campus Charité Mitte, Charité – Universitätsmedizin BerlinBerlinGermany
- German Center for Infection Research, Associated Partner CharitéBerlinGermany
| | - Christine Goffinet
- Institute of Virology, Campus Charité Mitte, Charité – Universitätsmedizin BerlinBerlinGermany
- Berlin Institute of HealthBerlinGermany
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22
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Bhattacharjee C, Mukhopadhyay A. Generation of fluorescent HCV pseudoparticles to study early viral entry events- involvement of Rab1a in HCV entry. Virusdisease 2022; 33:172-184. [PMID: 35855963 PMCID: PMC9275390 DOI: 10.1007/s13337-022-00770-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2022] [Accepted: 06/01/2022] [Indexed: 12/05/2022] Open
Abstract
Understanding the early events in viral biology holds the key to the development of potent preventives. In this study, fluorescent hepatitis C virus pseudoparticles (HCVpp) have been generated where the envelope glycoprotein of Hepatitis C virus (HCV) has an EGFP tag. Using these pseudoparticles, entry assays were conducted where their entry was tracked via confocal microscopy. Using this system, fusion of host and viral membranes is predicted to occur within 15 min of HCV entry. Using cells with a knockdown for Rab1a, HCV trafficking was observed to be altered, indicating a role of Rab1a in HCV trafficking. In conclusion, this study reports the generation and use of fluorescent HCVpp which may be used to understand the early events of viral entry. This system may be adapted for the study of other enveloped viruses as well.
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Affiliation(s)
- Chayan Bhattacharjee
- Molecular Virology Laboratory, Department of Life Sciences, Presidency University, 86/1 College Street, Kolkata, 700073 India
| | - Aparna Mukhopadhyay
- Molecular Virology Laboratory, Department of Life Sciences, Presidency University, 86/1 College Street, Kolkata, 700073 India
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23
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Heuss C, Rothhaar P, Burm R, Lee JY, Ralfs P, Haselmann U, Ströh LJ, Colasanti O, Tran CS, Schäfer N, Schnitzler P, Merle U, Bartenschlager R, Patel AH, Graw F, Krey T, Laketa V, Meuleman P, Lohmann V. A Hepatitis C virus genotype 1b post-transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo. PLoS Pathog 2022; 18:e1010472. [PMID: 35763545 PMCID: PMC9273080 DOI: 10.1371/journal.ppat.1010472] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2022] [Revised: 07/11/2022] [Accepted: 05/29/2022] [Indexed: 12/23/2022] Open
Abstract
Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes and isolates, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, likely due to cell-to-cell transmission, which appeared to substantially contribute to spreading of other isolates as well. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is an efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines.
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Affiliation(s)
- Christian Heuss
- Department of Infectious Diseases, Molecular Virology, Section virus-host interactions, Heidelberg University, Heidelberg, Germany
| | - Paul Rothhaar
- Department of Infectious Diseases, Molecular Virology, Section virus-host interactions, Heidelberg University, Heidelberg, Germany
| | - Rani Burm
- Laboratory of Liver Infectious Diseases, Ghent University, Gent, Belgium
| | - Ji-Young Lee
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
| | - Philipp Ralfs
- Department of Infectious Diseases, Molecular Virology, Section virus-host interactions, Heidelberg University, Heidelberg, Germany
| | - Uta Haselmann
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
| | - Luisa J. Ströh
- Institute of Virology, Hannover Medical School, Hannover, Germany
| | - Ombretta Colasanti
- Department of Infectious Diseases, Molecular Virology, Section virus-host interactions, Heidelberg University, Heidelberg, Germany
| | - Cong Si Tran
- Department of Infectious Diseases, Molecular Virology, Section virus-host interactions, Heidelberg University, Heidelberg, Germany
| | - Noemi Schäfer
- Department of Infectious Diseases, Molecular Virology, Section virus-host interactions, Heidelberg University, Heidelberg, Germany
| | - Paul Schnitzler
- Department of Infectious Diseases Virology, University Hospital Heidelberg, Heidelberg, Germany
| | - Uta Merle
- Department of Internal Medicine IV, University Hospital Heidelberg, Heidelberg, Germany
| | - Ralf Bartenschlager
- Department of Infectious Diseases, Molecular Virology, Heidelberg University, Heidelberg, Germany
- German Center for Infection Research, partner site Heidelberg, Heidelberg, Germany
- Division Virus-Associated Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg, Germany
| | - Arvind H. Patel
- MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom
| | - Frederik Graw
- BioQuant – Center for Quantitative Biology, Heidelberg University, Heidelberg, Germany
- Interdisciplinary Center for Scientific Computing, Heidelberg University, Heidelberg, Germany
| | - Thomas Krey
- Institute of Virology, Hannover Medical School, Hannover, Germany
- Center of Structural and Cell Biology in Medicine, Institute of Biochemistry, University of Lübeck, Lübeck, Germany
- Centre for Structural Systems Biology (CSSB), Hamburg, Germany
- German Center for Infection Research (DZIF), Partner Site Hamburg-Lübeck-Borstel-Riems, Lübeck, Germany
- Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany
| | - Vibor Laketa
- Department of Infectious Diseases Virology, University Hospital Heidelberg, Heidelberg, Germany
- German Center for Infection Research, partner site Heidelberg, Heidelberg, Germany
| | - Philip Meuleman
- Laboratory of Liver Infectious Diseases, Ghent University, Gent, Belgium
| | - Volker Lohmann
- Department of Infectious Diseases, Molecular Virology, Section virus-host interactions, Heidelberg University, Heidelberg, Germany
- German Center for Infection Research, partner site Heidelberg, Heidelberg, Germany
- * E-mail:
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24
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Kobayashi C, Watanabe Y, Oshima M, Hirose T, Yamasaki M, Iwamoto M, Iwatsuki M, Asami Y, Kuramochi K, Wakae K, Aizaki H, Muramatsu M, Sureau C, Sunazuka T, Watashi K. Fungal Secondary Metabolite Exophillic Acid Selectively Inhibits the Entry of Hepatitis B and D Viruses. Viruses 2022; 14:v14040764. [PMID: 35458494 PMCID: PMC9026752 DOI: 10.3390/v14040764] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2022] [Revised: 04/01/2022] [Accepted: 04/04/2022] [Indexed: 02/04/2023] Open
Abstract
Current anti-hepatitis B virus (HBV) drugs are suppressive but not curative for HBV infection, so there is considerable demand for the development of new anti-HBV agents. In this study, we found that fungus-derived exophillic acid inhibits HBV infection with a 50% maximal inhibitory concentration (IC50) of 1.1 µM and a 50% cytotoxic concentration (CC50) of >30 µM in primary human hepatocytes. Exophillic acid inhibited preS1-mediated viral attachment to cells but did not affect intracellular HBV replication. Exophillic acid appears to target the host cells to reduce their susceptibility to viral attachment rather than acting on the viral particles. We found that exophillic acid interacted with the HBV receptor, sodium taurocholate cotransporting polypeptide (NTCP). Exophillic acid impaired the uptake of bile acid, the original function of NTCP. Consistent with our hypothesis that it affects NTCP, exophillic acid inhibited infection with HBV and hepatitis D virus (HDV), but not that of hepatitis C virus. Moreover, exophillic acid showed a pan-genotypic anti-HBV effect. We thus identified the anti-HBV/HDV activity of exophillic acid and revealed its mode of action. Exophillic acid is expected to be a potential new lead compound for the development of antiviral agents.
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Affiliation(s)
- Chisa Kobayashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan; (C.K.); (M.O.); (M.Y.); (M.I.); (K.W.); (H.A.); (M.M.)
- Department of Applied Biological Science, Tokyo University of Science, Noda 278-8510, Japan;
| | - Yoshihiro Watanabe
- Graduate School of Infection Control Sciences, Kitasato University, Tokyo 108-8641, Japan; (Y.W.); (T.H.); (M.I.); (Y.A.); (T.S.)
- Ōmura Satoshi Memorial Institute, Kitasato University, Tokyo 108-8641, Japan
| | - Mizuki Oshima
- Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan; (C.K.); (M.O.); (M.Y.); (M.I.); (K.W.); (H.A.); (M.M.)
- Department of Applied Biological Science, Tokyo University of Science, Noda 278-8510, Japan;
| | - Tomoyasu Hirose
- Graduate School of Infection Control Sciences, Kitasato University, Tokyo 108-8641, Japan; (Y.W.); (T.H.); (M.I.); (Y.A.); (T.S.)
- Ōmura Satoshi Memorial Institute, Kitasato University, Tokyo 108-8641, Japan
| | - Masako Yamasaki
- Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan; (C.K.); (M.O.); (M.Y.); (M.I.); (K.W.); (H.A.); (M.M.)
- Department of Applied Biological Science, Tokyo University of Science, Noda 278-8510, Japan;
| | - Masashi Iwamoto
- Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan; (C.K.); (M.O.); (M.Y.); (M.I.); (K.W.); (H.A.); (M.M.)
| | - Masato Iwatsuki
- Graduate School of Infection Control Sciences, Kitasato University, Tokyo 108-8641, Japan; (Y.W.); (T.H.); (M.I.); (Y.A.); (T.S.)
- Ōmura Satoshi Memorial Institute, Kitasato University, Tokyo 108-8641, Japan
| | - Yukihiro Asami
- Graduate School of Infection Control Sciences, Kitasato University, Tokyo 108-8641, Japan; (Y.W.); (T.H.); (M.I.); (Y.A.); (T.S.)
