Li X H, Carrington J C
Department of Biology, Texas A&M University, College Station 77843.
Proc Natl Acad Sci U S A. 1995 Jan 17;92(2):457-61. doi: 10.1073/pnas.92.2.457.
A genetic complementation system was developed in which tobacco etch virus (TEV) polymerase (NIb)-expressing transgenic plants or protoplasts were inoculated with NIb-defective TEV mutants. A beta-glucuronidase (GUS) reporter gene integrated into the genomes of parental and four mutant viruses was used to assay RNA amplification. Two mutants (termed VNN and EDE) contained substitutions affecting the conserved "GDD" polymerase motif or a nuclear localization signal sequence, respectively; one (aD/b) contained a mutation debilitating the NIb N-terminal cleavage site, whereas the other (delta b) lacked the entire NIb sequence. Each mutant was unable to amplify in nontransformed tobacco protoplasts. In contrast, the VNN, EDE, and delta b mutants were complemented to various degrees in NIb-expressing cells, whereas the aD/b mutant was not complemented. The VNN mutant was complemented most efficiently, reaching an average of 11-12% the level of parental TEV-GUS, although in some experiments the level was near 100%. This mutant also replicated in, and spread through, whole transgenic plants to the same level as parental virus. The EDE mutant was complemented relatively poorly, reaching 1% or less of the level of parental TEV-GUS. Despite the close proximity of the EDE substitution to the N-terminal cleavage site, proteolytic processing of NIb was unaffected in an in vitro assay. The delta b mutant was complemented to an intermediate degree in protoplasts, reaching 3.5% the level of parental virus, and replicated and moved systemically in transgenic plants. These data indicate that free NIb supplied entirely in trans can provide all NIb functions essential for RNA amplification. The relative inefficient complementation of the EDE mutant suggests that the resulting mutant protein was transinhibitory.
构建了一种基因互补系统，用缺失NIb的烟草蚀纹病毒（TEV）突变体接种表达TEV聚合酶（NIb）的转基因植物或原生质体。将整合到亲本病毒和四种突变病毒基因组中的β-葡萄糖醛酸酶（GUS）报告基因用于检测RNA扩增。两个突变体（分别称为VNN和EDE），其中一个的取代影响保守的“GDD”聚合酶基序，另一个影响核定位信号序列；一个突变体（aD/b）的突变使NIb N端切割位点失活，而另一个突变体（delta b）缺失整个NIb序列。每个突变体在未转化的烟草原生质体中均无法扩增。相反，VNN、EDE和delta b突变体在表达NIb的细胞中得到不同程度的互补，而aD/b突变体未得到互补。VNN突变体的互补效率最高，平均达到亲本TEV-GUS水平的11%-12%，尽管在某些实验中该水平接近100%。该突变体也能在整个转基因植物中复制并传播，达到与亲本病毒相同的水平。EDE突变体的互补效果相对较差，达到亲本TEV-GUS水平的1%或更低。尽管EDE取代位点与N端切割位点非常接近，但在体外试验中NIb的蛋白水解加工未受影响。delta b突变体在原生质体中得到中等程度的互补，达到亲本病毒水平的3.5%，并在转基因植物中进行系统性复制和移动。这些数据表明，完全通过反式提供的游离NIb可以提供RNA扩增所需的所有NIb功能。EDE突变体相对低效的互补表明，产生的突变蛋白具有反式抑制作用。
