RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination
RNA-Dependent RNA Polymerase Speed and Fidelity are not the Only Determinants of the Mechanism or Efficiency of Recombination
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DOI：
10.3390/genes10120968
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发表时间：
2019-12-01
期刊：
影响因子：
3.5
通讯作者：
Cameron, Craig E.
中科院分区：
文献类型：
--
作者：
Kim, Hyejeong;Ellis, Victor D., III;Cameron, Craig E.
以脊髓灰质炎病毒（PV）的RNA依赖性RNA聚合酶（RdRp）为模型系统，我们证明了D基序中的Lys-359在核苷酸转移机制中起着普通酸的作用。K359 H（KH）RdRp衍生物相对于野生型酶是缓慢且忠实的。在KH病毒的背景下，RdRp编码序列进化，选择以下取代：1331 F（IF，基序-C）和P356 S（PS，基序-D）。我们已经评估了IF-KH、PS-KH和IF-PS-KH病毒和酶。每一个双重突变体的速度和保真度都是相等的。每一种都表现出独特的重组表型，IF-KH能够进行拷贝选择重组，PS-KH能够进行强制拷贝选择重组。尽管IF-PS-KH RdRp表现出两倍于野生型的生化特性，但病毒在细胞中的重组受到实质性损害。我们的结论是，有生化特性的RdRp除了速度和保真度，确定重组的机制和效率。速度、保真度、这里提出的未定义属性以及重组的交织性质使得不可能将RdRp的单一属性归因于适应性。然而，这里描述的衍生物可以允许在恒定聚合酶速度和保真度的背景下阐明重组对病毒群体适应性的重要性。
Using the RNA-dependent RNA polymerase (RdRp) from poliovirus (PV) as our model system, we have shown that Lys-359 in motif-D functions as a general acid in the mechanism of nucleotidyl transfer. A K359H (KH) RdRp derivative is slow and faithful relative to wild-type enzyme. In the context of the KH virus, RdRp-coding sequence evolves, selecting for the following substitutions: 1331F (IF, motif-C) and P356S (PS, motif-D). We have evaluated IF-KH, PS-KH, and IF-PS-KH viruses and enzymes. The speed and fidelity of each double mutant are equivalent. Each exhibits a unique recombination phenotype, with IF-KH being competent for copy-choice recombination and PS-KH being competent for forced-copy-choice recombination. Although the IF-PS-KH RdRp exhibits biochemical properties within twofold of wild type, the virus is impaired substantially for recombination in cells. We conclude that there are biochemical properties of the RdRp in addition to speed and fidelity that determine the mechanism and efficiency of recombination. The interwoven nature of speed, fidelity, the undefined property suggested here, and recombination makes it impossible to attribute a single property of the RdRp to fitness. However, the derivatives described here may permit elucidation of the importance of recombination on the fitness of the viral population in a background of constant polymerase speed and fidelity.