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J Biol Chem, Vol. 274, Issue 16, 10807-10815, April 16, 1999
,
From the NS5B of the hepatitis C virus is an RNA
template-dependent RNA polymerase and therefore the key
player of the viral replicase complex. Using a highly purified enzyme
expressed with recombinant baculoviruses in insect cells, we
demonstrate a stimulation of RNA synthesis up to 2 orders of magnitude
by high concentrations of GTP but not with ATP, CTP, UTP, GDP, or GMP.
Enhancement of RNA synthesis was found with various heteropolymeric RNA
templates, with poly(C)-oligo(G)12 but not with
poly(A)-oligo(U)12. Several amino acid substitutions in
polymerase motifs B, C, and D previously shown to be crucial for RdRp
activity were tested for GTP stimulation of RNA synthesis. Most of
these mutations, in particular those affecting the GDD motif (motif C)
strongly reduced or completely abolished activation by GTP, suggesting
that the same NTP-binding site is used for stimulation and RNA
synthesis. Since GTP did not affect the overall RNA binding properties
or the elongation rate, high concentrations of GTP appear to accelerate
a rate-limiting step at the level of initiation of RNA synthesis.
Finally, enhancement of RNA synthesis by high GTP concentrations was
also found with NS5B of the pestivirus classical swine fever virus, but
not with the 3D polymerase of poliovirus. Thus, stimulation of RdRp
activity by GTP is evolutionarily conserved between the closely related hepaciviruses and pestiviruses but not between these and the more distantly related picornaviruses.
Institute for Virology, Johannes-Gutenberg
University Mainz, Obere Zahlbacher Straße 67, 55131 Mainz, Germany
and § Roche Products Ltd., Welwyn Garden City,
Hertfordshire AL7 3AY, United Kingdom
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