Vaccine-derived Mutation in Motif D of Poliovirus RNA-dependent RNA Polymerase Lowers Nucleotide Incorporation Fidelity
Vaccine-derived Mutation in Motif D of Poliovirus RNA-dependent RNA Polymerase Lowers Nucleotide Incorporation Fidelity
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DOI：
10.1074/jbc.m113.484428
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发表时间：
2013-11-08
影响因子：
4.8
通讯作者：
Boehr, David D.
中科院分区：
文献类型：
--
作者：
Liu, Xinran;Yang, Xiaorong;Boehr, David D.
背景：脊髓灰质炎病毒 RNA 依赖性 RNA 聚合酶中的基序 D 环对于催化和保真度非常重要。结果：基序 D 中的疫苗衍生突变通过改变基序 D 构象动力学降低了 RdRp 保真度。结论：基序 D 的非保守残基可以改变 RdRp 功能。意义：RdRp 的基序 D 可能是一个通用靶标，允许创建保真度受到干扰的酶和毒力降低的病毒。所有病毒 RNA 依赖性 RNA 聚合酶 (RdRps) 都有一个称为基序 D 的保守结构元件。对脊髓灰质炎病毒 (PV) 的 RdRp 的研究表明，基序 D 的构象变化通过将 Lys-359 引入其充当活性位点，从而导致有效和忠实的核苷酸添加。一般酸。 Sabin I疫苗株的RdRp已将Thr-362改为Ile。如此接近 Lys-359 的剧烈变化可能会改变 RdRp 功能，并在某种程度上导致 Sabin I 型的减毒表型。在这里，我们介绍了 T362I RdRp 的表征。我们发现 T362I RdRp 在体外生化实验中表现出突变表型。使用NMR，我们表明核苷酸掺入保真度的这种变化与基序D的结构动力学的变化相关。表达T362I RdRp的重组PV在细胞培养物中表现出正常的生长特性，但在细胞中表达突变表型。例如，含有T362I的PV比野生型PV对利巴韦林的诱变活性更敏感。有趣的是，T362I 的变化足以导致病毒毒力在统计学上显着降低。总的来说，这些研究表明，当需要改变核苷酸掺入保真度时，可以靶向基序 D 的残基。鉴于保真突变体可以作为候选疫苗的观察结果，有可能为此目的使用基序 D 的工程。
Background: The motif D loop in poliovirus RNA-dependent RNA polymerase is important for catalysis and fidelity. Results: A vaccine-derived mutation in motif D decreases RdRp fidelity by changing motif D conformational dynamics. Conclusion: Non-conserved residues of motif D can alter RdRp function. Significance: Motif D of the RdRp may be a universal target permitting creation of enzymes with perturbed fidelity and viruses with reduced virulence.All viral RNA-dependent RNA polymerases (RdRps) have a conserved structural element termed motif D. Studies of the RdRp from poliovirus (PV) have shown that a conformational change of motif D leads to efficient and faithful nucleotide addition by bringing Lys-359 into the active site where it serves as a general acid. The RdRp of the Sabin I vaccine strain has Thr-362 changed to Ile. Such a drastic change so close to Lys-359 might alter RdRp function and contribute in some way to the attenuated phenotype of Sabin type I. Here we present our characterization of the T362I RdRp. We find that the T362I RdRp exhibits a mutator phenotype in biochemical experiments in vitro. Using NMR, we show that this change in nucleotide incorporation fidelity correlates with a change in the structural dynamics of motif D. A recombinant PV expressing the T362I RdRp exhibits normal growth properties in cell culture but expresses a mutator phenotype in cells. For example, the T362I-containing PV is more sensitive to the mutagenic activity of ribavirin than wild-type PV. Interestingly, the T362I change was sufficient to cause a statistically significant reduction in viral virulence. Collectively, these studies suggest that residues of motif D can be targeted when changes in nucleotide incorporation fidelity are desired. Given the observation that fidelity mutants can serve as vaccine candidates, it may be possible to use engineering of motif D for this purpose.