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Journal of Virology, November 2008, p. 10671-10683, Vol. 82, No. 21
0022-538X/08/$08.00+0     doi:10.1128/JVI.00875-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

A Genetic Interaction between Hepatitis C Virus NS4B and NS3 Is Important for RNA Replication{triangledown}

Anne M. Paredes and 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 24 April 2008/ Accepted 11 August 2008

Hepatitis C virus (HCV) nonstructural protein 4B (NS4B), a poorly characterized integral membrane protein, is thought to function as a scaffold for replication complex assembly; however, functional interactions with the other HCV nonstructural proteins within this complex have not been defined. We report that a Con1 chimeric subgenomic replicon containing the NS4B gene from the closely related H77 isolate is defective for RNA replication in a transient assay, suggesting that H77 NS4B is unable to productively interact with the Con1 replication machinery. The H77 NS4B sequences that proved detrimental for Con1 RNA replication resided in the predicted N- and C-terminal cytoplasmic domains as well as the central transmembrane region. Selection for Con1 derivatives that could utilize the entire H77 NS4B or hybrid Con1-H77 NS4B proteins yielded mutants containing single amino acid substitutions in NS3 and NS4A. The second-site mutations in NS3 partially restored the replication of Con1 chimeras containing the N-terminal or transmembrane domains of H77 NS4B. In contrast, the deleterious H77-specific sequences in the C terminus of NS4B, which mapped to a cluster of four amino acids, were completely suppressed by second-site substitutions in NS3. Collectively, these results provide the first evidence for a genetic interaction between NS4B and NS3 important for productive HCV RNA replication.

* Corresponding author. 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

{triangledown} Published ahead of print on 20 August 2008.

Journal of Virology, November 2008, p. 10671-10683, Vol. 82, No. 21
0022-538X/08/$08.00+0     doi:10.1128/JVI.00875-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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