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

Masanori Ikeda, Min Kyung Yi, Kui Li, Stanley M. Lemon

Research output: Contribution to journalArticle

326 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2997-3006
Number of pages10
JournalJournal of Virology
Volume76
Issue number6
DOIs
StatePublished - Mar 11 2002

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Hepatitis C virus
Hepacivirus
cultured cells
Cultured Cells
Clone Cells
Genome
RNA
clones
genome
transfection
Replicon
Transfection
replicon
Mutation
mutation
cells
Polyproteins
Cell Culture Techniques
cell culture
Gastroenterology

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

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. / Ikeda, Masanori; Yi, Min Kyung; Li, Kui; Lemon, Stanley M.

In: Journal of Virology, Vol. 76, No. 6, 11.03.2002, p. 2997-3006.

Research output: Contribution to journalArticle

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