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Journal of Virology, June 2007, p. 5724-5736, Vol. 81, No. 11
0022-538X/07/$08.00+0     doi:10.1128/JVI.02481-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Allelic Variation in the Hepatitis C Virus NS4B Protein Dramatically Influences RNA Replication

Keril J. Blight^*

Department of Molecular Microbiology, Center for Infectious Disease Research, Washington University School of Medicine, 660 South Euclid Ave., Campus Box 8230, St. Louis, Missouri 63110

Received 10 November 2006/ Accepted 5 March 2007

In the Huh-7.5 hepatoma cell line, replication of the genotype 1a H77 strain of hepatitis C virus (HCV) is attenuated compared to that of the genotype 1b Con1 strain. This study identifies the poorly characterized integral membrane protein, NS4B, as a major determinant for this replication difference. Chimeric H77 subgenomic replicons containing the entire NS4B gene from Con1 in place of the H77 NS4B sequence replicated approximately 10-fold better than the H77 parent and to levels similar to that of the adapted Con1 replicon. An intermediate level of replication enhancement was conferred by H77 chimeras containing the poorly conserved N-terminal 47 residues or the remaining less-divergent C terminus of Con1 NS4B. The replication-enhancing activity within the N terminus of NS4B was further mapped to two Con1-specific amino acids. Experiments to elucidate the mechanism of enhanced H77 replication revealed that Con1 NS4B primarily increased H77 RNA synthesis on a per cell basis, as indicated by the similar capacities of chimeric and parental replicons to establish replication in Huh-7.5 cells and the higher levels of both positive- and negative-strand RNAs for the chimeras than for the H77 parent. Additionally, enhanced H77 replication was not the result of Con1 NS4B-mediated effects on HCV translation efficiency or alterations in polyprotein processing. Expression of Con1 NS4B in trans did not improve the replication of the H77 parental replicon, suggesting a cis-dominant role for NS4B in HCV replication. These results provide the first evidence that allelic variation in the NS4B sequence between closely related isolates significantly impacts HCV replication in cell culture.

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* Mailing address: Department of Molecular Microbiology, Center for Infectious Disease Research, Washington University School of Medicine, 660 South Euclid Ave., Campus Box 8230, St. Louis, MO 63110. Phone: (314) 286-0065. Fax: (314) 362-1232. E-mail: blight{at}borcim.wustl.edu

Published ahead of print on 14 March 2007.

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Journal of Virology, June 2007, p. 5724-5736, Vol. 81, No. 11
0022-538X/07/$08.00+0     doi:10.1128/JVI.02481-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Jones, D. M., Patel, A. H., Targett-Adams, P., McLauchlan, J. (2009). The Hepatitis C Virus NS4B Protein Can trans-Complement Viral RNA Replication and Modulates Production of Infectious Virus. J. Virol. 83: 2163-2177 [Abstract] [Full Text]  
• Paredes, A. M., Blight, K. J. (2008). A Genetic Interaction between Hepatitis C Virus NS4B and NS3 Is Important for RNA Replication. J. Virol. 82: 10671-10683 [Abstract] [Full Text]