- Ōmura Satoshi Memorial Institute, Kitasato University, Tokyo 108-8641, Japan
| | - Kouji Kuramochi
- Department of Applied Biological Science, Tokyo University of Science, Noda 278-8510, Japan;
| | - Kousho Wakae
- Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan; (C.K.); (M.O.); (M.Y.); (M.I.); (K.W.); (H.A.); (M.M.)
| | - Hideki Aizaki
- Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan; (C.K.); (M.O.); (M.Y.); (M.I.); (K.W.); (H.A.); (M.M.)
| | - Masamichi Muramatsu
- Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan; (C.K.); (M.O.); (M.Y.); (M.I.); (K.W.); (H.A.); (M.M.)
| | - Camille Sureau
- Laboratoire de Virologie Moléculaire, Institut National de la Transfusion Sanguine, 75739 Paris, France;
| | - Toshiaki Sunazuka
- Graduate School of Infection Control Sciences, Kitasato University, Tokyo 108-8641, Japan; (Y.W.); (T.H.); (M.I.); (Y.A.); (T.S.)
- Ōmura Satoshi Memorial Institute, Kitasato University, Tokyo 108-8641, Japan
| | - Koichi Watashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan; (C.K.); (M.O.); (M.Y.); (M.I.); (K.W.); (H.A.); (M.M.)
- Department of Applied Biological Science, Tokyo University of Science, Noda 278-8510, Japan;
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
- MIRAI, JST, Saitama 332-0012, Japan
- Correspondence:
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25
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Deng L, Liang P, Cui H. Pseudotyped lentiviral vectors: Ready for translation into targeted cancer gene therapy? Genes Dis 2022. [PMID: 37492721 PMCID: PMC10363566 DOI: 10.1016/j.gendis.2022.03.007] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
Gene therapy holds great promise for curing cancer by editing the deleterious genes of tumor cells, but the lack of vector systems for efficient delivery of genetic material into specific tumor sites in vivo has limited its full therapeutic potential in cancer gene therapy. Over the past two decades, increasing studies have shown that lentiviral vectors (LVs) modified with different glycoproteins from a donating virus, a process referred to as pseudotyping, have altered tropism and display cell-type specificity in transduction, leading to selective tumor cell killing. This feature of LVs together with their ability to enable high efficient gene delivery in dividing and non-dividing mammalian cells in vivo make them to be attractive tools in future cancer gene therapy. This review is intended to summarize the status quo of some typical pseudotypings of LVs and their applications in basic anti-cancer studies across many malignancies. The opportunities of translating pseudotyped LVs into clinic use in cancer therapy have also been discussed.
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26
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Qian X, Wu B, Xu C, Qi Z. Hepatitis C Virus Infection Cycle-Specific MicroRNA Profiling Reveals Stage-Specific miR-4423-3p Targets RIG-I to Facilitate Infection. Front Cell Infect Microbiol 2022; 12:851917. [PMID: 35402303 PMCID: PMC8987439 DOI: 10.3389/fcimb.2022.851917] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2022] [Accepted: 02/22/2022] [Indexed: 11/13/2022] Open
Abstract
Hepatitis C virus (HCV) infection is one of the main causes of chronic liver diseases, the disorders of which involve multiple pathological processes and elements including host factors such as non-coding small RNAs. Although several genes have been reported to be correlated with HCV infection, the potential regulatory network has not been deciphered clearly. By small RNA sequencing, we clarified the expression profile of microRNAs (miRNAs) in HCV-infected Huh7 and Huh7.5.1 cells and identified 6 dysregulated miRNAs with the same expression trend and 32 dysregulated miRNAs with different expression trends during different stages of HCV life cycle. By looking into each infection stage, we found that 6 miRNAs were entry stage specific, 4 miRNAs were replication stage specific, and 1 miRNA was related to the transmission stage. Moreover, due to the fact that Huh7.5.1 cells have a retinoic acid-inducible gene 1 (RIG-I) mutation which causes reduced production of interferons (IFNs), we here focused on the miRNAs of different trends to decipher the RIG-I/IFN specific miRNAs. Among them, miR-4423-3p showed a significant promotive effect on HCV infection by suppressing RIG-I/IFN pathway through direct binding to RIG-I mRNA. Together, the results displayed novel insights into the miRNA regulatory networks in HCV infection and progression, thus providing a prosperous perspective into the establishment of novel therapeutic and diagnostic targets of the disease.
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Affiliation(s)
- Xijing Qian
- Department of Microbiology, Faculty of Naval Medicine, Naval Medical University, Shanghai, China
| | - Bingan Wu
- Department of Microbiology, Faculty of Naval Medicine, Naval Medical University, Shanghai, China
| | - Chen Xu
- Spine Center, Department of Orthopedics, Shanghai Changzheng Hospital Affiliated to Naval Medical University, Shanghai, China
| | - Zhongtian Qi
- Department of Microbiology, Faculty of Naval Medicine, Naval Medical University, Shanghai, China
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27
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Lázaro-Frías A, Pérez P, Zamora C, Sánchez-Cordón PJ, Guzmán M, Luczkowiak J, Delgado R, Casasnovas JM, Esteban M, García-Arriaza J. Full efficacy and long-term immunogenicity induced by the SARS-CoV-2 vaccine candidate MVA-CoV2-S in mice. NPJ Vaccines 2022; 7:17. [PMID: 35140227 PMCID: PMC8828760 DOI: 10.1038/s41541-022-00440-w] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2021] [Accepted: 01/14/2022] [Indexed: 02/06/2023] Open
Abstract
Two doses of the MVA-CoV2-S vaccine candidate expressing the SARS-CoV-2 spike (S) protein protected K18-hACE2 transgenic mice from a lethal dose of SARS-CoV-2. This vaccination regimen prevented virus replication in the lungs, reduced lung pathology, and diminished levels of pro-inflammatory cytokines. High titers of IgG antibodies against S and receptor-binding domain (RBD) proteins and of neutralizing antibodies were induced against parental virus and variants of concern, markers that correlated with protection. Similar SARS-CoV-2-specific antibody responses were observed at prechallenge and postchallenge in the two-dose regimen, while the single-dose treatment does not avoid vaccine breakthrough infection. All vaccinated animals survived infection and were also protected to SARS-CoV-2 reinfection. Furthermore, two MVA-CoV2-S doses induced long-term memory S-specific humoral and cellular immune responses in C57BL/6 mice, 6 months after immunization. The efficacy and immunological benefits of the MVA-CoV2-S vaccine candidate against COVID-19 supports its consideration for human clinical trials.
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Affiliation(s)
- Adrián Lázaro-Frías
- Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), 28049, Madrid, Spain.,Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Madrid, Spain
| | - Patricia Pérez
- Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), 28049, Madrid, Spain.,Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Madrid, Spain
| | - Carmen Zamora
- Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), 28049, Madrid, Spain
| | - Pedro J Sánchez-Cordón
- Pathology Department, Centro de Investigación en Sanidad Animal (CISA), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Consejo Superior de Investigaciones Científicas (CSIC), 28130, Valdeolmos, Madrid, Spain
| | - María Guzmán
- Department of Macromolecular Structures, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), 28049, Madrid, Spain
| | - Joanna Luczkowiak
- Instituto de Investigación Hospital Universitario 12 de Octubre (imas12), 28041, Madrid, Spain
| | - Rafael Delgado
- Instituto de Investigación Hospital Universitario 12 de Octubre (imas12), 28041, Madrid, Spain.,Universidad Complutense School of Medicine, 28040, Madrid, Spain
| | - José M Casasnovas
- Department of Macromolecular Structures, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), 28049, Madrid, Spain
| | - Mariano Esteban
- Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), 28049, Madrid, Spain.
| | - Juan García-Arriaza
- Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB), Consejo Superior de Investigaciones Científicas (CSIC), 28049, Madrid, Spain. .,Centro de Investigación Biomédica en Red de Enfermedades Infecciosas (CIBERINFEC), Madrid, Spain.
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28
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Salas JH, Urbanowicz RA, Guest JD, Frumento N, Figueroa A, Clark KE, Keck Z, Cowton VM, Cole SJ, Patel AH, Fuerst TR, Drummer HE, Major M, Tarr AW, Ball JK, Law M, Pierce BG, Foung SKH, Bailey JR. An Antigenically Diverse, Representative Panel of Envelope Glycoproteins for Hepatitis C Virus Vaccine Development. Gastroenterology 2022; 162:562-574. [PMID: 34655573 PMCID: PMC8792218 DOI: 10.1053/j.gastro.2021.10.005] [Citation(s) in RCA: 29] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/27/2021] [Revised: 10/05/2021] [Accepted: 10/06/2021] [Indexed: 12/21/2022]
Abstract
BACKGROUND & AIMS Development of a prophylactic hepatitis C virus (HCV) vaccine will require accurate and reproducible measurement of neutralizing breadth of vaccine-induced antibodies. Currently available HCV panels may not adequately represent the genetic and antigenic diversity of circulating HCV strains, and the lack of standardization of these panels makes it difficult to compare neutralization results obtained in different studies. Here, we describe the selection and validation of a genetically and antigenically diverse reference panel of 15 HCV pseudoparticles (HCVpps) for neutralization assays. METHODS We chose 75 envelope (E1E2) clones to maximize representation of natural polymorphisms observed in circulating HCV isolates, and 65 of these clones generated functional HCVpps. Neutralization sensitivity of these HCVpps varied widely. HCVpps clustered into 15 distinct groups based on patterns of relative sensitivity to 7 broadly neutralizing monoclonal antibodies. We used these data to select a final panel of 15 antigenically representative HCVpps. RESULTS Both the 65 and 15 HCVpp panels span 4 tiers of neutralization sensitivity, and neutralizing breadth measurements for 7 broadly neutralizing monoclonal antibodies were nearly equivalent using either panel. Differences in neutralization sensitivity between HCVpps were independent of genetic distances between E1E2 clones. CONCLUSIONS Neutralizing breadth of HCV antibodies should be defined using viruses spanning multiple tiers of neutralization sensitivity rather than panels selected solely for genetic diversity. We propose that this multitier reference panel could be adopted as a standard for the measurement of neutralizing antibody potency and breadth, facilitating meaningful comparisons of neutralization results from vaccine studies in different laboratories.
