Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 201 Althouse Laboratory, University Park, PA 16802, USA.
Genes (Basel). 2019 Nov 25;10(12):968. doi: 10.3390/genes10120968.
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.
利用脊髓灰质炎病毒 (PV) 的 RNA 依赖性 RNA 聚合酶 (RdRp) 作为我们的模型系统，我们已经表明， motif-D 中的 Lys-359 在核苷酸转移机制中充当通用酸。K359H (KH) RdRp 衍生物相对于野生型酶较慢但忠实。在 KH 病毒的背景下， RdRp 编码序列进化，选择以下取代：I331F (IF， motif-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都相等。每个都表现出独特的重组表型，IF-KH 能够进行拷贝选择重组，而 PS-KH 能够进行强制拷贝选择重组。尽管 IF-PS-KH RdRp 的生化特性在两倍于野生型，但该病毒在细胞中的重组能力大大受损。我们得出结论，除了速度和保真度之外， RdRp 还有其他生化特性决定了重组的机制和效率。RdRp 的速度、保真度、这里提出的未定义性质的交织性质使得不可能将 RdRp 的单一性质归因于适合度。然而，这里描述的衍生物可能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种群适合度的重要性。
