| Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells. | |
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MedLine Citation:
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PMID: 11861865 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Dicistronic, selectable subgenomic replicons derived from the Con1 strain of hepatitis C virus (HCV) are capable of autonomous replication in cultured Huh7 cells (Lohmann et al., Science 285:110-113, 1999). However, adaptive mutations in the NS3, NS5A, and/or NS5B proteins are required for efficient replication of these RNAs and increase by orders of magnitude the numbers of G418-resistant colonies selected following transfection of Huh7 cells. Here, we demonstrate that a subgenomic replicon (NNeo/3-5B) derived from an infectious molecular clone of a second genotype 1b virus, HCV-N (Beard et al., Hepatology 30:316-324, 1999) is also capable of efficient replication in Huh7 cells. G418-resistant cells selected following transfection with NNeo/3-5B RNA contained abundant NS5A antigen and HCV RNA detectable by Northern analysis. Replicon RNA in one of three clonally isolated cell lines contained no mutations in the NS3-NS5B polyprotein, confirming that adaptive mutations are not required for efficient replication in these cells. However, the deletion of a unique 4-amino-acid insertion that is present within the interferon sensitivity-determining region (ISDR) of the NS5A protein in wild-type HCV-N drastically decreased the number of G418-resistant colonies obtained following transfection of Huh7 cells. This effect could be reversed by inclusion of a previously described Con1 cell culture-adaptive mutation (S2005-->I), confirming that this natural insertion has a controlling role in determining the replication capacity of wild-type HCV-N RNA in Huh7 cells. Additional selectable, dicistronic RNAs encoding NS2-NS5B, E1-NS5B, or the full-length HCV polyprotein were also capable of replication and gave rise to G418-resistant cell clones following transfection of Huh7 cells. We conclude that RNA derived from this documented infectious molecular clone has a unique capacity for replication in Huh7 cells in the absence of additional cell culture-adaptive mutations. |
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Authors:
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Masanori Ikeda; MinKyung Yi; Kui Li; Stanley M Lemon |
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Publication Detail:
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Type: Journal Article; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Journal of virology Volume: 76 ISSN: 0022-538X ISO Abbreviation: J. Virol. Publication Date: 2002 Mar |
Date Detail:
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Created Date: 2002-02-25 Completed Date: 2002-03-29 Revised Date: 2013-04-18 |
Medline Journal Info:
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Nlm Unique ID: 0113724 Medline TA: J Virol Country: United States |
Other Details:
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Languages: eng Pagination: 2997-3006 Citation Subset: IM |
Affiliation:
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Department of Microbiology & Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019, USA. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Amino Acid Sequence Cell Line Cloning, Molecular* Genome, Viral* Hepacivirus / genetics, pathogenicity, physiology* Hepatitis C / virology Humans Kanamycin Kinase / genetics, metabolism Molecular Sequence Data RNA, Viral / genetics*, metabolism Reverse Transcriptase Polymerase Chain Reaction Transcription, Genetic Transfection Viral Nonstructural Proteins / genetics, metabolism Virus Replication* |
| Grant Support | |
ID/Acronym/Agency:
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U19-AI40035/AI/NIAID NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/RNA, Viral; 0/Viral Nonstructural Proteins; EC 2.7.1.95/Kanamycin Kinase |
| Comments/Corrections | |
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