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Affiliation(s)
- Jordan H Salas
- Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Richard A Urbanowicz
- School of Life Sciences, Faculty of Medicine and Health Sciences, The University of Nottingham, Nottingham, United Kingdom; Wolfson Centre for Global Virus Research, The University of Nottingham, Nottingham, United Kingdom; National Institute for Health Research Nottingham Biomedical Research Centre, Nottingham University Hospitals National Health Service Trust, Nottingham, United Kingdom
| | - Johnathan D Guest
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland; Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland
| | - Nicole Frumento
- Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Alexis Figueroa
- Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Kaitlyn E Clark
- Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland
| | - Zhenyong Keck
- Department of Pathology, Stanford University School of Medicine, Stanford, California
| | - Vanessa M Cowton
- Medical Research Council-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom
| | - Sarah J Cole
- Medical Research Council-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom
| | - Arvind H Patel
- Medical Research Council-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom
| | - Thomas R Fuerst
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland; Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland
| | - Heidi E Drummer
- Viral Entry and Vaccines Group, Burnet Institute, Melbourne, Victoria, Australia; Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia; Department of Microbiology, Monash University, Clayton, Victoria, Australia
| | - Marian Major
- Division of Viral Products, Center for Biologics Evaluation and Research, United States Food and Drug Administration, Silver Spring, Maryland
| | - Alexander W Tarr
- School of Life Sciences, Faculty of Medicine and Health Sciences, The University of Nottingham, Nottingham, United Kingdom; Wolfson Centre for Global Virus Research, The University of Nottingham, Nottingham, United Kingdom; National Institute for Health Research Nottingham Biomedical Research Centre, Nottingham University Hospitals National Health Service Trust, Nottingham, United Kingdom
| | - Jonathan K Ball
- School of Life Sciences, Faculty of Medicine and Health Sciences, The University of Nottingham, Nottingham, United Kingdom; Wolfson Centre for Global Virus Research, The University of Nottingham, Nottingham, United Kingdom; National Institute for Health Research Nottingham Biomedical Research Centre, Nottingham University Hospitals National Health Service Trust, Nottingham, United Kingdom
| | - Mansun Law
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, California
| | - Brian G Pierce
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland; Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland
| | - Steven K H Foung
- Department of Pathology, Stanford University School of Medicine, Stanford, California
| | - Justin R Bailey
- Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland.
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29
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Eichler M, Aksi E, Pfeilschifter J, Imre G. Application of pseudotyped virus particles to monitor Ebola virus and SARS-CoV-2 viral entry in human cell lines. STAR Protoc 2021; 2:100818. [PMID: 34467222 PMCID: PMC8390363 DOI: 10.1016/j.xpro.2021.100818] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022] Open
Abstract
Experimental work on highly pathogenic viruses such as Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus-2 requires high-level biosafety facilities. Here, we provide a detailed step-by-step protocol which details the production and application of replication-incompetent murine leukemia virus-based pseudotyped particles to monitor and quantify the viral entry efficiency in human cell lines under biosafety level-2 conditions. We describe the use of viral particles encoding luciferase gene and the quantification of transduction efficiency by measuring luciferase activity. For complete details on the use and execution of this protocol, please refer to Imre et al. (2021).
MLV pseudotyped viruses are suitable for studies on viral entry in human cell lines Replication incompetence of pseudoviruses enables bio safety level-2 conditions Quantification of transduction efficiency by measurement of luciferase activity
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Affiliation(s)
- Madeleine Eichler
- Institute of General Pharmacology and Toxicology, Goethe University Frankfurt, 60590 Frankfurt am Main, Germany
| | - Ebru Aksi
- Institute of General Pharmacology and Toxicology, Goethe University Frankfurt, 60590 Frankfurt am Main, Germany
| | - Josef Pfeilschifter
- Institute of General Pharmacology and Toxicology, Goethe University Frankfurt, 60590 Frankfurt am Main, Germany
| | - Gergely Imre
- Institute of General Pharmacology and Toxicology, Goethe University Frankfurt, 60590 Frankfurt am Main, Germany
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30
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Chan SW, Shafi T, Ford RC. Kite-Shaped Molecules Block SARS-CoV-2 Cell Entry at a Post-Attachment Step. Viruses 2021; 13:v13112306. [PMID: 34835112 PMCID: PMC8619434 DOI: 10.3390/v13112306] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2021] [Revised: 11/02/2021] [Accepted: 11/15/2021] [Indexed: 12/13/2022] Open
Abstract
Anti-viral small molecules are currently lacking for treating coronavirus infection. The long development timescales for such drugs are a major problem, but could be shortened by repurposing existing drugs. We therefore screened a small library of FDA-approved compounds for potential severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antivirals using a pseudovirus system that allows a sensitive read-out of infectivity. A group of structurally-related compounds, showing moderate inhibitory activity with IC50 values in the 2–5 μM range, were identified. Further studies demonstrated that these “kite-shaped” molecules were surprisingly specific for SARS-CoV-1 and SARS-CoV-2 and that they acted early in the entry steps of the viral infectious cycle, but did not affect virus attachment to the cells. Moreover, the compounds were able to prevent infection in both kidney- and lung-derived human cell lines. The structural homology of the hits allowed the production of a well-defined pharmacophore that was found to be highly accurate in predicting the anti-viral activity of the compounds in the screen. We discuss the prospects of repurposing these existing drugs for treating current and future coronavirus outbreaks.
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31
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Dong J, Wu T, Zhang Y, Xie Z, He J. Transcriptome and MicroRNAs Profiling Analysis of Huh7.5.1 Cells in Response to Hepatitis C Virus Infection. HEPATITIS MONTHLY 2021; 21. [DOI: 10.5812/hepatmon.118724] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
Abstract
Background: There is a great need for further study on the mechanism of HCV infection or its pathopoiesis mechanism. Therefore, an HCV infection model was used to analyze the mechanisms of transcriptional and post-transcriptional regulation of gene expression. Methods: The detections of transcriptome and microRNAs expressions in Huh7.5.1 cells infected with JFH-1 were conducted with next-generation sequencing. Moreover, bioinformatics data were obtained. Results: There were 21,827,299, and 42,588,251 reads qualified Illumina read pairs obtained from JFH-1-infected (HCV) and non-infected (blank) Huh7.5.1 cells, respectively. Moreover, 678 and 1,041 mRNAs data with a length of 101 bp from HCV and blank Huh7.5.1 cells cDNA sequence were generated, respectively. The results of comparative transcriptome sequencing analysis declared 460 differentially expressed mRNAs in HCV-infected cells, including 152 upregulated mRNAs and 308 downregulated mRNAs (HCV vs. blank). Gene Ontology (GO) and KEGG pathway enrichment analyses indicated the involved pathways, such as MAPK, p53, and PI3K/Akt signaling pathways, as well as oocyte meiosis and pathways in cancer. Conclusions: Our work confirmed the transcriptome and microRNA data profiling from the cell model of HCV infection with JFH-1 using next-generation sequencing (NGS). Furthermore, the gene expression and regulation information or signaling pathways associated with the pathopoiesis mechanism of HCV infection were identified.
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32
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Echeverría N, Comas V, Aldunate F, Perbolianachis P, Moreno P, Cristina J. In the era of rapid mRNA-based vaccines: Why is there no effective hepatitis C virus vaccine yet? World J Hepatol 2021; 13:1234-1268. [PMID: 34786164 PMCID: PMC8568586 DOI: 10.4254/wjh.v13.i10.1234] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/04/2021] [Revised: 05/14/2021] [Accepted: 09/10/2021] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) is responsible for no less than 71 million people chronically infected and is one of the most frequent indications for liver transplantation worldwide. Despite direct-acting antiviral therapies fuel optimism in controlling HCV infections, there are several obstacles regarding treatment accessibility and reinfection continues to remain a possibility. Indeed, the majority of new HCV infections in developed countries occur in people who inject drugs and are more plausible to get reinfected. To achieve global epidemic control of this virus the development of an effective prophylactic or therapeutic vaccine becomes a must. The coronavirus disease 19 (COVID-19) pandemic led to auspicious vaccine development against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, which has renewed interest on fighting HCV epidemic with vaccination. The aim of this review is to highlight the current situation of HCV vaccine candidates designed to prevent and/or to reduce HCV infectious cases and their complications. We will emphasize on some of the crossroads encountered during vaccine development against this insidious virus, together with some key aspects of HCV immunology which have, so far, hampered the progress in this area. The main focus will be on nucleic acid-based as well as recombinant viral vector-based vaccine candidates as the most novel vaccine approaches, some of which have been recently and successfully employed for SARS-CoV-2 vaccines. Finally, some ideas will be presented on which methods to explore for the design of live-attenuated vaccines against HCV.
