Document Detail


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.
MedLine Citation:
PMID:  11861865     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Masanori Ikeda; MinKyung Yi; Kui Li; Stanley M Lemon
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Journal of virology     Volume:  76     ISSN:  0022-538X     ISO Abbreviation:  J. Virol.     Publication Date:  2002 Mar 
Date Detail:
Created Date:  2002-02-25     Completed Date:  2002-03-29     Revised Date:  2013-04-18    
Medline Journal Info:
Nlm Unique ID:  0113724     Medline TA:  J Virol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  2997-3006     Citation Subset:  IM    
Affiliation:
Department of Microbiology & Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019, USA.
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MeSH Terms
Descriptor/Qualifier:
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:
U19-AI40035/AI/NIAID NIH HHS
Chemical
Reg. No./Substance:
0/RNA, Viral; 0/Viral Nonstructural Proteins; EC 2.7.1.95/Kanamycin Kinase
Comments/Corrections

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