Thompson Jeremy R, Buratti Emanuele, de Wispelaere Mélissanne, Tepfer Mark
Plant Virology Group, ICGEB Biosafety Outstation, Via Piovega 23, 31056 Ca' Tron di Roncade, Italy.
Molecular Pathology Group, ICGEB, AREA Science Park, Padriciano 99, 34012 Trieste, Italy.
J Gen Virol. 2008 Jul;89(Pt 7):1729-1738. doi: 10.1099/vir.0.2008/001057-0.
The uncapped and ORF-less subgenomic RNA5 is produced in subgroup II strains of cucumber mosaic virus (CMV), but not in subgroup I strains. Its initiation nucleotide (nt 1903) is in a 21 nt conserved sequence (Box1) that is absent in CMV subgroup I. Putative non-coding RNA structural elements surrounding Box1 in the plus and minus strand were identified in silico and by in vitro RNase probing. Four main stem-loop structures (SLM, SLL, SLK and SLJ) were identified between nt 1887 and 1999 of isolate R-CMV (subgroup II), with notable differences within SLM and SLL between the two strands. Mutation of a stem-loop within SLM, even when the predicted wild-type structure was maintained, showed significant reduction in RNA5 levels in planta. Three mutants containing 3-4 nt substitutions between positions -39 and +49 showed significantly reduced levels of RNA5, while another similar mutant at positions 80-83 had RNA5 levels comparable to wild-type. Deletion of Box1 resulted in similar levels of RNA3 and 4 as wild-type, while eliminating RNA5. Insertion of Box1 into a subgroup I isolate was not sufficient to produce RNA5. However, in a mutant with an additional 21 nt of R-CMV 3' of Box1 (positions -1 to +41), low levels of RNA5 were detected. Taken together, these results have identified regions of the viral genome responsible for RNA5 production and in addition provide strong evidence for the existence of newly identified conserved structural elements in the 5' part of the 3' untranslated region.
无帽且无开放阅读框的亚基因组RNA5在黄瓜花叶病毒（CMV）的II亚组毒株中产生，但不在I亚组毒株中产生。其起始核苷酸（第1903位核苷酸）位于一个21个核苷酸的保守序列（Box1）中，该序列在CMV I亚组中不存在。通过计算机模拟和体外核糖核酸酶探测，确定了正义链和反义链中围绕Box1的推定非编码RNA结构元件。在分离株R-CMV（II亚组）的第1887位核苷酸和1999位核苷酸之间鉴定出四个主要的茎环结构（SLM、SLL、SLK和SLJ），两条链的SLM和SLL内存在显著差异。即使保持预测的野生型结构，SLM内一个茎环的突变也显示植物中RNA5水平显著降低。三个在-39位至+49位之间含有3至4个核苷酸替换的突变体显示RNA5水平显著降低，而另一个在80至83位的类似突变体的RNA5水平与野生型相当。删除Box1导致RNA3和RNA4水平与野生型相似，但消除了RNA5。将Box1插入I亚组分离株中不足以产生RNA5。然而，在一个在Box1 3'端额外含有21个R-CMV核苷酸（-1位至+41位）的突变体中，检测到了低水平的RNA5。综上所述，这些结果确定了病毒基因组中负责RNA5产生的区域，此外还为3'非翻译区5'部分新鉴定的保守结构元件的存在提供了有力证据。