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Affiliation(s)
- Natalia Echeverría
- Laboratorio de Virología Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Montevideo 11400, Uruguay
| | - Victoria Comas
- Departamento de Desarrollo Biotecnológico, Instituto de Higiene, Facultad de Medicina, Universidad de la República, Montevideo 11600, Uruguay
| | - Fabián Aldunate
- Laboratorio de Virología Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Montevideo 11400, Uruguay
| | - Paula Perbolianachis
- Laboratorio de Virología Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Montevideo 11400, Uruguay
| | - Pilar Moreno
- Laboratorio de Virología Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Montevideo 11400, Uruguay
| | - Juan Cristina
- Laboratorio de Virología Molecular, Centro de Investigaciones Nucleares, Facultad de Ciencias, Universidad de la República, Montevideo 11400, Uruguay.
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Evidence that two instead of one defective interfering RNA in influenza A virus-derived defective interfering particles (DIPs) does not enhance antiviral activity. Sci Rep 2021; 11:20477. [PMID: 34650149 PMCID: PMC8516915 DOI: 10.1038/s41598-021-99691-1] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2021] [Accepted: 09/23/2021] [Indexed: 11/08/2022] Open
Abstract
Influenza A virus (IAV) infection constitutes a significant health threat. Defective interfering particles (DIPs) can arise during IAV infection and inhibit spread of wild type (WT) IAV. DIPs harbor defective RNA segments, termed DI RNAs, that usually contain internal deletions and interfere with replication of WT viral RNA segments. Here, we asked whether DIPs harboring two instead of one DI RNA exert increased antiviral activity. For this, we focused on DI RNAs derived from segments 1 and 3, which encode the polymerase subunits PB2 and PA, respectively. We demonstrate the successful production of DIPs harboring deletions in segments 1 and/or 3, using cell lines that co-express PB2 and PA. Further, we demonstrate that DIPs harboring two instead of one DI RNA do not exhibit increased ability to inhibit replication of a WT RNA segment. Similarly, the presence of two DI RNAs did not augment the induction of the interferon-stimulated gene MxA and the inhibition of IAV infection. Collectively, our findings suggest that the presence of multiple DI RNAs derived from genomic segments encoding polymerase subunits might not result in increased antiviral activity.
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Dobrica M, van Eerde A, Tucureanu C, Onu A, Paruch L, Caras I, Vlase E, Steen H, Haugslien S, Alonzi D, Zitzmann N, Bock R, Dubuisson J, Popescu C, Stavaru C, Liu Clarke J, Branza‐Nichita N. Hepatitis C virus E2 envelope glycoprotein produced in Nicotiana benthamiana triggers humoral response with virus-neutralizing activity in vaccinated mice. PLANT BIOTECHNOLOGY JOURNAL 2021; 19:2027-2039. [PMID: 34002936 PMCID: PMC8486241 DOI: 10.1111/pbi.13631] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/23/2020] [Revised: 04/27/2021] [Accepted: 05/13/2021] [Indexed: 05/03/2023]
Abstract
Chronic infection with hepatitis C virus (HCV) remains a leading cause of liver-related pathologies and a global health problem, currently affecting more than 71 million people worldwide. The development of a prophylactic vaccine is much needed to complement the effective antiviral treatment available and achieve HCV eradication. Current strategies focus on increasing the immunogenicity of the HCV envelope glycoprotein E2, the major target of virus-neutralizing antibodies, by testing various expression systems or manipulating the protein conformation and the N-glycosylation pattern. Here we report the first evidence of successful production of the full-length HCV E2 glycoprotein in Nicotiana benthamiana, by using the Agrobacterium-mediated transient expression technology. Molecular and functional analysis showed that the viral protein was correctly processed in plant cells and achieved the native folding required for binding to CD81, one of the HCV receptors. N-glycan analysis of HCV-E2 produced in N. benthamiana and mammalian cells indicated host-specific trimming of mannose residues and possibly, protein trafficking. Notably, the plant-derived viral antigen triggered a significant immune response in vaccinated mice, characterized by the presence of antibodies with HCV-neutralizing activity. Together, our study demonstrates that N. benthamiana is a viable alternative to costly mammalian cell cultures for the expression of complex viral antigens and supports the use of plants as cost-effective production platforms for the development of HCV vaccines.
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Affiliation(s)
| | | | - Catalin Tucureanu
- Cantacuzino” Medico‐Military National Research InstituteBucharestRomania
| | - Adrian Onu
- Cantacuzino” Medico‐Military National Research InstituteBucharestRomania
| | - Lisa Paruch
- NIBIO ‐ Norwegian Institute of Bioeconomy ResearchÅsNorway
| | - Iuliana Caras
- Cantacuzino” Medico‐Military National Research InstituteBucharestRomania
| | - Ene Vlase
- Cantacuzino” Medico‐Military National Research InstituteBucharestRomania
| | - Hege Steen
- NIBIO ‐ Norwegian Institute of Bioeconomy ResearchÅsNorway
| | | | - Dominic Alonzi
- Oxford Glycobiology InstituteDepartment of BiochemistryUniversity of OxfordOxfordUK
| | - Nicole Zitzmann
- Oxford Glycobiology InstituteDepartment of BiochemistryUniversity of OxfordOxfordUK
| | - Ralph Bock
- Max Planck Institute of Molecular Plant PhysiologyPotsdam‐GolmGermany
| | - Jean Dubuisson
- Université LilleCNRSINSERMCHU LilleInstitut Pasteur de LilleU1019‐UMR 9017‐CIIL‐Center for Infection and Immunity of LilleLilleFrance
| | | | - Crina Stavaru
- Cantacuzino” Medico‐Military National Research InstituteBucharestRomania
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Zheng X, Guo R, Liu Q, Wakae K, Watanabe N, Fukano K, Que L, Li Y, Aly HH, Watashi K, Suzuki R, Murayama A, Kato T, Aizaki H, Wakita T, Huang X, Yan Y, Song SJ, Muramatsu M. Identification of natural compounds extracted from crude drugs as novel inhibitors of hepatitis C virus. Biochem Biophys Res Commun 2021; 567:1-8. [PMID: 34130179 DOI: 10.1016/j.bbrc.2021.06.022] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2021] [Accepted: 06/04/2021] [Indexed: 12/12/2022]
Abstract
Natural product-derived crude drugs are expected to yield an abundance of new drugs to treat infectious diseases. Hepatitis C virus (HCV) is an oncogenic virus that significantly impacts public health. In this study, we sought to identify anti-HCV compounds in extracts of natural products. A total of 110 natural compounds extracted from several herbal medicine plants were examined for antiviral activity against HCV. Using a Huh7-mCherry-NLS-IPS reporter system for HCV infection, we first performed a rapid screening for anti-HCV compounds extracted from crude drugs. The compounds threo-2,3-bis(4-hydroxy-3-methoxyphenyl)-3-butoxypropan-1-ol (#106) and medioresinol (#110), which were extracted from Crataegus cuneate, exhibited anti-HCV activity and significantly inhibited HCV production in a dose-dependent manner. Analyses using HCV pseudoparticle and subgenomic replicon systems indicated that compounds #106 and #110 specifically inhibit HCV RNA replication but not viral entry or translation. Interestingly, compound #106 also inhibited the replication and production of hepatitis A virus. Our findings suggest that C. cuneate is a new source for novel anti-hepatitis virus drug development.
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Affiliation(s)
- Xin Zheng
- Department of Cardiology, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, China; Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
| | - Rui Guo
- Key Laboratory of Computational Chemistry Based Natural Antitumor Drug Research & Development, Liaoning Province, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, People's Republic of China
| | - Qingbo Liu
- Key Laboratory of Computational Chemistry Based Natural Antitumor Drug Research & Development, Liaoning Province, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, People's Republic of China
| | - Kousho Wakae
- Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
| | - Noriyuki Watanabe
- Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
| | - Kento Fukano
- Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
| | - Lusheng Que
- Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
| | - Yingfang Li
- Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
| | - Hussein H Aly
- Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
| | - Koichi Watashi
- Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan; Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
| | - Ryosuke Suzuki
- Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
| | - Asako Murayama
- Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
| | - Takanobu Kato
- Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
| | - Hideki Aizaki
- Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
| | - Xiaoxiao Huang
- Key Laboratory of Computational Chemistry Based Natural Antitumor Drug Research & Development, Liaoning Province, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, People's Republic of China
| | - Yi Yan
- Department of Cardiology, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, China
| | - Shao-Jiang Song
- Key Laboratory of Computational Chemistry Based Natural Antitumor Drug Research & Development, Liaoning Province, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, People's Republic of China.
| | - Masamichi Muramatsu
- Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan.
