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J. Virol. doi:10.1128/JVI.02088-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

NS3 helicase and NS5B to 3' X regions are important for efficient JFH-1 replication in Huh7 cells

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

^* To whom correspondence should be addressed. Email: wakita{at}nih.go.jp.

	Abstract

The JFH-1 strain of hepatitis C virus 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 non-structural 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 ability in Huh7 cells. Via subgenomic RNA replication assays, we found that both the JFH-1 NS5B to 3'X (N5BX) and 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.

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