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Journal of Virology, December 2000, p. 11121-11128, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Template Requirements for RNA Synthesis by a Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase

C. Cheng Kao,1 Xueyong Yang,2 Allen Kline,2 Q. May Wang,3 Donna Barket,3 and Beverly A. Heinz3,*

Department of Biology, Indiana University, Bloomington, Indiana 47405,1 and Structural Biology Division2 and Infectious Disease Division,3 Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285

Received 8 June 2000/Accepted 26 August 2000

The RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV), nonstructural protein 5B (NS5B), has recently been shown to direct de novo initiation using a number of complex RNA templates. In this study, we analyzed the features in simple RNA templates that are required to direct de novo initiation of RNA synthesis by HCV NS5B. NS5B was found to protect RNA fragments of 8 to 10 nucleotides (nt) from RNase digestion. However, NS5B could not direct RNA synthesis unless the template contained a stable secondary structure and a single-stranded sequence that contained at least one 3' cytidylate. The structure of a 25-nt template, named SLD3, was determined by nuclear magnetic resonance spectroscopy to contain an 8-bp stem and a 6-nt single-stranded sequence. Systematic analysis of changes in SLD3 revealed which features in the stem, loop, and 3' single-stranded sequence were required for efficient RNA synthesis. Also, chimeric molecules composed of DNA and RNA demonstrated that a DNA molecule containing a 3'-terminal ribocytidylate was able to direct RNA synthesis as efficiently as a sequence composed entirely of RNA. These results define the template sequence and structure sufficient to direct the de novo initiation of RNA synthesis by HCV RdRp.

* Corresponding author. Mailing address: Infectious Disease Division, Eli Lilly and Company, Indianapolis, IN 46285. Phone: (317) 276-6911. Fax: (317) 276-1743. E-mail: HEINZ_BEVERLY_A{at}Lilly.com.

Journal of Virology, December 2000, p. 11121-11128, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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