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Journal of Virology, October 2004, p. 10865-10877, Vol. 78, No. 20
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.20.10865-10877.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

cis-Acting RNA Signals in the NS5B C-Terminal Coding Sequence of the Hepatitis C Virus Genome

Haekyung Lee, Hyukwoo Shin, Eckard Wimmer, and Aniko V. Paul*

Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York

Received 21 January 2004/ Accepted 4 June 2004

The cis-replicating RNA elements in the 5' and 3' nontranslated regions (NTRs) of the hepatitis C virus (HCV) genome have been thoroughly studied before. However, no cis-replicating elements have been identified in the coding sequences of the HCV polyprotein until very recently. The existence of highly conserved and stable stem-loop structures in the RNA polymerase NS5B coding sequence, however, has been previously predicted (A. Tuplin, J. Wood, D. J. Evans, A. H. Patel, and P. Simmonds, RNA 8:824-841, 2002). We have selected for our studies a 249-nt-long RNA segment in the C-terminal NS5B coding region (NS5BCR), which is predicted to form four stable stem-loop structures (SL-IV to SL-VII). By deletion and mutational analyses of the RNA structures, we have determined that two of the stem-loops (SL-V and SL-VI) are essential for replication of the HCV subgenomic replicon in Huh-7 cells. Mutations in the loop and the top of the stem of these RNA elements abolished replicon RNA synthesis but had no effect on translation. In vitro gel shift and filter-binding assays revealed that purified NS5B specifically binds to SL-V. The NS5B-RNA complexes were specifically competed away by unlabeled homologous RNA, to a small extent by 3' NTR RNA, and only poorly by 5' NTR RNA. The other two stem-loops (SL-IV and SL-VII) of the NS5BCR domain were found to be important but not essential for colony formation by the subgenomic replicon. The precise function(s) of these cis-acting RNA elements is not known.

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794. Phone: (631) 632-9777. Fax: (631) 632-8891. E-mail: apaul{at}notes.cc.sunysb.edu.

Journal of Virology, October 2004, p. 10865-10877, Vol. 78, No. 20
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.20.10865-10877.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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