Kim K H, Lommel S A
Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina, 27695-7616, USA.
Virology. 1998 Oct 10;250(1):50-9. doi: 10.1006/viro.1998.9358.
The RNA-1 of the bipartite red clover necrotic mosaic dianthovirus (RCNMV) genome encodes the 88-kDa polymerase. The polymerase is translated from both 5' proximal and internal open reading frames by a -1 ribosomal frameshifting event. A shifty heptanucleotide conforming to the simultaneous slippage model is identified, and a downstream stem-loop structure and atypical pseudoknot are predicted. A beta-glucuronidase reporter assay identified a 118-nucleotide element containing both the shifty heptanucleotide and the predicted secondary structures that were required for efficient -1 ribosomal frameshift expression in vivo. A series of site-directed and compensatory mutations affecting the base-paired regions of the predicted secondary structure were introduced into a RCNMV RNA-1 cDNA clone from which infectious transcripts were derived. Mutations that destroyed the predicted pseudoknot had no effect on frameshifting efficiency in vitro or infectivity of the virus, whereas mutations destabilizing the stem-loop structure abolished both ribosomal frameshifting in vitro and biological activity. These results demonstrate the essential role of a predicted secondary structure that does not involve a pseudoknot in the expression of the RCNMV polymerase by ribosomal frameshifting.
二分体红三叶草坏死花叶病毒（RCNMV）基因组的RNA-1编码88 kDa的聚合酶。该聚合酶通过-1核糖体移码事件从5'近端和内部开放阅读框进行翻译。确定了符合同时滑移模型的一个移位七核苷酸，并预测了下游茎环结构和非典型假结。β-葡萄糖醛酸酶报告基因检测鉴定出一个118核苷酸元件，其包含移位七核苷酸和预测的二级结构，这些是体内有效-1核糖体移码表达所必需的。一系列影响预测二级结构碱基配对区域的定点突变和补偿性突变被引入到一个RCNMV RNA-1 cDNA克隆中，从中获得感染性转录本。破坏预测假结的突变对体外移码效率或病毒感染性没有影响，而使茎环结构不稳定的突变则消除了体外核糖体移码和生物活性。这些结果证明了一种不涉及假结的预测二级结构在RCNMV聚合酶通过核糖体移码表达中的重要作用。