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36
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Yu T, Yang Q, Tian F, Chang H, Hu Z, Yu B, Han L, Xing Y, Jiu Y, He Y, Zhong J. Glycometabolism regulates hepatitis C virus release. PLoS Pathog 2021; 17:e1009746. [PMID: 34297778 PMCID: PMC8301660 DOI: 10.1371/journal.ppat.1009746] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Accepted: 06/23/2021] [Indexed: 01/01/2023] Open
Abstract
HCV cell-culture system uses hepatoma-derived cell lines for efficient virus propagation. Tumor cells cultured in glucose undergo active aerobic glycolysis, but switch to oxidative phosphorylation for energy production when cultured in galactose. Here, we investigated whether modulation of glycolysis in hepatocytes affects HCV infection. We showed HCV release, but not entry, genome replication or virion assembly, is significantly blocked when cells are cultured in galactose, leading to accumulation of intracellular infectious virions within multivesicular body (MVB). Blockade of the MVB-lysosome fusion or treatment with pro-inflammatory cytokines promotes HCV release in galactose. Furthermore, we found this glycometabolic regulation of HCV release is mediated by MAPK-p38 phosphorylation. Finally, we showed HCV cell-to-cell transmission is not affected by glycometabolism, suggesting that HCV cell-to-supernatant release and cell-to-cell transmission are two mechanistically distinct pathways. In summary, we demonstrated glycometabolism regulates the efficiency and route of HCV release. We proposed HCV may exploit the metabolic state in hepatocytes to favor its spread through the cell-to-cell transmission in vivo to evade immune response. Hepatitis C virus (HCV) is a positive-stranded RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. HCV infectious cycle comprises viral entry, uncoating, translation and replication of viral RNA, assembly into new virions and release. Establishment of HCV cell culture system (HCVcc) has yielded many insights into complete HCV infectious cycle in Huh7 cell and Huh7-derived human hepatoma cell lines. However, because hepatoma-derived cell lines and hepatocytes vary in metabolism, HCV infectious cycle in tumor cell lines and the patient’s liver may also be different. Therefore, we explored the alterations of HCV infectious cycle by forcing the tumor cell lines to switch their glycometabolic pathways. We found that HCV release can be blocked by culturing cells in galactose-containing medium, leading to accumulation of intracellular infectious virions within MVB. Moreover, we provided new evidence to suggest that HCV cell-to-cell transmission may be mechanistically distinct from cell-to-supernatant release. Finally, we proposed a new concept that HCV release from hepatocytes into circulation may be naturally inefficient due to the metabolic state in liver that may favor more HCV cell-to-cell transmission. This strategy would allow HCV to effectively evade neutralizing antibodies to establish persistent infection.
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Affiliation(s)
- Tao Yu
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Qiankun Yang
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Fangling Tian
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
- ShanghaiTech University, Shanghai, China
| | - Haishuang Chang
- Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai
| | - Zhenzheng Hu
- Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai
| | - Bowen Yu
- Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai
| | - Lin Han
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
- ShanghaiTech University, Shanghai, China
| | - Yifan Xing
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
- Cell Biology and Imaging Study of Pathogen Host Interaction Unit, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
| | - Yaming Jiu
- Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai
- Cell Biology and Imaging Study of Pathogen Host Interaction Unit, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
| | - Yongning He
- Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai
- State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, Shanghai
| | - Jin Zhong
- Unit of Viral Hepatitis, Institut Pasteur of Shanghai, CAS Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, Shanghai, China
- University of Chinese Academy of Sciences, Beijing, China
- ShanghaiTech University, Shanghai, China
- * E-mail:
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Olesen CH, Augestad EH, Troise F, Bukh J, Prentoe J. In vitro adaptation and characterization of attenuated hypervariable region 1 swap chimeras of hepatitis C virus. PLoS Pathog 2021; 17:e1009720. [PMID: 34280245 PMCID: PMC8321405 DOI: 10.1371/journal.ppat.1009720] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2021] [Revised: 07/29/2021] [Accepted: 06/15/2021] [Indexed: 12/18/2022] Open
Abstract
Hepatitis C virus (HCV) chronically infects 70 million people worldwide with an estimated annual disease-related mortality of 400,000. A vaccine could prevent spread of this pervasive human pathogen, but has proven difficult to develop, partly due to neutralizing antibody evasion mechanisms that are inherent features of the virus envelope glycoproteins, E1 and E2. A central actor is the E2 motif, hypervariable region 1 (HVR1), which protects several non-overlapping neutralization epitopes through an incompletely understood mechanism. Here, we show that introducing different HVR1-isolate sequences into cell-culture infectious JFH1-based H77 (genotype 1a) and J4 (genotype 1b) Core-NS2 recombinants can lead to severe viral attenuation. Culture adaptation of attenuated HVR1-swapped recombinants permitted us to identify E1/E2 substitutions at conserved positions both within and outside HVR1 that increased the infectivity of attenuated HVR1-swapped recombinants but were not adaptive for original recombinants. H77 recombinants with HVR1 from multiple other isolates consistently acquired substitutions at position 348 in E1 and position 385 in HVR1 of E2. Interestingly, HVR1-swapped J4 recombinants primarily acquired other substitutions: F291I (E1), F438V (E2), F447L/V/I (E2) and V710L (E2), indicating a different adaptation pathway. For H77 recombinants, the adaptive E1/E2 substitutions increased sensitivity to the neutralizing monoclonal antibodies AR3A and AR4A, whereas for J4 recombinants, they increased sensitivity to AR3A, while having no effect on sensitivity to AR4A. To evaluate effects of the substitutions on AR3A and AR4A binding, we performed ELISAs on extracted E1/E2 protein and performed immunoprecipitation of relevant viruses. However, extracted E1/E2 protein and immunoprecipitation of HCV particles only reproduced the neutralization phenotypes of the J4 recombinants. Finally, we found that the HVR1-swap E1/E2 substitutions decrease virus entry dependency on co-receptor SR-BI. Our study identifies E1/E2 positions that could be critical for intra-complex HVR1 interactions while emphasizing the need for developing novel tools for molecular studies of E1/E2 interactions.
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Affiliation(s)
- Christina Holmboe Olesen
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Elias H. Augestad
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Fulvia Troise
- Ceinge Biotecnologie Avanzate Via Gaetano Salvatore, Napoli, Italy
| | - Jens Bukh
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Jannick Prentoe
- Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, Hvidovre, Denmark
- Copenhagen Hepatitis C Program (CO-HEP), Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
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38
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Franz S, Pott F, Zillinger T, Schüler C, Dapa S, Fischer C, Passos V, Stenzel S, Chen F, Döhner K, Hartmann G, Sodeik B, Pessler F, Simmons G, Drexler JF, Goffinet C. Human IFITM3 restricts chikungunya virus and Mayaro virus infection and is susceptible to virus-mediated counteraction. Life Sci Alliance 2021; 4:e202000909. [PMID: 34078739 PMCID: PMC8200292 DOI: 10.26508/lsa.202000909] [Citation(s) in RCA: 11] [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: 09/16/2020] [Revised: 05/21/2021] [Accepted: 05/21/2021] [Indexed: 11/29/2022] Open
Abstract
Interferon-induced transmembrane (IFITM) proteins restrict membrane fusion and virion internalization of several enveloped viruses. The role of IFITM proteins during alphaviral infection of human cells and viral counteraction strategies are insufficiently understood. Here, we characterized the impact of human IFITMs on the entry and spread of chikungunya virus and Mayaro virus and provide first evidence for a CHIKV-mediated antagonism of IFITMs. IFITM1, 2, and 3 restricted infection at the level of alphavirus glycoprotein-mediated entry, both in the context of direct infection and cell-to-cell transmission. Relocalization of normally endosomal IFITM3 to the plasma membrane resulted in loss of antiviral activity. rs12252-C, a naturally occurring variant of IFITM3 that may associate with severe influenza in humans, restricted CHIKV, MAYV, and influenza A virus infection as efficiently as wild-type IFITM3 Antivirally active IFITM variants displayed reduced cell surface levels in CHIKV-infected cells involving a posttranscriptional process mediated by one or several nonstructural protein(s) of CHIKV. Finally, IFITM3-imposed reduction of specific infectivity of nascent particles provides a rationale for the necessity of a virus-encoded counteraction strategy against this restriction factor.
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Affiliation(s)
- Sergej Franz
- Institute of Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a Joint Venture Between the Hannover Medical School (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
- Vitalant Research Institute, San Francisco, CA, USA
| | - Fabian Pott
- Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Virology, Berlin, Germany
- Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Berlin, Germany
| | - Thomas Zillinger
- Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital, Venusberg-Campus 1, Bonn, Germany
| | - Christiane Schüler
- Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Virology, Berlin, Germany
- Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Berlin, Germany
| | - Sandra Dapa
- Institute of Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a Joint Venture Between the Hannover Medical School (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Carlo Fischer
- Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Virology, Berlin, Germany
| | - Vânia Passos
- Institute of Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a Joint Venture Between the Hannover Medical School (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
| | - Saskia Stenzel
- Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Virology, Berlin, Germany
- Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Berlin, Germany
| | - Fangfang Chen
- Research Group Biomarkers for Infectious Diseases, TWINCORE, Centre for Experimental and Clinical Infection Research, a Joint Venture Between the Hanover Medical School (MHH) and the Helmholtz Centre for Infection Research (HZI), Hanover, Germany
| | - Katinka Döhner
- Institute of Virology, Hannover Medical School, Hanover, Germany
| | - Gunther Hartmann
- Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Virology, Berlin, Germany
| | - Beate Sodeik
- Institute of Virology, Hannover Medical School, Hanover, Germany
- Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany
| | - Frank Pessler
- Research Group Biomarkers for Infectious Diseases, TWINCORE, Centre for Experimental and Clinical Infection Research, a Joint Venture Between the Hanover Medical School (MHH) and the Helmholtz Centre for Infection Research (HZI), Hanover, Germany
| | | | - Jan Felix Drexler
- Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Virology, Berlin, Germany
| | - Christine Goffinet
- Institute of Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a Joint Venture Between the Hannover Medical School (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany
- Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Virology, Berlin, Germany
- Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Berlin, Germany
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Interdependent Impact of Lipoprotein Receptors and Lipid-Lowering Drugs on HCV Infectivity. Cells 2021; 10:cells10071626. [PMID: 34209751 PMCID: PMC8303410 DOI: 10.3390/cells10071626] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2021] [Accepted: 06/25/2021] [Indexed: 12/19/2022] Open
Abstract
The HCV replication cycle is tightly associated with host lipid metabolism: Lipoprotein receptors SR-B1 and LDLr promote entry of HCV, replication is associated with the formation of lipid-rich membranous organelles and infectious particle assembly highjacks the very-low-density lipoprotein (VLDL) secretory pathway. Hence, medications that interfere with the lipid metabolism of the cell, such as statins, may affect HCV infection. Here, we study the interplay between lipoprotein receptors, lipid homeostasis, and HCV infection by genetic and pharmacological interventions. We found that individual ablation of the lipoprotein receptors SR-B1 and LDLr did not drastically affect HCV entry, replication, or infection, but double lipoprotein receptor knock-outs significantly reduced HCV infection. Furthermore, we could show that this effect was neither due to altered expression of additional HCV entry factors nor caused by changes in cellular cholesterol content. Strikingly, whereas lipid-lowering drugs such as simvastatin or fenofibrate did not affect HCV entry or infection of immortalized hepatoma cells expressing SR-B1 and/or LDLr or primary human hepatocytes, ablation of these receptors rendered cells more susceptible to these drugs. Finally, we observed no significant differences between statin users and control groups with regards to HCV viral load in a cohort of HCV infected patients before and during HCV antiviral treatment. Interestingly, statin treatment, which blocks the mevalonate pathway leading to decreased cholesterol levels, was associated with mild but appreciable lower levels of liver damage markers before HCV therapy. Overall, our findings confirm the role of lipid homeostasis in HCV infection and highlight the importance of the mevalonate pathway in the HCV replication cycle.
