使用来自脊髓灰质炎病毒(PV)的RNA依赖性RNA聚合酶(RdRp)作为我们的模型系统,我们已经证明,基序-D中的Lys-359在核苷酸转移机制中起着一般酸的作用。相对于野生型酶,K359H(KH)RdRp衍生物是缓慢且忠实的。在KH病毒的背景下,RdRp编码序列演变,选择以下替换:I331F(IF,基序-C)和P356S(PS,motif-D).我们已经评估了IF-KH,PS-KH,和IF-PS-KH病毒和酶。每个双突变体的速度和保真度是相等的。每个都表现出独特的重组表型,IF-KH能胜任拷贝选择重组,PS-KH能胜任强制拷贝选择重组。尽管IF-PS-KHRdRp具有野生型两倍的生化特性,病毒在细胞中的重组基本上受损。我们得出的结论是,除了速度和保真度外,RdRp还具有生化特性,这些特性决定了重组的机制和效率。速度的交织性质,保真度,这里建议的未定义属性,和重组使得不可能将RdRp的单个属性归因于适合性。然而,这里描述的衍生物可以在恒定的聚合酶速度和保真度的背景下阐明重组对病毒种群适应性的重要性。
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: I331F (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.