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J. Biol. Chem., Vol. 276, Issue 43, 39926-39937, October 26, 2001
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and
From the Department of Biochemistry and Molecular Biology,
University of Kansas Medical Center,
Kansas City, Kansas 66160-7421
Replication of positive strand flaviviruses is
mediated by the viral RNA-dependent RNA polymerases (RdRP).
To study replication of dengue virus (DEN), a flavivirus family member,
an in vitro RdRP assay was established using cytoplasmic
extracts of DEN-infected mosquito cells and viral subgenomic RNA
templates containing 5'- and 3'-terminal regions (TRs). Evidence
supported that an interaction between the TRs containing conserved
stem-loop, cyclization motifs, and pseudoknot structural elements is
required for RNA synthesis. Two RNA products, a template size and a
hairpin, twice that of the template, were formed. To isolate the
function of the viral RdRP (NS5) from that of other host or viral
factors present in the cytoplasmic extracts, the NS5 protein was
expressed and purified from Escherichia coli. In this
study, we show that the purified NS5 alone is sufficient for the
synthesis of the two products and that the template-length RNA is the
product of de novo initiation. Furthermore, the incubation
temperature during initiation, but not elongation phase of RNA
synthesis modulates the relative amounts of the hairpin and de
novo RNA products. A model is proposed that a specific
conformation of the viral polymerase and/or structure at the 3'
end of the template RNA is required for de novo initiation.
Supported by a Madison and Lila Self graduate fellowship.
§
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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