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40
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Zhang H, Zheng X, Li J, Liu Q, Huang XX, Ding H, Suzuki R, Muramatsu M, Song SJ. Flavonoid-triazolyl hybrids as potential anti-hepatitis C virus agents: Synthesis and biological evaluation. Eur J Med Chem 2021; 218:113395. [PMID: 33838584 DOI: 10.1016/j.ejmech.2021.113395] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2020] [Revised: 02/06/2021] [Accepted: 03/17/2021] [Indexed: 12/15/2022]
Abstract
A series of flavonoid-triazolyl hybrids were synthesized and evaluated as novel inhibitors of hepatitis C virus (HCV). The results of anti-HCV activity assays showed that most of the synthesized derivatives at a concentration of 100 μg/mL inhibited the generation of progeny virus. Among these derivatives, 10m and 10r exhibited the most potent anti-HCV activity and inhibited the production of HCV in a dose-dependent manner. Interestingly, 10m and 10r had no significant inhibitory effect on viral translation or replication. Additional action mechanism studies revealed that the most potent compounds, 10m and 10r, significantly inhibited viral entry to 34.0% and 52.0%, respectively, at 10 μM. These results suggest further effective application of 10m and 10r as potential HCV preventive agents.
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Affiliation(s)
- Han Zhang
- Key Laboratory of Computational Chemistry Based Natural Antitumor Drug Research & Development, Liaoning Province, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, People's Republic of China
| | - Xin Zheng
- Department of Cardiology, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510150, China; Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
| | - Jichong Li
- Key Laboratory of Computational Chemistry Based Natural Antitumor Drug Research & Development, Liaoning Province, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, People's Republic of China
| | - Qingbo Liu
- Key Laboratory of Computational Chemistry Based Natural Antitumor Drug Research & Development, Liaoning Province, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, People's Republic of China.
| | - Xiao-Xiao Huang
- Key Laboratory of Computational Chemistry Based Natural Antitumor Drug Research & Development, Liaoning Province, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, People's Republic of China
| | - Huaiwei Ding
- Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, China.
| | - Ryosuke Suzuki
- Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan
| | - Masamichi Muramatsu
- Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan.
| | - Shao-Jiang Song
- Key Laboratory of Computational Chemistry Based Natural Antitumor Drug Research & Development, Liaoning Province, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, People's Republic of China.
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41
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LeBlanc EV, Kim Y, Capicciotti CJ, Colpitts CC. Hepatitis C Virus Glycan-Dependent Interactions and the Potential for Novel Preventative Strategies. Pathogens 2021; 10:pathogens10060685. [PMID: 34205894 PMCID: PMC8230238 DOI: 10.3390/pathogens10060685] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2021] [Revised: 05/25/2021] [Accepted: 05/28/2021] [Indexed: 12/15/2022] Open
Abstract
Chronic hepatitis C virus (HCV) infections continue to be a major contributor to liver disease worldwide. HCV treatment has become highly effective, yet there are still no vaccines or prophylactic strategies available to prevent infection and allow effective management of the global HCV burden. Glycan-dependent interactions are crucial to many aspects of the highly complex HCV entry process, and also modulate immune evasion. This review provides an overview of the roles of viral and cellular glycans in HCV infection and highlights glycan-focused advances in the development of entry inhibitors and vaccines to effectively prevent HCV infection.
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Affiliation(s)
- Emmanuelle V. LeBlanc
- Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada; (E.V.L.); (Y.K.); (C.J.C.)
| | - Youjin Kim
- Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada; (E.V.L.); (Y.K.); (C.J.C.)
| | - Chantelle J. Capicciotti
- Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada; (E.V.L.); (Y.K.); (C.J.C.)
- Department of Chemistry, Queen’s University, Kingston, ON K7L 3N6, Canada
- Department of Surgery, Queen’s University, Kingston, ON K7L 3N6, Canada
| | - Che C. Colpitts
- Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON K7L 3N6, Canada; (E.V.L.); (Y.K.); (C.J.C.)
- Correspondence:
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42
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Chan S, Shafi T, Ford RC. Kite-shaped molecules block SARS-CoV-2 cell entry at a post-attachment step.. [DOI: 10.1101/2021.05.29.446272] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/01/2023]
Abstract
ABSTRACTAnti-viral small molecules are currently lacking for treating coronavirus infection. The long development timescales for such drugs are a major problem, but could be shortened by repurposing existing drugs. We therefore screened a small library of FDA-approved compounds for potential severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antivirals using a pseudovirus system that allows a sensitive read-out of infectivity. A group of structurally-related compounds, showing moderate inhibitory activity with IC50values in the 1-5µM range, were identified. Further studies demonstrated that these ‘kite-shaped’ molecules were surprisingly specific for SARS-CoV and SARS-CoV-2 and that they acted early in the entry steps of the viral infectious cycle, but did not affect virus attachment to the cells. Moreover the compounds were able to prevent infection in both kidney- and lung-derived human cell lines. The structural homology of the hits allowed the production of a well-defined pharmacophore that was found to be highly accurate in predicting the anti-viral activity of the compounds in the screen. We discuss the prospects of repurposing these existing drugs for treating current and future coronavirus outbreaks.
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43
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Andrianov AK, Fuerst TR. Immunopotentiating and Delivery Systems for HCV Vaccines. Viruses 2021; 13:v13060981. [PMID: 34070543 PMCID: PMC8227888 DOI: 10.3390/v13060981] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2021] [Revised: 05/16/2021] [Accepted: 05/19/2021] [Indexed: 12/13/2022] Open
Abstract
Development of preventive vaccines against hepatitis C virus (HCV) remains one of the main strategies in achieving global elimination of the disease. The effort is focused on the quest for vaccines capable of inducing protective cross-neutralizing humoral and cellular immune responses, which in turn dictate the need for rationally designed cross-genotype vaccine antigens and potent immunoadjuvants systems. This review provides an assessment of the current state of knowledge on immunopotentiating compounds and vaccine delivery systems capable of enhancing HCV antigen-specific immune responses, while focusing on the synergy and interplay of two modalities. Structural, physico-chemical, and biophysical features of these systems are discussed in conjunction with the analysis of their in vivo performance. Extreme genetic diversity of HCV-a well-known hurdle in the development of an HCV vaccine, may also present a challenge in a search for an effective immunoadjuvant, as the effort necessitates systematic and comparative screening of rationally designed antigenic constructs. The progress may be accelerated if the preference is given to well-defined molecular immunoadjuvants with greater formulation flexibility and adaptability, including those capable of spontaneous self-assembly behavior, while maintaining their robust immunopotentiating and delivery capabilities.
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Affiliation(s)
- Alexander K. Andrianov
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850, USA;
- Correspondence:
| | - Thomas R. Fuerst
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850, USA;
- Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA
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44
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Kalemera MD, Capella-Pujol J, Chumbe A, Underwood A, Bull RA, Schinkel J, Sliepen K, Grove J. Optimized cell systems for the investigation of hepatitis C virus E1E2 glycoproteins. J Gen Virol 2021; 102. [PMID: 33147126 PMCID: PMC8116788 DOI: 10.1099/jgv.0.001512] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Great strides have been made in understanding and treating hepatitis C virus (HCV) thanks to the development of various experimental systems including cell-culture-proficient HCV, the HCV pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterizing neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones produce pseudoparticles that are either non-infectious or exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate-binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293TCD81KO). Clones made in 293TCD81KO cells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T cells and Freestyle 293-F (293-F) cells exhibit important differences. We found that 293-F-produced sE2 harbours mostly complex-type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex-type glycans and high-mannose or hybrid-type glycans. Moreover, sE2 produced in 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293-F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.
