Biochemical characterization of enzyme fidelity of influenza A virus RNA polymerase complex.
Biochemical characterization of enzyme fidelity of influenza A virus RNA polymerase complex.
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DOI：
10.1371/journal.pone.0010372
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发表时间：
2010-04-29
期刊：
影响因子：
3.7
通讯作者：
Kim B
中科院分区：
文献类型：
--
作者：
Aggarwal S;Bradel-Tretheway B;Takimoto T;Dewhurst S;Kim B
人们普遍认为，甲型流感病毒（IAV）的高度易错复制过程，以及病毒基因组的分类，促进了IAV的有效进化能力。因此，从逻辑上假设负责病毒RNA复制过程的酶，A型流感病毒RNA聚合酶（IAV Pol），是一种高度易错的聚合酶，其提供病毒进化和宿主适应所必需的基因组突变。然而，重要的是，IAV RNA聚合酶的实际酶保真度从未被表征。在这里，我们建立了新的生化检测条件，使我们能够评估聚合酶活性与生理NTP池和酶保真度的IAV波尔。我们报告说，IAV波尔显示高度活跃的RNA依赖性RNA聚合酶活性在无偏见的生理NTP底物浓度。有了这种强大的酶活性，我们第一次能够比较IAV Pol复合物的酶保真度与细菌噬菌体T7 RNA聚合酶和人类免疫缺陷病毒（HIV-1）和小鼠白血病病毒（MuLV）的逆转录酶（RT），这是已知的低和高保真酶，分别。我们观察到，IAV Pol显示显着更高的保真度比HIV-1 RT和T7 RNA聚合酶和同等或更高的保真度比MuLV RT。此外，IAV Pol复合物显示在较低的温度下保真度增加。此外，在用Mn++替换Mg++后，IAV Pol显示聚合酶活性增加，但持续合成能力显著降低，并且在Mn++存在下错误掺入略微升高。最后，当IAV核蛋白（NP）被包括在反应中，IAV Pol复合物表现出增强的聚合酶活性与增加的保真度。我们的研究表明IAV Pol是一种高保真酶。我们设想IAV Pol的高保真性质对于平衡每个感染周期的多轮IAV基因组扩增可能是重要的，这为IAV Pol提供了产生和扩增基因组创始突变的充足机会，从而实现其进化的最佳病毒诱变。
It is widely accepted that the highly error prone replication process of influenza A virus (IAV), together with viral genome assortment, facilitates the efficient evolutionary capacity of IAV. Therefore, it has been logically assumed that the enzyme responsible for viral RNA replication process, influenza virus type A RNA polymerase (IAV Pol), is a highly error-prone polymerase which provides the genomic mutations necessary for viral evolution and host adaptation. Importantly, however, the actual enzyme fidelity of IAV RNA polymerase has never been characterized. Here we established new biochemical assay conditions that enabled us to assess both polymerase activity with physiological NTP pools and enzyme fidelity of IAV Pol. We report that IAV Pol displays highly active RNA-dependent RNA polymerase activity at unbiased physiological NTP substrate concentrations. With this robust enzyme activity, for the first time, we were able to compare the enzyme fidelity of IAV Pol complex with that of bacterial phage T7 RNA polymerase and the reverse transcriptases (RT) of human immunodeficiency virus (HIV-1) and murine leukemia virus (MuLV), which are known to be low and high fidelity enzymes, respectively. We observed that IAV Pol displayed significantly higher fidelity than HIV-1 RT and T7 RNA polymerase and equivalent or higher fidelity than MuLV RT. In addition, the IAV Pol complex showed increased fidelity at lower temperatures. Moreover, upon replacement of Mg++ with Mn++, IAV Pol displayed increased polymerase activity, but with significantly reduced processivity, and misincorporation was slightly elevated in the presence of Mn++. Finally, when the IAV nucleoprotein (NP) was included in the reactions, the IAV Pol complex exhibited enhanced polymerase activity with increased fidelity. Our study indicates that IAV Pol is a high fidelity enzyme. We envision that the high fidelity nature of IAV Pol may be important to counter-balance the multiple rounds of IAV genome amplification per infection cycle, which provides IAV Pol with ample opportunities to generate and amplify genomic founder mutations, and thus achieve optimal viral mutagenesis for its evolution.
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