Fidelity variants of RNA dependent RNA polymerases uncover an indirect, mutagenic activity of amiloride compounds.
Fidelity variants of RNA dependent RNA polymerases uncover an indirect, mutagenic activity of amiloride compounds.
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DOI：
10.1371/journal.ppat.1001163
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发表时间：
2010-10-28
期刊：
影响因子：
6.7
通讯作者：
Vignuzzi M
中科院分区：
文献类型：
--
作者：
Levi LI;Gnädig NF;Beaucourt S;McPherson MJ;Baron B;Arnold JJ;Vignuzzi M
在RNA诱变剂抗性筛选中，我们分离到柯萨奇病毒B3(CVB3)的高保真RNA依赖RNA聚合酶(RdRp)变异体。奇怪的是，这种变种A372V也对阿米洛利具有抗药性。我们假设阿米洛利具有以前未被描述的诱变活性。事实上，阿米洛利化合物会增加CVB3和脊髓灰质炎病毒的突变频率，这两种病毒的高保真变体对这种影响更具抵抗力。我们推测，这种突变活性是通过改变细胞内离子，如镁和锰，进而通过影响RdRp保真度来增加病毒突变频率来介导的。此外，我们还证明了另一种抗阿米洛利的RdRp变异体S299T完全抵抗这种突变活性，并且不受离子浓度变化的影响。我们发现RdRp变异体通过两种不同的机制来抵抗阿米洛利的诱变活性：1)增加保真度，产生基础突变频率较低的病毒群体；2)抵抗影响聚合酶保真度的二价阳离子浓度的变化。我们的结果揭示了一种基于突变的新的抗病毒方法。RNA病毒具有极高的突变频率，这在很大程度上是由于复制其基因组的病毒RNA依赖RNA聚合酶(RdRp)的错误性质。由于RdRp缺乏校对和修复机制，使用碱基类似物作为RNA诱变剂来增加致命突变和消灭病毒种群是一种很有前途的抗病毒策略。最近，对这种抗病毒治疗的耐药性筛查发现，脊髓灰质炎病毒的RdRp变种具有更高的保真度，这表明RdRp的保真度可以通过单一氨基酸的替换来调节。为了将这些观察扩展到其他病毒，我们使用柯萨奇病毒B3(CVB3)进行了类似的筛查。我们鉴定了一种新的高保真RdRp变异体，该变异体也对阿米洛利化合物具有抗药性，这些化合物没有已知的诱变活性。使用脊髓灰质炎病毒和CVB3的野生型和RdRp保真度变体，我们证明了阿米洛利化合物确实具有诱变活性，并通过改变影响聚合酶保真度的细胞内离子浓度间接作用于RNA病毒群体。我们的结果确定了一种通过增加突变频率来靶向病毒的新方法，这种方法使用非核苷化合物来改变病毒复制的细胞环境。
In a screen for RNA mutagen resistance, we isolated a high fidelity RNA dependent RNA polymerase (RdRp) variant of Coxsackie virus B3 (CVB3). Curiously, this variant A372V is also resistant to amiloride. We hypothesize that amiloride has a previously undescribed mutagenic activity. Indeed, amiloride compounds increase the mutation frequencies of CVB3 and poliovirus and high fidelity variants of both viruses are more resistant to this effect. We hypothesize that this mutagenic activity is mediated through alterations in intracellular ions such as Mg2+ and Mn2+, which in turn increase virus mutation frequency by affecting RdRp fidelity. Furthermore, we show that another amiloride-resistant RdRp variant, S299T, is completely resistant to this mutagenic activity and unaffected by changes in ion concentrations. We show that RdRp variants resist the mutagenic activity of amiloride via two different mechanisms: 1) increased fidelity that generates virus populations presenting lower basal mutation frequencies or 2) resisting changes in divalent cation concentrations that affect polymerase fidelity. Our results uncover a new antiviral approach based on mutagenesis. RNA viruses have extreme mutation frequencies, due in large part to the erroneous nature of the viral RNA dependent RNA polymerases (RdRp) that replicate their genomes. Since RdRp lack proofreading and repair mechanisms, the use of base analogs as RNA mutagens to increase lethal mutations and extinguish the virus population is a promising antiviral strategy. Recently, a screen for resistance to this antiviral treatment identified a higher fidelity RdRp variant of poliovirus, indicating that RdRp fidelity can be modulated by single amino acid substitutions. To extend these observations to other viruses, we performed a similar screen using Coxsackie virus B3 (CVB3). We identified a new high fidelity RdRp variant which was also resistant to amiloride compounds that have no known mutagenic activity. Using wild type and RdRp fidelity variants of poliovirus and CVB3, we show that amiloride compounds do have mutagenic activity and act on RNA virus populations indirectly, by altering intracellular ion concentrations that affect polymerase fidelity. Our results identify a new means of targeting viruses through increases in mutation frequency using non-nucleoside compounds that alter the cellular environment in which the virus replicates.
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影响因子：
4.8
作者：
Arnold, JJ;Cameron, CE
通讯作者：
Cameron, CE
影响因子：
7.6
作者：
Gazina, EV;Harrison, DN;Petrou, S
通讯作者：
Petrou, S
影响因子：
2.9
作者：
Arnold, JJ;Cameron, CE
通讯作者：
Cameron, CE
DOI：
10.1073/pnas.0608952104
发表时间：
2007-03-13
影响因子：
11.1
作者：
Castro, Christian;Smidansky, Eric;Cameron, Craig E.
通讯作者：
Cameron, Craig E.
影响因子：
2.9
作者：
Arnold, JJ;Gohara, DW;Cameron, CE
通讯作者：
Cameron, CE