Sequence-Specific Fidelity Alterations Associated with West Nile Virus Attenuation in Mosquitoes.
Sequence-Specific Fidelity Alterations Associated with West Nile Virus Attenuation in Mosquitoes.
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DOI：
10.1371/journal.ppat.1005009
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发表时间：
2015-06
期刊：
影响因子：
6.7
通讯作者：
Ciota AT
中科院分区：
文献类型：
--
作者：
Van Slyke GA;Arnold JJ;Lugo AJ;Griesemer SB;Moustafa IM;Kramer LD;Cameron CE;Ciota AT
容易出错的高复制率导致RNA病毒的遗传多样性迅速积累。最近的研究表明，选择变异率是为了获得最佳的病毒适合度，复制酶保真度的轻微变化可能与病毒衰减有关。节肢动物传播的病毒(虫媒病毒)对寄主循环的要求是独特的，可能需要大量的遗传和表型可塑性。为了更深入地研究虫媒病毒保真度的相关性、机制和后果，我们选择了西尼罗河病毒(西尼罗河病毒；黄病毒科，黄病毒)的保真度变异体，在诱变剂存在下进行选择。我们发现了与保真度增加相关的两个西尼罗河病毒RNA依赖RNA聚合酶突变V793I和G806R，以及与保真度降低相关的西尼罗河病毒甲基转移酶T248I的一个突变。深度测序和体外生化分析都证实了不同菌株在保真度和突变偏差方面的差异。西尼罗河病毒保真度变异株在体外表现出宿主特定的复制适合性变化，在蚊子细胞培养中有轻微的衰减，但在脊椎动物细胞培养中没有。中华按蚊定植种群和野外种群的实验感染Ququiefaciatus证明，西尼罗河病毒的保真度变化与在蚊子中建立存活感染的能力显著受损有关。综上所述，这些研究(I)证明了变构相互作用在调节突变率方面的重要性，(Ii)确立了突变谱可以是序列和菌株相关的，以及(Iii)展示了与黄病毒复制复杂功能改变相关的深刻的表型后果。西尼罗河病毒(WNV)是世界上分布最广的节肢动物传播病毒(虫媒病毒)。像大多数虫媒病毒一样，西尼罗河病毒是一种高度变异的RNA病毒，在自然界中以遗传多样性的突变群存在。虽然最近的许多研究已经调查了病毒突变率和病毒适合性之间的关系，但以前还没有对西尼罗河病毒或其他黄病毒确定这一关系。我们通过在诱变剂存在下的细胞培养传代鉴定了与突变率变异相关的西尼罗河病毒突变，并将这些突变改造成具有感染性的西尼罗河病毒克隆，以调查保真度变化的原因和后果。我们的结果表明，组成西尼罗河病毒复制复合体的蛋白质之间的相互作用可以显著改变发生突变的程度和类型。此外，我们还发现，西尼罗河病毒保真度的增加和降低都对细胞培养中的复制有宿主特异性的影响，并与几乎完全消除西尼罗河病毒在蚊媒中的感染有关。这些结果对我们了解蚊虫虫媒病毒的进化、复制复合体功能和虫媒病毒适合性具有重要意义，并为研究媒介能力和虫媒病毒保真度的决定因素和机制提供了重要的靶点。
High rates of error-prone replication result in the rapid accumulation of genetic diversity of RNA viruses. Recent studies suggest that mutation rates are selected for optimal viral fitness and that modest variations in replicase fidelity may be associated with viral attenuation. Arthropod-borne viruses (arboviruses) are unique in their requirement for host cycling and may necessitate substantial genetic and phenotypic plasticity. In order to more thoroughly investigate the correlates, mechanisms and consequences of arbovirus fidelity, we selected fidelity variants of West Nile virus (WNV; Flaviviridae, Flavivirus) utilizing selection in the presence of a mutagen. We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity. Both deep-sequencing and in vitro biochemical assays confirmed strain-specific differences in both fidelity and mutational bias. WNV fidelity variants demonstrated host-specific alterations to replicative fitness in vitro, with modest attenuation in mosquito but not vertebrate cell culture. Experimental infections of colonized and field populations of Cx. quinquefaciatus demonstrated that WNV fidelity alterations are associated with a significantly impaired capacity to establish viable infections in mosquitoes. Taken together, these studies (i) demonstrate the importance of allosteric interactions in regulating mutation rates, (ii) establish that mutational spectra can be both sequence and strain-dependent, and (iii) display the profound phenotypic consequences associated with altered replication complex function of flaviviruses. West Nile virus (WNV) is the most geographically widespread arthropod-borne virus (arbovirus) in the world. Like most arboviruses, WNV is a RNA virus which is highly mutable and exists in nature as genetically diverse mutant swarms. Although many recent studies have investigated the relationship between virus mutation rate and viral fitness, this had not previously been determined for WNV or other flaviviruses. We identified WNV mutations associated with variation in mutation rate using cell culture passage in the presence of a mutagen and engineered these mutations into an infectious WNV clone in order to investigate the causes and consequences of altered fidelity. Our results demonstrate that interactions among proteins which comprise the WNV replication complex can significantly alter both the extent and types of mutations that occur. In addition, we show that both increasing and decreasing WNV fidelity has host-specific effects on replication in cell culture and is associated with nearly complete ablation of WNV infection in mosquito vectors. These results have significant implications for our understanding of arbovirus evolution, replication complex function and arboviral fitness in mosquitoes, and identify important targets to study the determinants and mechanisms of vector competence and arbovirus fidelity.
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影响因子：
6.8
作者：
Cameron, Craig E.;Moustafa, Ibrahim M.;Arnold, Jamie J.
通讯作者：
Arnold, Jamie J.
影响因子：
3.8
作者：
Ciota, Alexander T.;Ngo, Kiet A.;Kramer, Laura D.
通讯作者：
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DOI：
10.1073/pnas.1111650108
发表时间：
2011-09-20
影响因子：
11.1
作者：
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通讯作者：
Vignuzzi, Marco
影响因子：
5.4
作者：
Bressanelli, S;Tomei, L;De Francesco, R
通讯作者：
De Francesco, R
影响因子：
3.8
作者：
Ciota, Alexander T.;Payne, Anne F.;Kramer, Laura D.
通讯作者：
Kramer, Laura D.