A new model to produce infectious hepatitis C virus without the replication requirement.

Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5' and 3' termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes. Ausführliche Beschreibung

Gespeichert in:

Autor*in:	Miriam Triyatni [verfasserIn] Edward A Berger [verfasserIn] Bertrand Saunier [verfasserIn]

Format:	E-Artikel
Sprache:	Englisch

Erschienen:	2011

Übergeordnetes Werk:	In: PLoS Pathogens - Public Library of Science (PLoS), 2005, 7(2011), 4, p e1001333
Übergeordnetes Werk:	volume:7 ; year:2011 ; number:4, p e1001333

Links:	Link aufrufen Link aufrufen Link aufrufen Journal toc Journal toc

DOI / URN:	10.1371/journal.ppat.1001333

Katalog-ID:	DOAJ024546224

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allfields	10.1371/journal.ppat.1001333 doi (DE-627)DOAJ024546224 (DE-599)DOAJ660626157d944ef0b7e10446eb3e16c1 DE-627 ger DE-627 rakwb eng RC581-607 QH301-705.5 Miriam Triyatni verfasserin aut A new model to produce infectious hepatitis C virus without the replication requirement. 2011 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5' and 3' termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes. Immunologic diseases. Allergy Biology (General) Edward A Berger verfasserin aut Bertrand Saunier verfasserin aut In PLoS Pathogens Public Library of Science (PLoS), 2005 7(2011), 4, p e1001333 (DE-627)501074422 (DE-600)2205412-1 15537374 nnns volume:7 year:2011 number:4, p e1001333 https://doi.org/10.1371/journal.ppat.1001333 kostenfrei https://doaj.org/article/660626157d944ef0b7e10446eb3e16c1 kostenfrei http://europepmc.org/articles/PMC3077361?pdf=render kostenfrei https://doaj.org/toc/1553-7366 Journal toc kostenfrei https://doaj.org/toc/1553-7374 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_2522 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2011 4, p e1001333
spelling	10.1371/journal.ppat.1001333 doi (DE-627)DOAJ024546224 (DE-599)DOAJ660626157d944ef0b7e10446eb3e16c1 DE-627 ger DE-627 rakwb eng RC581-607 QH301-705.5 Miriam Triyatni verfasserin aut A new model to produce infectious hepatitis C virus without the replication requirement. 2011 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5' and 3' termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes. Immunologic diseases. Allergy Biology (General) Edward A Berger verfasserin aut Bertrand Saunier verfasserin aut In PLoS Pathogens Public Library of Science (PLoS), 2005 7(2011), 4, p e1001333 (DE-627)501074422 (DE-600)2205412-1 15537374 nnns volume:7 year:2011 number:4, p e1001333 https://doi.org/10.1371/journal.ppat.1001333 kostenfrei https://doaj.org/article/660626157d944ef0b7e10446eb3e16c1 kostenfrei http://europepmc.org/articles/PMC3077361?pdf=render kostenfrei https://doaj.org/toc/1553-7366 Journal toc kostenfrei https://doaj.org/toc/1553-7374 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_2522 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2011 4, p e1001333
allfields_unstemmed	10.1371/journal.ppat.1001333 doi (DE-627)DOAJ024546224 (DE-599)DOAJ660626157d944ef0b7e10446eb3e16c1 DE-627 ger DE-627 rakwb eng RC581-607 QH301-705.5 Miriam Triyatni verfasserin aut A new model to produce infectious hepatitis C virus without the replication requirement. 2011 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5' and 3' termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes. Immunologic diseases. Allergy Biology (General) Edward A Berger verfasserin aut Bertrand Saunier verfasserin aut In PLoS Pathogens Public Library of Science (PLoS), 2005 7(2011), 4, p e1001333 (DE-627)501074422 (DE-600)2205412-1 15537374 nnns volume:7 year:2011 number:4, p e1001333 https://doi.org/10.1371/journal.ppat.1001333 kostenfrei https://doaj.org/article/660626157d944ef0b7e10446eb3e16c1 kostenfrei http://europepmc.org/articles/PMC3077361?pdf=render kostenfrei https://doaj.org/toc/1553-7366 Journal toc kostenfrei https://doaj.org/toc/1553-7374 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_2522 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2011 4, p e1001333
allfieldsGer	10.1371/journal.ppat.1001333 doi (DE-627)DOAJ024546224 (DE-599)DOAJ660626157d944ef0b7e10446eb3e16c1 DE-627 ger DE-627 rakwb eng RC581-607 QH301-705.5 Miriam Triyatni verfasserin aut A new model to produce infectious hepatitis C virus without the replication requirement. 2011 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5' and 3' termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes. Immunologic diseases. Allergy Biology (General) Edward A Berger verfasserin aut Bertrand Saunier verfasserin aut In PLoS Pathogens Public Library of Science (PLoS), 2005 7(2011), 4, p e1001333 (DE-627)501074422 (DE-600)2205412-1 15537374 nnns volume:7 year:2011 number:4, p e1001333 https://doi.org/10.1371/journal.ppat.1001333 kostenfrei https://doaj.org/article/660626157d944ef0b7e10446eb3e16c1 kostenfrei http://europepmc.org/articles/PMC3077361?pdf=render kostenfrei https://doaj.org/toc/1553-7366 Journal toc kostenfrei https://doaj.org/toc/1553-7374 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_2522 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2011 4, p e1001333
