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Journal of Virology, November 2000, p. 10312-10322, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Template Nucleotide Moieties Required for De Novo
Initiation of RNA Synthesis by a Recombinant Viral RNA-Dependent
RNA Polymerase
Min-Ju
Kim,1
Weidong
Zhong,2,
Zhi
Hong,2,
and
C. Cheng
Kao1,*
Department of Biology, Indiana University,
Bloomington, Indiana 47405,1 and
Antiviral Therapy, Schering-Plough Research Institute,
Kenilworth, New Jersey 070332
Received 12 May 2000/Accepted 18 August 2000
The recombinant RNA-dependent RNA polymerase of the bovine viral
diarrhea virus specifically requires a cytidylate at the 3' end for the
de novo initiation of RNA synthesis (C. C. Kao, A. M. Del
Vecchio, and W. Zhong, Virology 253:1-7, 1999). Using RNAs containing
nucleotide analogs, we found that the N3 and C4-amino group at the
initiation cytidine were required for RNA synthesis. However, the
ribose C2'-hydroxyl of the initiating cytidylate can accept several
modifications and retain the ability to direct synthesis. The only
unacceptable modification is a protonated C2'-amino group. Quite
strikingly, the recognition of the functional groups for the initiation
cytidylate and other template nucleotides are different. For example, a
C5-methyl group in cytidine can direct RNA synthesis at all template
positions except at the initiation cytidylate and C2'-amino
modifications are tolerated better after the +11 position. When a
4-thiouracil (4sU) base analog that allows only imperfect base pairing
with the nascent RNA is placed at different positions in the template,
the efficiency of synthesis is correlated with the calculated stability
of the template-nascent RNA duplex adjacent to the position of the 4sU.
These results define the requirements for the specific interactions
required for the initiation of RNA synthesis and will be compared to
the mechanisms of initiation by other RNA-dependent and DNA-dependent RNA polymerases.
*
Corresponding author. Mailing address: Department of
Biology, Jordan Hall 138, Indiana University, Bloomington, IN 47405. Phone: (812) 855-7959. Fax: (812) 855-6705. E-mail:
ckao{at}bio.indiana.edu.

Present address: ICN, Costa Mesa, CA
92626.
Journal of Virology, November 2000, p. 10312-10322, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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