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Journal of Virology, September 2001, p. 8615-8623, Vol. 75, No. 18
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.18.8615-8623.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Terminal Nucleotidyl Transferase Activity of Recombinant Flaviviridae RNA-Dependent RNA Polymerases: Implication for Viral RNA Synthesis

C. T. Ranjith-Kumar,1 J. Gajewski,1 L. Gutshall,2 D. Maley,2 R. T. Sarisky,2 and C. Cheng Kao1,*

Department of Biology, Indiana University, Bloomington, Indiana 47405,1 and Department of Host Defense, The Antimicrobial and Host Defense Center of Excellence for Drug Discovery, GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania 194262

Received 22 March 2001/Accepted 14 June 2001

Recombinant hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was reported to possess terminal transferase (TNTase) activity, the ability to add nontemplated nucleotides to the 3' end of viral RNAs. However, this TNTase was later purported to be a cellular enzyme copurifying with the HCV RdRp. In this report, we present evidence that TNTase activity is an inherent function of HCV and bovine viral diarrhea virus RdRps highly purified from both prokaryotic and eukaryotic cells. A change of the highly conserved GDD catalytic motif in the HCV RdRp to GAA abolished both RNA synthesis and TNTase activity. Furthermore, the nucleotides added via this TNTase activity are strongly influenced by the sequence near the 3' terminus of the viral template RNA, perhaps accounting for the previous discrepant observations between RdRp preparations. Last, the RdRp TNTase activity was shown to restore the ability to direct initiation of RNA synthesis in vitro on an initiation-defective RNA substrate, thereby implicating this activity in maintaining the integrity of the viral genome termini.

* Corresponding author. Mailing address: Department of Biology, Indiana University, 1001 E. Third St., Bloomington, IN 47405. Phone: (812) 855-7959. Fax: (812) 855-6705. E-mail: ckao{at}bio.indiana.edu.

Journal of Virology, September 2001, p. 8615-8623, Vol. 75, No. 18
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.18.8615-8623.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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