allfieldsSound	10.1371/journal.ppat.1001333 doi (DE-627)DOAJ024546224 (DE-599)DOAJ660626157d944ef0b7e10446eb3e16c1 DE-627 ger DE-627 rakwb eng RC581-607 QH301-705.5 Miriam Triyatni verfasserin aut A new model to produce infectious hepatitis C virus without the replication requirement. 2011 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5' and 3' termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes. Immunologic diseases. Allergy Biology (General) Edward A Berger verfasserin aut Bertrand Saunier verfasserin aut In PLoS Pathogens Public Library of Science (PLoS), 2005 7(2011), 4, p e1001333 (DE-627)501074422 (DE-600)2205412-1 15537374 nnns volume:7 year:2011 number:4, p e1001333 https://doi.org/10.1371/journal.ppat.1001333 kostenfrei https://doaj.org/article/660626157d944ef0b7e10446eb3e16c1 kostenfrei http://europepmc.org/articles/PMC3077361?pdf=render kostenfrei https://doaj.org/toc/1553-7366 Journal toc kostenfrei https://doaj.org/toc/1553-7374 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2031 GBV_ILN_2038 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2061 GBV_ILN_2111 GBV_ILN_2113 GBV_ILN_2190 GBV_ILN_2522 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 7 2011 4, p e1001333
language	English
source	In PLoS Pathogens 7(2011), 4, p e1001333 volume:7 year:2011 number:4, p e1001333
sourceStr	In PLoS Pathogens 7(2011), 4, p e1001333 volume:7 year:2011 number:4, p e1001333
format_phy_str_mv	Article
institution	findex.gbv.de
topic_facet	Immunologic diseases. Allergy Biology (General)
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container_title	PLoS Pathogens
authorswithroles_txt_mv	Miriam Triyatni @@aut@@ Edward A Berger @@aut@@ Bertrand Saunier @@aut@@
publishDateDaySort_date	2011-01-01T00:00:00Z
hierarchy_top_id	501074422
id	DOAJ024546224
language_de	englisch
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callnumber-first	R - Medicine
author	Miriam Triyatni
spellingShingle	Miriam Triyatni misc RC581-607 misc QH301-705.5 misc Immunologic diseases. Allergy misc Biology (General) A new model to produce infectious hepatitis C virus without the replication requirement.
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topic_title	RC581-607 QH301-705.5 A new model to produce infectious hepatitis C virus without the replication requirement
topic	misc RC581-607 misc QH301-705.5 misc Immunologic diseases. Allergy misc Biology (General)
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title	A new model to produce infectious hepatitis C virus without the replication requirement.
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title_full	A new model to produce infectious hepatitis C virus without the replication requirement
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title_sort	new model to produce infectious hepatitis c virus without the replication requirement
callnumber	RC581-607
title_auth	A new model to produce infectious hepatitis C virus without the replication requirement.
abstract	Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5' and 3' termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes.
abstractGer	Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5' and 3' termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes.
abstract_unstemmed	Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5' and 3' termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes.
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container_issue	4, p e1001333
title_short	A new model to produce infectious hepatitis C virus without the replication requirement.
url	https://doi.org/10.1371/journal.ppat.1001333 https://doaj.org/article/660626157d944ef0b7e10446eb3e16c1 http://europepmc.org/articles/PMC3077361?pdf=render https://doaj.org/toc/1553-7366 https://doaj.org/toc/1553-7374
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Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. 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A new model to produce infectious hepatitis C virus without the replication requirement.

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