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Journal of Virology, February 2000, p. 2017-2022, Vol. 74, No. 4
Department of Antiviral Therapy,
Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539
Received 14 July 1999/Accepted 18 November 1999
RNA-dependent RNA polymerase (RdRp) encoded by positive-strand RNA
viruses is critical to the replication of viral RNA genome. Like other
positive-strand RNA viruses, replication of hepatitis C virus (HCV) RNA
is mediated through a negative-strand intermediate, which is generated
through copying the positive-strand genomic RNA. Although it has been
demonstrated that HCV NS5B alone can direct RNA replication through a
copy-back primer at the 3' end, de novo initiation of RNA synthesis is
likely to be the mode of RNA replication in infected cells. In this
study, we demonstrate that a recombinant HCV NS5B protein has the
ability to initiate de novo RNA synthesis in vitro. The NS5B used HCV
3' X-tail RNA (98 nucleotides) as the template to synthesize an RNA
product of monomer size, which can be labeled by
[
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
De Novo Initiation of RNA Synthesis by Hepatitis C
Virus Nonstructural Protein 5B Polymerase
-32P]nucleoside triphosphate. The de novo initiation
activity was further confirmed by using small synthetic RNAs ending
with dideoxynucleotides at the 3' termini. In addition, HCV NS5B
preferred GTP as the initiation nucleotide. The optimal conditions for
the de novo initiation activity have been determined. Identification
and characterization of the de novo priming or initiation activity by
HCV NS5B provides an opportunity to screen for inhibitors that
specifically target the initiation step.
*
Corresponding author. Mailing address: Department of
Antiviral Therapy, K-15-4/4945, Schering-Plough Research Institute,
2015 Galloping Hill Rd., Kenilworth, NJ 07033-0539. Phone: (908)
740-3025. Fax: (908) 740-3032. E-mail:
weidong.zhong{at}spcorp.com.
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