A Drosophila toolkit for the visualization and quantification of viral replication launched from transgenic genomes.
A Drosophila toolkit for the visualization and quantification of viral replication launched from transgenic genomes.
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DOI：
10.1371/journal.pone.0112092
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Clandinin TR
中科院分区：
文献类型：
--
作者：
Wernet MF;Klovstad M;Clandinin TR
节肢动物RNA病毒对人类健康构成严重威胁，但其复制周期的许多方面仍未完全了解。在这里，我们描述了来自Sindbis病毒的转基因、自我复制基因组（“复制子”）的多功能果蝇工具包，可以快速可视化和量化病毒在体内的复制。我们生成了表达荧光素酶的复制子，用于病毒复制的量化，作为大规模遗传筛选的有用新工具，用于识别影响病毒复制的细胞途径。我们还提出了一种新的二元系统，其中复制缺陷病毒基因组可以“反式”激活，通过共同表达一个完整的复制子来促进RNA依赖的RNA聚合酶。当在同一细胞启动子控制下共表达不同荧光蛋白时，观察到表达不同荧光蛋白的复制子之间的随机排斥现象，证明了该工具在研究病毒生物学方面的实用性。这个过程类似于细胞培养中病毒颗粒之间的“重复感染排斥”，这是一个尚未完全了解的过程。我们表明，病毒聚合酶强烈倾向于复制编码它们的基因组，并且几乎总是只有一个病毒基因组被随机选择在每个细胞中进行复制。我们的体内系统现在使这一过程适合于详细的基因解剖。因此，该工具包允许在遗传模式生物中对病毒复制进行细胞类型特异性定量研究，为病毒生物学的分子、遗传和药理学解剖和工具开发开辟了新的途径。
Arthropod RNA viruses pose a serious threat to human health, yet many aspects of their replication cycle remain incompletely understood. Here we describe a versatile Drosophila toolkit of transgenic, self-replicating genomes (‘replicons’) from Sindbis virus that allow rapid visualization and quantification of viral replication in vivo. We generated replicons expressing Luciferase for the quantification of viral replication, serving as useful new tools for large-scale genetic screens for identifying cellular pathways that influence viral replication. We also present a new binary system in which replication-deficient viral genomes can be activated ‘in trans’, through co-expression of an intact replicon contributing an RNA-dependent RNA polymerase. The utility of this toolkit for studying virus biology is demonstrated by the observation of stochastic exclusion between replicons expressing different fluorescent proteins, when co-expressed under control of the same cellular promoter. This process is analogous to ‘superinfection exclusion’ between virus particles in cell culture, a process that is incompletely understood. We show that viral polymerases strongly prefer to replicate the genome that encoded them, and that almost invariably only a single virus genome is stochastically chosen for replication in each cell. Our in vivo system now makes this process amenable to detailed genetic dissection. Thus, this toolkit allows the cell-type specific, quantitative study of viral replication in a genetic model organism, opening new avenues for molecular, genetic and pharmacological dissection of virus biology and tool development.
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影响因子：
3.7
作者：
Costa A;Jan E;Sarnow P;Schneider D
通讯作者：
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影响因子：
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作者：
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DOI：
10.1073/pnas.92.10.4477
发表时间：
1995-05-09
影响因子：
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通讯作者：
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通讯作者：
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