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Journal of Virology, August 2007, p. 8030-8040, Vol. 81, No. 15
0022-538X/07/$08.00+0     doi:10.1128/JVI.02088-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Asako Murayama,1 Tomoko Date,1 Kenichi Morikawa,1 Daisuke Akazawa,1,2 Michiko Miyamoto,3 Minako Kaga,1 Koji Ishii,1 Tetsuro Suzuki,1 Takanobu Kato,4,5 Masashi Mizokami,4 and Takaji Wakita1*

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.

* Corresponding author. Mailing address: Department of Virology II, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku, Tokyo 162-8640, Japan. Phone: 81-3-5285-1111. Fax: 81-3-5285-1161. E-mail: wakita{at}nih.go.jp

{triangledown} Published ahead of print on 23 May 2007.

Journal of Virology, August 2007, p. 8030-8040, Vol. 81, No. 15
0022-538X/07/$08.00+0     doi:10.1128/JVI.02088-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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