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J. Biol. Chem., Vol. 276, Issue 19, 15581-15591, May 11, 2001
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From the Viral replicases of many positive-strand RNA
viruses are membrane-bound complexes of cellular and viral proteins
that include viral RNA-dependent RNA polymerase (RdRP).
The in vitro RdRP assay system that utilizes cytoplasmic
extracts from dengue viral-infected cells and exogenous RNA templates
was developed to understand the mechanism of viral replication in
vivo. Our results indicated that in vitro RNA
synthesis at the 3'-untranslated region (UTR) required the presence of
the 5'-terminal region (TR) and the two cyclization (CYC) motifs
suggesting a functional interaction between the TRs. In this study,
using a psoralen-UV cross-linking method and an in vitro
RdRP assay, we analyzed structural determinants for physical and
functional interactions. Exogenous RNA templates that were used in the
assays contained deletion mutations in the 5'-TR and substitution
mutations in the 3'-stem-loop structure including those that would
disrupt the predicted pseudoknot structure. Our results indicate that
there is physical interaction between the 5'-TR and 3'-UTR that
requires only the CYC motifs. RNA synthesis at the 3'-UTR, however,
requires long range interactions involving the 5'-UTR, CYC motifs, and
the 3'-stem-loop region that includes the tertiary pseudoknot structure.
In Vitro RNA Synthesis from Exogenous Dengue Viral
RNA Templates Requires Long Range Interactions between 5'- and
3'-Terminal Regions That Influence RNA Structure*
§,
Department of Biochemistry and Molecular
Biology, University of Kansas Medical Center,
Kansas City, Kansas 66160-7421 and ¶ Division of Viral
Products, Center for Biologics Evaluation and Research, Food and Drug
Administration, Rockville, Maryland 20852-1448
*
This research was supported in part by National Institutes
of Health Grants AI-32078 and AI-45623.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, University of Kansas Medical
Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7421. Tel.:
913-588-7018; Fax: 913-588-7440; E-mail: rpadmana@kumc.edu.
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