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Affiliation(s)
- Mphatso D Kalemera
- Institute of Immunity and Transplantation, Division of Infection and Immunity, The Royal Free Hospital, University College London, London, UK
| | - Joan Capella-Pujol
- Department of Medical Microbiology, Amsterdam Infection & Immunity Institute, Amsterdam UMC, Amsterdam, University of Amsterdam, Amsterdam, The Netherlands
| | - Ana Chumbe
- Department of Medical Microbiology, Amsterdam Infection & Immunity Institute, Amsterdam UMC, Amsterdam, University of Amsterdam, Amsterdam, The Netherlands
| | - Alexander Underwood
- Viral Immunology Systems Program, The Kirby Institute, School of Medical Sciences, Faculty of Medicine, The University of New South Wales, Sydney, Australia
| | - Rowena A Bull
- Viral Immunology Systems Program, The Kirby Institute, School of Medical Sciences, Faculty of Medicine, The University of New South Wales, Sydney, Australia
| | - Janke Schinkel
- Department of Medical Microbiology, Amsterdam Infection & Immunity Institute, Amsterdam UMC, Amsterdam, University of Amsterdam, Amsterdam, The Netherlands
| | - Kwinten Sliepen
- Department of Medical Microbiology, Amsterdam Infection & Immunity Institute, Amsterdam UMC, Amsterdam, University of Amsterdam, Amsterdam, The Netherlands
| | - Joe Grove
- Institute of Immunity and Transplantation, Division of Infection and Immunity, The Royal Free Hospital, University College London, London, UK
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45
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Shionoya K, Yamasaki M, Iwanami S, Ito Y, Fukushi S, Ohashi H, Saso W, Tanaka T, Aoki S, Kuramochi K, Iwami S, Takahashi Y, Suzuki T, Muramatsu M, Takeda M, Wakita T, Watashi K. Mefloquine, a Potent Anti-severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2) Drug as an Entry Inhibitor in vitro. Front Microbiol 2021; 12:651403. [PMID: 33995308 PMCID: PMC8119653 DOI: 10.3389/fmicb.2021.651403] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2021] [Accepted: 04/06/2021] [Indexed: 12/30/2022] Open
Abstract
Coronavirus disease 2019 (COVID-19) has caused serious public health, social, and economic damage worldwide and effective drugs that prevent or cure COVID-19 are urgently needed. Approved drugs including Hydroxychloroquine, Remdesivir or Interferon were reported to inhibit the infection or propagation of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), however, their clinical efficacies have not yet been well demonstrated. To identify drugs with higher antiviral potency, we screened approved anti-parasitic/anti-protozoal drugs and identified an anti-malarial drug, Mefloquine, which showed the highest anti-SARS-CoV-2 activity among the tested compounds. Mefloquine showed higher anti-SARS-CoV-2 activity than Hydroxychloroquine in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 1.28 μM, IC90 = 2.31 μM, and IC99 = 4.39 μM in VeroE6/TMPRSS2 cells. Mefloquine inhibited viral entry after viral attachment to the target cell. Combined treatment with Mefloquine and Nelfinavir, a replication inhibitor, showed synergistic antiviral activity. Our mathematical modeling based on the drug concentration in the lung predicted that Mefloquine administration at a standard treatment dosage could decline viral dynamics in patients, reduce cumulative viral load to 7% and shorten the time until virus elimination by 6.1 days. These data cumulatively underscore Mefloquine as an anti-SARS-CoV-2 entry inhibitor.
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Affiliation(s)
- Kaho Shionoya
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.,Department of Applied Biological Science, Tokyo University of Science, Tokyo, Japan
| | - Masako Yamasaki
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.,Department of Applied Biological Science, Tokyo University of Science, Tokyo, Japan
| | - Shoya Iwanami
- Interdisciplinary Biology Laboratory (iBLab), Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan.,Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka, Japan
| | - Yusuke Ito
- Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka, Japan
| | - Shuetsu Fukushi
- Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan
| | - Hirofumi Ohashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.,Department of Applied Biological Science, Tokyo University of Science, Tokyo, Japan
| | - Wakana Saso
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.,The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.,AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan
| | - Tomohiro Tanaka
- Faculty of Pharmaceutical Sciences, Tokyo University of Science, Tokyo, Japan
| | - Shin Aoki
- Research Institute for Science and Technology, Tokyo University of Science, Tokyo, Japan
| | - Kouji Kuramochi
- Department of Applied Biological Science, Tokyo University of Science, Tokyo, Japan
| | - Shingo Iwami
- Interdisciplinary Biology Laboratory (iBLab), Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan.,Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka, Japan.,MIRAI, JST, Saitama, Japan.,Institute of Mathematics for Industry, Kyushu University, Fukuoka, Japan.,Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, Japan.,NEXT-Ganken Program, Japanese Foundation for Cancer Research (JFCR), Tokyo, Japan.,Science Groove Inc., Fukuoka, Japan
| | - Yoshimasa Takahashi
- Department of Immunology, National Institute of Infectious Diseases, Tokyo, Japan.,Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan
| | - Tadaki Suzuki
- Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan
| | - Masamichi Muramatsu
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Makoto Takeda
- Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan
| | - Takaji Wakita
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
| | - Koichi Watashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.,Department of Applied Biological Science, Tokyo University of Science, Tokyo, Japan.,MIRAI, JST, Saitama, Japan.,Research Center for Drug and Vaccine Development, National Institute of Infectious Diseases, Tokyo, Japan.,Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
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46
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To Include or Occlude: Rational Engineering of HCV Vaccines for Humoral Immunity. Viruses 2021; 13:v13050805. [PMID: 33946211 PMCID: PMC8146105 DOI: 10.3390/v13050805] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2021] [Revised: 04/16/2021] [Accepted: 04/28/2021] [Indexed: 02/07/2023] Open
Abstract
Direct-acting antiviral agents have proven highly effective at treating existing hepatitis C infections but despite their availability most countries will not reach the World Health Organization targets for elimination of HCV by 2030. A prophylactic vaccine remains a high priority. Whilst early vaccines focused largely on generating T cell immunity, attention is now aimed at vaccines that generate humoral immunity, either alone or in combination with T cell-based vaccines. High-resolution structures of hepatitis C viral glycoproteins and their interaction with monoclonal antibodies isolated from both cleared and chronically infected people, together with advances in vaccine technologies, provide new avenues for vaccine development.
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47
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Hoffmann M, Arora P, Groß R, Seidel A, Hörnich BF, Hahn AS, Krüger N, Graichen L, Hofmann-Winkler H, Kempf A, Winkler MS, Schulz S, Jäck HM, Jahrsdörfer B, Schrezenmeier H, Müller M, Kleger A, Münch J, Pöhlmann S. SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies. Cell 2021; 184:2384-2393.e12. [PMID: 33794143 PMCID: PMC7980144 DOI: 10.1016/j.cell.2021.03.036] [Citation(s) in RCA: 692] [Impact Index Per Article: 173.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2021] [Revised: 02/26/2021] [Accepted: 03/16/2021] [Indexed: 02/07/2023]
Abstract
The global spread of SARS-CoV-2/COVID-19 is devastating health systems and economies worldwide. Recombinant or vaccine-induced neutralizing antibodies are used to combat the COVID-19 pandemic. However, the recently emerged SARS-CoV-2 variants B.1.1.7 (UK), B.1.351 (South Africa), and P.1 (Brazil) harbor mutations in the viral spike (S) protein that may alter virus-host cell interactions and confer resistance to inhibitors and antibodies. Here, using pseudoparticles, we show that entry of all variants into human cells is susceptible to blockade by the entry inhibitors soluble ACE2, Camostat, EK-1, and EK-1-C4. In contrast, entry of the B.1.351 and P.1 variant was partially (Casirivimab) or fully (Bamlanivimab) resistant to antibodies used for COVID-19 treatment. Moreover, entry of these variants was less efficiently inhibited by plasma from convalescent COVID-19 patients and sera from BNT162b2-vaccinated individuals. These results suggest that SARS-CoV-2 may escape neutralizing antibody responses, which has important implications for efforts to contain the pandemic.
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Affiliation(s)
- Markus Hoffmann
- Infection Biology Unit, German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany; Faculty of Biology and Psychology, Georg-August-University Göttingen, Wilhelmsplatz 1, 37073 Göttingen, Germany.
