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Journal of Virology, February 2004, p. 1352-1366, Vol. 78, No. 3
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.3.1352-1366.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A cis-Acting Replication Element in the Sequence Encoding the NS5B RNA-Dependent RNA Polymerase Is Required for Hepatitis C Virus RNA Replication

Shihyun You,1 Decherd D. Stump,2 Andrea D. Branch,2 and Charles M. Rice1*

Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York 10021,1 Division of Liver Disease, Department of Medicine, Mount Sinai School of Medicine, New York, New York 100292

Received 23 July 2003/ Accepted 10 October 2003

RNA structures play key roles in the replication of RNA viruses. Sequence alignment software, thermodynamic RNA folding programs, and classical comparative phylogenetic analysis were used to build models of six RNA elements in the coding region of the hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B. The importance of five of these elements was evaluated by site-directed mutagenesis of a subgenomic HCV replicon. Mutations disrupting one of the predicted stem-loop structures, designated 5BSL3.2, blocked RNA replication, implicating it as an essential cis-acting replication element (CRE). 5BSL3.2 is about 50 bases in length and is part of a larger predicted cruciform structure (5BSL3). As confirmed by RNA structure probing, 5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop, and an 8-base internal loop. Mutational analysis and structure probing were used to explore the importance of these features. Primary sequences in the loops were shown to be important for HCV RNA replication, and the upper helix appears to serve as an essential scaffold that helps maintain the overall RNA structure. Unlike certain picornavirus CREs, whose function is position independent, 5BSL3.2 function appears to be context dependent. Understanding the role of 5BSL3.2 and determining how this new CRE functions in the context of previously identified elements at the 5' and 3' ends of the RNA genome should provide new insights into HCV RNA replication.

* Corresponding author. Mailing address: Laboratory of Virology and Infectious Disease, Center for the Study of Hepatitis C, The Rockefeller University, 1230 York Ave., New York, NY 10021. Phone: (212) 327-7046. Fax: (212) 327-7048. E-mail: ricec{at}rockefeller.edu.

Journal of Virology, February 2004, p. 1352-1366, Vol. 78, No. 3
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.3.1352-1366.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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