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Journal of Virology, March 2002, p. 2997-3006, Vol. 76, No. 6
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.6.2997-3006.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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, MinKyung Yi, Kui Li, and Stanley M. Lemon*

Department of Microbiology & Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019

Received 3 October 2001/ Accepted 20 December 2001

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.

* Corresponding author. Mailing address: Department of Microbiology & Immunology, The University of Texas Medical Branch at Galveston, Galveston, TX 77555-1019. Phone: (409) 772-4793. Fax: (409) 772-2684. E-mail: smlemon{at}utmb.edu.

Journal of Virology, March 2002, p. 2997-3006, Vol. 76, No. 6
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.6.2997-3006.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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