| | - Prerna Arora
- Infection Biology Unit, German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany; Faculty of Biology and Psychology, Georg-August-University Göttingen, Wilhelmsplatz 1, 37073 Göttingen, Germany
| | - Rüdiger Groß
- Institute of Molecular Virology, Ulm University Medical Center, Meyerhofstr. 1, 89081 Ulm, Germany
| | - Alina Seidel
- Institute of Molecular Virology, Ulm University Medical Center, Meyerhofstr. 1, 89081 Ulm, Germany
| | - Bojan F Hörnich
- Junior Research Group Herpesviruses - Infection Biology Unit, German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany
| | - Alexander S Hahn
- Junior Research Group Herpesviruses - Infection Biology Unit, German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany
| | - Nadine Krüger
- Infection Biology Unit, German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany
| | - Luise Graichen
- Infection Biology Unit, German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany
| | - Heike Hofmann-Winkler
- Infection Biology Unit, German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany
| | - Amy Kempf
- Infection Biology Unit, German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany; Faculty of Biology and Psychology, Georg-August-University Göttingen, Wilhelmsplatz 1, 37073 Göttingen, Germany
| | - Martin S Winkler
- Department of Anaesthesiology, University of Göttingen Medical Center, Göttingen, Georg-August University of Göttingen, Robert-Koch-Straße 40, 37075 Göttingen, Germany
| | - Sebastian Schulz
- Division of Molecular Immunology, Department of Internal Medicine 3, Friedrich-Alexander University of Erlangen-Nürnberg, Glückstraße 6, 91054 Erlangen, Germany
| | - Hans-Martin Jäck
- Division of Molecular Immunology, Department of Internal Medicine 3, Friedrich-Alexander University of Erlangen-Nürnberg, Glückstraße 6, 91054 Erlangen, Germany
| | - Bernd Jahrsdörfer
- Department of Transfusion Medicine, Ulm University, Helmholtzstraße 10, 89081 Ulm, Germany; Institute for Clinical Transfusion Medicine and Immunogenetics, German Red Cross Blood Transfusion Service Baden-Württemberg - Hessen and University Hospital Ulm, Helmholtzstraße 10, 89081 Ulm, Germany
| | - Hubert Schrezenmeier
- Department of Transfusion Medicine, Ulm University, Helmholtzstraße 10, 89081 Ulm, Germany; Institute for Clinical Transfusion Medicine and Immunogenetics, German Red Cross Blood Transfusion Service Baden-Württemberg - Hessen and University Hospital Ulm, Helmholtzstraße 10, 89081 Ulm, Germany
| | - Martin Müller
- Department of Internal Medicine 1, Ulm University Hospital, Albert-Einstein-Allee 23, 89081 Ulm, Germany
| | - Alexander Kleger
- Department of Internal Medicine 1, Ulm University Hospital, Albert-Einstein-Allee 23, 89081 Ulm, Germany
| | - Jan Münch
- Institute of Molecular Virology, Ulm University Medical Center, Meyerhofstr. 1, 89081 Ulm, Germany; Core Facility Functional Peptidomics, Ulm University Medical Center, Meyerhofstr. 4, 89081 Ulm, Germany
| | - Stefan Pöhlmann
- Infection Biology Unit, German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany; Faculty of Biology and Psychology, Georg-August-University Göttingen, Wilhelmsplatz 1, 37073 Göttingen, Germany.
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48
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Arez F, Rodrigues AF, Brito C, Alves PM. Bioengineered Liver Cell Models of Hepatotropic Infections. Viruses 2021; 13:773. [PMID: 33925701 PMCID: PMC8146083 DOI: 10.3390/v13050773] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2021] [Revised: 04/22/2021] [Accepted: 04/23/2021] [Indexed: 02/07/2023] Open
Abstract
Hepatitis viruses and liver-stage malaria are within the liver infections causing higher morbidity and mortality rates worldwide. The highly restricted tropism of the major human hepatotropic pathogens-namely, the human hepatitis B and C viruses and the Plasmodium falciparum and Plasmodium vivax parasites-has hampered the development of disease models. These models are crucial for uncovering the molecular mechanisms underlying the biology of infection and governing host-pathogen interaction, as well as for fostering drug development. Bioengineered cell models better recapitulate the human liver microenvironment and extend hepatocyte viability and phenotype in vitro, when compared with conventional two-dimensional cell models. In this article, we review the bioengineering tools employed in the development of hepatic cell models for studying infection, with an emphasis on 3D cell culture strategies, and discuss how those tools contributed to the level of recapitulation attained in the different model layouts. Examples of host-pathogen interactions uncovered by engineered liver models and their usefulness in drug development are also presented. Finally, we address the current bottlenecks, trends, and prospect toward cell models' reliability, robustness, and reproducibility.
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MESH Headings
- Animals
- Bioengineering/methods
- Cell Culture Techniques
- Disease Models, Animal
- Disease Susceptibility
- Drug Discovery
- Hepatitis/drug therapy
- Hepatitis/etiology
- Hepatitis/metabolism
- Hepatitis/pathology
- Hepatitis, Viral, Human/etiology
- Hepatitis, Viral, Human/metabolism
- Hepatitis, Viral, Human/pathology
- Hepatocytes/metabolism
- Hepatocytes/parasitology
- Hepatocytes/virology
- Host-Pathogen Interactions
- Humans
- Liver/metabolism
- Liver/parasitology
- Liver/virology
- Liver Diseases, Parasitic/etiology
- Liver Diseases, Parasitic/metabolism
- Liver Diseases, Parasitic/pathology
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Affiliation(s)
- Francisca Arez
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; (F.A.); (A.F.R.); (C.B.)
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
| | - Ana F. Rodrigues
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; (F.A.); (A.F.R.); (C.B.)
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
| | - Catarina Brito
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; (F.A.); (A.F.R.); (C.B.)
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
- The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Av. da República, 2780-157 Oeiras, Portugal
| | - Paula M. Alves
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2781-901 Oeiras, Portugal; (F.A.); (A.F.R.); (C.B.)
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
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49
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Galvez J, Zanni R, Galvez-Llompart M, Benlloch JM. Macrolides May Prevent Severe Acute Respiratory Syndrome Coronavirus 2 Entry into Cells: A Quantitative Structure Activity Relationship Study and Experimental Validation. J Chem Inf Model 2021; 61:2016-2025. [PMID: 33734704 PMCID: PMC7986980 DOI: 10.1021/acs.jcim.0c01394] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Indexed: 02/07/2023]
Abstract
The global pandemic caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is threatening the health and economic systems worldwide. Despite the enormous efforts of scientists and clinicians around the world, there is still no drug or vaccine available worldwide for the treatment and prevention of the infection. A rapid strategy for the identification of new treatments is based on repurposing existing clinically approved drugs that show antiviral activity against SARS-CoV-2 infection. In this study, after developing a quantitative structure activity relationship analysis based on molecular topology, several macrolide antibiotics are identified as promising SARS-CoV-2 spike protein inhibitors. To confirm the in silico results, the best candidates were tested against two human coronaviruses (i.e., 229E-GFP and SARS-CoV-2) in cell culture. Time-of-addition experiments and a surrogate model of viral cell entry were used to identify the steps in the virus life cycle inhibited by the compounds. Infection experiments demonstrated that azithromycin, clarithromycin, and lexithromycin reduce the intracellular accumulation of viral RNA and virus spread as well as prevent virus-induced cell death, by inhibiting the SARS-CoV-2 entry into cells. Even though the three macrolide antibiotics display a narrow antiviral activity window against SARS-CoV-2, it may be of interest to further investigate their effect on the viral spike protein and their potential in combination therapies for the coronavirus disease 19 early stage of infection.
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Affiliation(s)
- Jorge Galvez
- Molecular Topology and Drug Design
Unit, Department of Physical Chemistry, Universitat de
Valencia, Burjassot 46100,
Spain
| | - Riccardo Zanni
- Molecular Topology and Drug Design
Unit, Department of Physical Chemistry, Universitat de
Valencia, Burjassot 46100,
Spain
| | - Maria Galvez-Llompart
- Molecular Topology and Drug Design
Unit, Department of Physical Chemistry, Universitat de
Valencia, Burjassot 46100,
Spain
- Instituto de Tecnología
Química, UPV-CSIC, Universidad Politícnica
de Valencia, Valencia 46022,
Spain
| | - Jose Maria Benlloch
- Instituto de Instrumentación para
Imagen Molecular, Centro Mixto CSIC—Universitat
Politècnica de València, Valencia
46022, Spain
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50
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Chen F, Tzarum N, Lin X, Giang E, Velázquez-Moctezuma R, Augestad EH, Nagy K, He L, Hernandez M, Fouch ME, Grinyó A, Chavez D, Doranz BJ, Prentoe J, Stanfield RL, Lanford R, Bukh J, Wilson IA, Zhu J, Law M. Functional convergence of a germline-encoded neutralizing antibody response in rhesus macaques immunized with HCV envelope glycoproteins. Immunity 2021; 54:781-796.e4. [PMID: 33675683 PMCID: PMC8046733 DOI: 10.1016/j.immuni.2021.02.013] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Revised: 12/14/2020] [Accepted: 02/10/2021] [Indexed: 12/14/2022]
Abstract
Human IGHV1-69-encoded broadly neutralizing antibodies (bnAbs) that target the hepatitis C virus (HCV) envelope glycoprotein (Env) E2 are important for protection against HCV infection. An IGHV1-69 ortholog gene, VH1.36, is preferentially used for bnAbs isolated from HCV Env-immunized rhesus macaques (RMs). Here, we studied the genetic, structural, and functional properties of VH1.36-encoded bnAbs generated by vaccination, in comparison to IGHV1-69-encoded bnAbs from HCV patients. Global B cell repertoire analysis confirmed the expansion of VH1.36-derived B cells in immunized animals. Most E2-specific, VH1.36-encoded antibodies cross-neutralized HCV. Crystal structures of two RM bnAbs with E2 revealed that the RM bnAbs engaged conserved E2 epitopes using similar molecular features as human bnAbs but with a different binding mode. Longitudinal analyses of the RM antibody repertoire responses during immunization indicated rapid lineage development of VH1.36-encoded bnAbs with limited somatic hypermutation. Our findings suggest functional convergence of a germline-encoded bnAb response to HCV Env with implications for vaccination in humans.
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Affiliation(s)
- Fang Chen
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Netanel Tzarum
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Xiaohe Lin
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Erick Giang
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Rodrigo Velázquez-Moctezuma
- 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
| | - Elias H Augestad
- 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
| | - Kenna Nagy
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Linling He
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | | | | | | | - Deborah Chavez
- Southwest National Primate Research Center at Texas Biomedical Research Institute, San Antonio, TX 788227, USA
| | | | - Jannick Prentoe
- 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
| | - Robyn L Stanfield
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
| | - Robert Lanford
- Southwest National Primate Research Center at Texas Biomedical Research Institute, San Antonio, TX 788227, USA
| | - 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
| | - Ian A Wilson
- Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA; Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
| | - Jiang Zhu
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA; Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
| | - Mansun Law
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA 92037, USA.
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