Structure-function relationships of the viral RNA-dependent RNA polymerase - Fidelity, replication speed, and initiation mechanism determined by a residue in the ribose-binding pocket
Structure-function relationships of the viral RNA-dependent RNA polymerase - Fidelity, replication speed, and initiation mechanism determined by a residue in the ribose-binding pocket
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DOI：
10.1074/jbc.m610090200
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发表时间：
2007-06-01
影响因子：
4.8
通讯作者：
Cameron, Craig E.
中科院分区：
文献类型：
--
作者：
Korneeva, Victoria S.;Cameron, Craig E.
对脊髓灰质炎病毒（PV）的RNA依赖性RNA聚合酶（RdRp） 3Dpol的研究表明，Asn-297允许这种酶区分核糖和2'-脱氧核糖。所有动物RNA病毒在其聚合酶的结构同源位置上都有Asn，表明该残基具有保守功能。然而，所有的原核RNA病毒在这个位置上都有Glu。在Mg2+存在下，与野生型相比，Glu-297 3Dpol对2’-脱氧核糖核苷酸的表观亲和力降低了6倍，但对2’-dNMP结合的保真度没有显著差异。与野生型相比，Glu-297 3Dpol的核糖核苷酸错误掺入保真度降低了14倍。观察到核糖核苷酸掺入率降低了4至11倍。Glu-297 PV无法在HeLa细胞中生长，原因是其复制缺陷相当于在编码失活聚合酶的突变PV中观察到的缺陷。对蛋白（VPg）引发起始反应的评价表明，只有一半的Glu-297 3Dpol起始复合物能够产生VPg- pupu产物，并且与野生型酶相比，尿苷化VPg产物的总产量降低了20倍，这是由于对UTP的亲和力降低所致。这些研究确定了第一个具有突变表型的RdRp衍生物，并为在培养中观察到的RNA噬菌体相对于动物RNA病毒的突变频率升高提供了机制基础。虽然蛋白质引发的起始复合物和rna引发的延伸复合物使用相同的聚合酶活性位点，但这里报道的功能差异意味着这些复合物之间存在显著的结构差异。
Studies of the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV), 3Dpol, have shown that Asn-297 permits this enzyme to distinguish ribose from 2'-deoxyribose. All animal RNA viruses have Asn at the structurally homologous position of their polymerases, suggesting a conserved function for this residue. However, all prokaryotic RNA viruses have Glu at this position. In the presence of Mg2+, the apparent affinity of Glu-297 3Dpol for 2'-deoxyribonucleotides was decreased by 6-fold relative to wild type without a substantial difference in the fidelity of 2'-dNMP incorporation. The fidelity of ribonucleotide misincorporation for Glu-297 3Dpol was reduced by 14-fold relative to wild type. A 4- to 11-fold reduction in the rate of ribonucleotide incorporation was observed. Glu-297 PV was unable to grow in HeLa cells due to a replication defect equivalent to that observed for a mutant PV encoding an inactive polymerase. Evaluation of the protein-(VPg)-primed initiation reaction showed that only half of the Glu-297 3Dpol initiation complexes were capable of producing VPg-pUpU product and that the overall yield of uridylylated VPg products was reduced by 20-fold relative to wild-type enzyme, a circumstance attributable to a reduced affinity for UTP. These studies identify the first RdRp derivative with a mutator phenotype and provide a mechanistic basis for the elevated mutation frequency of RNA phage relative to animal RNA viruses observed in culture. Although protein-primed initiation and RNA-primed elongation complexes employ the same polymerase active site, the functional differences reported here imply significant structural differences between these complexes.