Boccard F, Baulcombe D
Sainsbury Laboratory, Norwich Research Park, United Kingdom.
Virology. 1993 Apr;193(2):563-78. doi: 10.1006/viro.1993.1165.
RNA3 of the Kin strain of the tripartite (+)-strand cucumber mosaic virus has 2199 nucleotide residues. Two open reading frames encoding 3a protein (a putative movement protein) and coat protein (CP) are separated by a 286-nucleotide inter-cistronic region (IR). This IR contains a subgenomic promoter for production of a subgenomic RNA (RNA4), from which CP is synthesized. Using transcripts generated from mutant forms of a cDNA clone of RNA3, we have characterized the cis-acting sequences necessary for RNA3 accumulation and RNA4 synthesis and analyzed the role of the RNA3-encoded proteins. Efficient accumulation of RNA3 derivatives in tobacco protoplasts required 92 nucleotides at the 5' end, 250 nucleotides in the IR, and 275 nucleotides at the 3' end of the RNA molecule. The 250-nucleotide IR includes a 90-nucleotide sequence which is necessary for subgenomic promoter activity. Although common regions are involved in RNA3 accumulation and RNA4 synthesis, the modes of action of IR for these two phenomena are different. The analysis of forms of RNA3 with internal duplications demonstrated that RNA3 accumulation depended on the context of the IR. Subgenomic promoter activity was more position dependent and was always stronger from the promoter closer to the 3' end of the (+)-strand RNA. A mutation in IR specifically affected (+)-strand RNA accumulation, indicating a role for that region in (+)-strand synthesis. The role of the RNA3-encoded proteins was analyzed by mutation and inoculation either to plants or to protoplasts. Mutation of the 3a protein had no effect on RNA3 accumulation in protoplasts, whereas CP mutations caused reduced CMV RNA accumulation. This reduction was more pronounced for (+)- than for (-)-strand accumulation. RNA of CP mutations was undetectable in inoculated leaves, whereas RNA of 3a protein mutants accumulated, albeit at levels several orders of magnitude lower than with wild-type CMV. The conclusion from these data is that both proteins are required for efficient spread of CMV from the site of infection.
三分体正义黄瓜花叶病毒金氏株系的RNA3含有2199个核苷酸残基。两个编码3a蛋白（一种假定的运动蛋白）和外壳蛋白（CP）的开放阅读框被一个286个核苷酸的顺反子间区域（IR）隔开。这个IR包含一个用于产生亚基因组RNA（RNA4）的亚基因组启动子，CP就是从该亚基因组RNA合成的。利用从RNA3的cDNA克隆的突变形式产生的转录本，我们已经鉴定了RNA3积累和RNA4合成所需的顺式作用序列，并分析了RNA3编码蛋白的作用。RNA3衍生物在烟草原生质体中的高效积累需要RNA分子5'端的92个核苷酸、IR中的250个核苷酸和3'端的275个核苷酸。这250个核苷酸的IR包含一个90个核苷酸的序列，该序列对于亚基因组启动子活性是必需的。尽管共同区域参与了RNA3积累和RNA4合成，但IR对这两种现象的作用方式不同。对具有内部重复的RNA3形式的分析表明，RNA3积累取决于IR的上下文。亚基因组启动子活性更依赖于位置，并且从更靠近正义链RNA 3'端的启动子处总是更强。IR中的一个突变特异性地影响正义链RNA积累，表明该区域在正义链合成中起作用。通过对植物或原生质体进行突变和接种来分析RNA3编码蛋白的作用。3a蛋白的突变对原生质体中RNA3的积累没有影响，而CP突变导致CMV RNA积累减少。这种减少在正义链积累方面比在负义链积累方面更明显。接种叶片中未检测到CP突变的RNA，而3a蛋白突变体的RNA积累，尽管其水平比野生型CMV低几个数量级。这些数据得出的结论是，两种蛋白都是CMV从感染部位有效传播所必需的。
