Department of Plant Pathology & Microbiology, University of California, Riverside, California, USA.
J Virol. 2012 May;86(9):5204-20. doi: 10.1128/JVI.00351-12. Epub 2012 Feb 22.
Despite overwhelming interest in the impact exerted by recombination during evolution of RNA viruses, the relative contribution of the polarity of inoculum templates remains poorly understood. Here, by agroinfiltrating Nicotiana benthamiana leaves, we show that brome mosaic virus (BMV) replicase is competent to initiate positive-strand [(+)-strand] synthesis on an ectopically expressed RNA3 negative strand [(-) strand] and faithfully complete the replication cycle. Consequently, we sought to examine the role of RNA polarity in BMV recombination by expressing a series of replication-defective mutants of BMV RNA3 in (+) or (-) polarity. Temporal analysis of progeny sequences revealed that the genetic makeup of the primary recombinant pool is determined by the polarity of the inoculum template. When the polarity of the inoculum template was (+), the recombinant pool that accumulated during early phases of replication was a mixture of nonhomologous recombinants. These are longer than the inoculum template length, and a nascent 3' untranslated region (UTR) of wild-type (WT) RNA1 or RNA2 was added to the input mutant RNA3 3' UTR due to end-to-end template switching by BMV replicase during (-)-strand synthesis. In contrast, when the polarity of the inoculum was (-), the progeny contained a pool of native-length homologous recombinants generated by template switching of BMV replicase with a nascent UTR from WT RNA1 or RNA2 during (+)-strand synthesis. Repair of a point mutation caused by polymerase error occurred only when the polarity of the inoculum template was (+). These results contribute to the explanation of the functional role of RNA polarity in recombination mediated by copy choice mechanisms.
尽管人们对重组在 RNA 病毒进化过程中产生的影响非常感兴趣，但接种模板极性的相对贡献仍知之甚少。在这里，通过 agroinfiltration 侵染 Nicotiana benthamiana 叶片，我们表明，雀麦花叶病毒（BMV）复制酶有能力在异位表达的 RNA3 负链 [(-) 链] 上启动正链 [(+) 链] 合成，并准确完成复制周期。因此，我们试图通过在 (+) 或 (-) 极性上表达一系列具有复制缺陷的 BMV RNA3 突变体来研究 RNA 极性在 BMV 重组中的作用。对后代序列的时间分析表明，初级重组库的遗传构成取决于接种模板的极性。当接种模板的极性为 (+) 时，在复制的早期阶段积累的重组库是异源重组体的混合物。这些重组体比接种模板长，并且由于 BMV 复制酶在 (-) 链合成期间进行端到端模板转换，新生的 3'非翻译区 (UTR) 被添加到输入突变 RNA3 的 3'UTR 中。相比之下，当接种模板的极性为 (-) 时，由于 BMV 复制酶在 (+) 链合成期间用 WT RNA1 或 RNA2 的新生 UTR 进行模板转换，产生了一组同源全长重组体。只有当接种模板的极性为 (+) 时，才能发生由聚合酶错误引起的点突变修复。这些结果有助于解释 RNA 极性在由复制选择机制介导的重组中的功能作用。
