Department of Plant Pathology, University of Kentucky, Lexington, Kentucky, USA.
J Virol. 2012 Jan;86(1):156-71. doi: 10.1128/JVI.00404-11. Epub 2011 Oct 19.
In addition to its central role as a template for replication and translation, the viral plus-strand RNA genome also has nontemplate functions, such as recruitment to the site of replication and assembly of the viral replicase, activities that are mediated by cis-acting RNA elements within viral genomes. Two noncontiguous RNA elements, RII(+)-SL (located internally in the tombusvirus genome) and RIV (located at the 3'-terminus), are involved in template recruitment into replication and replicase assembly; however, the importance of each of these RNA elements for these two distinct functions is not fully elucidated. We used an in vitro replicase assembly assay based on yeast cell extract and purified recombinant tombusvirus replication proteins to show that RII(+)-SL, in addition to its known requirement for recruitment of the plus-strand RNA into replication, is also necessary for assembly of an active viral replicase complex. Additional studies using a novel two-component RNA system revealed that the recruitment function of RII(+)-SL can be provided in trans by a separate RNA and that the replication silencer element, located within RIV, defines the template that is used for initiation of minus-strand synthesis. Collectively, this work has revealed new functions for tombusvirus cis-acting RNA elements and provided insights into the pioneering round of minus-strand synthesis.
除了作为复制和翻译模板的核心作用外，病毒正链 RNA 基因组还具有非模板功能，例如招募到复制部位和病毒复制酶的组装，这些活动由病毒基因组内的顺式作用 RNA 元件介导。两个不连续的 RNA 元件，RII(+)-SL（位于弹状病毒基因组内部）和 RIV（位于 3'-末端），参与模板招募到复制和复制酶组装；然而，这些 RNA 元件对这两个不同功能的重要性尚未完全阐明。我们使用基于酵母细胞提取物和纯化的重组弹状病毒复制蛋白的体外复制酶组装测定法表明，RII(+)-SL 除了已知需要招募正链 RNA 进入复制外，对于组装活性病毒复制酶复合物也是必需的。使用新型双组分 RNA 系统的进一步研究表明，RII(+)-SL 的招募功能可以通过单独的 RNA 在转位提供，并且位于 RIV 内的复制沉默元件定义了用于起始负链合成的模板。总的来说，这项工作揭示了弹状病毒顺式作用 RNA 元件的新功能，并深入了解了开创性的负链合成。
