Selective stimulation of hepatitis C virus and pestivirus NS5B RNA polymerase activity by GTP

被引:83
作者
Lohmann, V
Overton, H
Bartenschlager, R
机构
[1] Univ Mainz, Inst Virol, D-55131 Mainz, Germany
[2] Roche Prod Ltd, Welwyn Garden City AL7 3AY, Herts, England
关键词
D O I
10.1074/jbc.274.16.10807
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 C;Mg, 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.
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页码:10807 / 10815
页数:9
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