Ayllón María A, Gowda Siddarame, Satyanarayana Tatineni, Dawson William O
Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, USA.
Virology. 2004 Apr 25;322(1):41-50. doi: 10.1016/j.virol.2004.01.005.
Citrus tristeza virus (CTV), a member of the Closteroviridae with a plus-stranded genomic RNA of approximately 20 kb, produces 10 3'-coterminal subgenomic (sg) RNAs that serve as messenger (m)RNAs for its internal genes. In addition, a population of 5'-terminal sgRNAs of approximately 700 nts are highly abundant in infected cells. Previous analysis demonstrated that the controller elements (CE) are responsible for the 3'-terminal mRNAs and the small 5'-terminal sgRNAs differ in the number of additional sgRNAs produced. A feature of both types of CE is production of 5'- and 3'-terminal positive-stranded sgRNAs, but the 3' CEs additionally produce a negative-stranded complement of the 3'-terminal mRNAs. Here, we found that the termination (for 5'-terminal sgRNAs) and initiation (for 3'-terminal sgRNAs) sites of the 5' vs. the 3' CEs occur at opposite ends of the respective minimal active CEs. The initiation site for the 3' CE of the major coat protein gene, and probably those of the p20 and p23 genes, was outside (3' in terms of the genomic RNA) the minimal unit, whereas the termination sites were located within the minimal CE, 30-50 nts upstream of the initiation site (referring to the positive-strand sequence). In contrast, the initiation site for the 5' CE was in the 5' region of the minimal unit, with the termination sites 20-35 nts downstream (referring to the positive-strand sequence). Furthermore, the CEs differ in initiation nucleotide and response to mutagenesis of that nucleotide. The 3' CE initiates sgRNA synthesis from a uridylate, whereas the 5' CE initiates from a cytidylate. We previously found that the 3' CEs were unusually tolerant to mutagenesis of the initiation sites, with initiation proceeding from alternative sites. Mutagenesis of the initiation site of the 5' CE prevented synthesis of either the 5'- or 3'-terminal sgRNAs. Thus, the cis-acting elements at opposite ends of the genome are remarkably different, perhaps having arisen from different origins and or with different functions in the life cycle of this virus.
柑橘衰退病毒（CTV）是长线形病毒科的成员，其基因组为正义单链RNA，大小约为20 kb，可产生10种3' 共末端亚基因组（sg）RNA，作为其内部基因的信使（m）RNA。此外，在受感染细胞中，大量存在一群约700 nt的5' 末端sgRNA。先前的分析表明，调控元件（CE）负责3' 末端mRNA的产生，而小的5' 末端sgRNA在产生的额外sgRNA数量上有所不同。这两种类型的CE的一个特点是都产生5' 和3' 末端的正义链sgRNA，但3' CE还额外产生3' 末端mRNA的负链互补序列。在这里，我们发现5' CE与3' CE的终止（针对5' 末端sgRNA）和起始（针对3' 末端sgRNA）位点分别位于各自最小活性CE的两端。主要外壳蛋白基因的3' CE的起始位点，可能还有p20和p23基因的起始位点，位于最小单元之外（就基因组RNA而言在3' 端），而终止位点位于最小CE内，在起始位点（参考正链序列）上游30 - 50 nt处。相比之下，5' CE的起始位点在最小单元的5' 区域，终止位点在下游20 - 35 nt处（参考正链序列）。此外，CE在起始核苷酸以及对该核苷酸诱变的反应方面存在差异。3' CE从尿苷酸起始sgRNA合成，而5' CE从胞苷酸起始。我们先前发现3' CE对起始位点的诱变异常耐受，起始可从替代位点进行。5' CE起始位点的诱变会阻止5' 或3' 末端sgRNA的合成。因此，基因组两端的顺式作用元件明显不同，可能源于不同的起源，或者在该病毒的生命周期中具有不同的功能。
