Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
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DOI：
10.3791/2953
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发表时间：
2011-06-01
影响因子：
1.2
通讯作者：
Vignuzzi, Marco
中科院分区：
文献类型：
--
作者：
Beaucourt, Stephanie;Bordera, Antonio V.;Vignuzzi, Marco
RNA病毒使用依赖RNA的RNA聚合酶来复制它们的基因组。这些酶固有的高错误率是产生极端种群多样性的一个重要因素，这种多样性促进了病毒的适应和进化。越来越多的证据表明，RNA病毒的内在错误率和由此产生的突变频率可以通过病毒聚合酶的细微氨基酸变化来调节。尽管存在一些允许定量测量整合保真度的病毒RNA聚合酶的生化分析，但在这里，我们描述了一种测量RNA病毒突变频率的简单方法，该方法已被证明在识别保真度改变突变方面与生化方法一样准确。该方法使用传统的病毒学和测序技术，可在大多数生物实验室进行。根据我们对许多不同病毒的经验，我们确定了必须优化的关键步骤，以增加分离保真度变体和生成统计显著性数据的可能性。保真度改变突变的分离和表征可以为聚合酶的结构和功能提供新的见解（1-3）。此外，这些保真度变异可以成为表征病毒适应和进化机制的有用工具（4-7）。
RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function(1-3). Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution(4-7).