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The NS3 Helicase and NS5B-to-3'X Regions Are Important for Efficient Hepatitis C Virus Strain JFH-1 Replication in Huh7 Cells

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan,¹ Pharmaceutical Research Lab, Toray Industries, Inc., Kanagawa, Japan,² Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan,³ Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan,⁴ Liver Disease Branch, NIDDK, National Institutes of Health, Bethesda, Maryland⁵

Received 23 September 2006/ Accepted 8 May 2007

The JFH-1 strain of hepatitis C virus (HCV) is a genotype 2a strain that can replicate autonomously in Huh7 cells. The J6 strain is also a genotype 2a strain, but its full genomic RNA does not replicate in Huh7 cells. However, chimeric J6/JFH-1 RNA that has J6 structural-protein-coding regions and JFH-1 nonstructural-protein-coding regions can replicate autonomously and produce infectious HCV particles. In order to determine the mechanisms underlying JFH-1 RNA replication, we constructed various J6/JFH-1 chimeras and tested their RNA replication and virus particle production abilities in Huh7 cells. Via subgenomic-RNA-replication assays, we found that both the JFH-1 NS5B-to-3'X (N5BX) and the NS3 helicase (N3H) regions are important for the replication of the J6CF replicon. We applied these results to full-length genomic RNA replication and analyzed replication using Northern blotting. We found that a chimeric J6 clone with JFH-1 N3H and N5BX could replicate autonomously but that a chimeric J6 clone with only JFH-1 N5BX had no replication ability. Finally, we tested the virus production abilities of these clones and found that a chimeric J6 clone with JFH-1 N3H and N5BX could produce infectious HCV particles. In conclusion, the JFH-1 NS3 helicase and NS5B-to-3'X regions are important for efficient replication and virus particle formation of HCV genotype 2a strains.

Published ahead of print on 23 May 2007.

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