Department of Microbiology and Immunology, University of California, San Francisco, California, United States of America.
PLoS Pathog. 2010 Jun 3;6(6):e1000936. doi: 10.1371/journal.ppat.1000936.
RNA structures present throughout RNA virus genomes serve as scaffolds to organize multiple factors involved in the initiation of RNA synthesis. Several of these RNA elements play multiple roles in the RNA replication pathway. An RNA structure formed around the 5'- end of the poliovirus genomic RNA has been implicated in the initiation of both negative- and positive-strand RNA synthesis. Dissecting the roles of these multifunctional elements is usually hindered by the interdependent nature of the viral replication processes and often pleiotropic effects of mutations. Here, we describe a novel approach to examine RNA elements with multiple roles. Our approach relies on the duplication of the RNA structure so that one copy is dedicated to the initiation of negative-strand RNA synthesis, while the other mediates positive-strand synthesis. This allows us to study the function of the element in promoting positive-strand RNA synthesis, independently of its function in negative-strand initiation. Using this approach, we demonstrate that the entire 5'-end RNA structure that forms on the positive-strand is required for initiation of new positive-strand RNAs. Also required to initiate positive-strand RNA synthesis are the binding sites for the viral polymerase precursor, 3CD, and the host factor, PCBP. Furthermore, we identify specific nucleotide sequences within "stem a" that are essential for the initiation of positive-strand RNA synthesis. These findings provide direct evidence for a trans-initiation model, in which binding of proteins to internal sequences of a pre-existing positive-strand RNA affects the synthesis of subsequent copies of that RNA, most likely by organizing replication factors around the initiation site.
RNA 结构存在于 RNA 病毒基因组的各个部位，充当组织与 RNA 合成起始相关的多种因素的支架。其中一些 RNA 元件在 RNA 复制途径中发挥多种作用。在脊髓灰质炎病毒基因组 RNA 的 5' 端形成的 RNA 结构被认为参与了负链和正链 RNA 合成的起始。解析这些多功能元件的作用通常受到病毒复制过程相互依存的性质以及突变的多效性的阻碍。在这里，我们描述了一种检查具有多种作用的 RNA 元件的新方法。我们的方法依赖于 RNA 结构的复制，以便一个副本专门用于负链 RNA 合成的起始，而另一个副本介导正链合成。这使我们能够研究该元件在促进正链 RNA 合成中的功能，而无需考虑其在负链起始中的功能。使用这种方法，我们证明了在正链上形成的整个 5' 端 RNA 结构对于新的正链 RNA 的起始是必需的。启动正链 RNA 合成还需要病毒聚合酶前体 3CD 和宿主因子 PCBP 的结合位点。此外，我们确定了“茎 a”内的特定核苷酸序列对于正链 RNA 合成的起始是必需的。这些发现为转起始模型提供了直接证据，其中蛋白质与预先存在的正链 RNA 的内部序列结合会影响该 RNA 后续拷贝的合成，这很可能通过围绕起始位点组织复制因子来实现。
