Department of Crop Sciences, Section Plant Virology, University of Göttingen, Grisebachstrasse 6, D-37077 Göttingen, Germany.
J Gen Virol. 2010 Feb;91(Pt 2):552-62. doi: 10.1099/vir.0.014712-0. Epub 2009 Oct 28.
Virus RNA recombination, one of the main factors for genetic variability and evolution, is thought to be based on different mechanisms. Here, the recently described in vivo potato virus X (PVX) recombination assay [Draghici, H.-K. & Varrelmann, M. (2009). J Virol 83, 7761-7769] was applied to characterize structural parameters of recombination. The assay uses an Agrobacterium-mediated expression system incorporating a PVX green fluorescent protein (GFP)-labelled full-length clone. The clone contains a partial coat protein (CP) deletion that causes defectiveness in cell-to-cell movement, together with a functional CP+3' non-translated region (ntr) transcript, in Nicotiana benthamiana leaf tissue. The structural parameters assessed were the length of sequence overlap, the distance between mutations and the degree of sequence similarity. The effects on the observed frequency of reconstitution and the composition of the recombination products were characterized. Application of four different type X intact PVX CP genes with variable composition allowed the estimation of the junction sites of precise homologous recombination. Although one template switch would have been sufficient for functional reconstitution, between one and seven template switches were observed. Use of PVX-GFP mutants with CP deletions of variable length resulted in a linear decrease of the reconstitution frequency. The critical length observed for homologous recombination was 20-50 nt. Reduction of the reconstitution frequency was obtained when a phylogenetically distant PVX type Bi CP gene was used. Finally, the prediction of CP and 3'-ntr RNA secondary structure demonstrated that recombination-junction sites were located mainly in regions of stem-loop structures, allowing the recombination observed to be categorized as similarity-assisted.
病毒 RNA 重组是遗传变异和进化的主要因素之一，据认为它基于不同的机制。在这里，我们应用最近描述的马铃薯 X 病毒（PVX）体内重组检测方法[Draghici, H.-K. & Varrelmann, M. (2009). J Virol 83, 7761-7769]来研究重组的结构参数。该检测方法使用一种农杆菌介导的表达系统，其中包含一个 PVX 绿色荧光蛋白（GFP）标记的全长克隆。该克隆包含一个部分外壳蛋白（CP）缺失，导致细胞间运动缺陷，以及一个功能性 CP+3'非翻译区（ntr）转录本，在 Nicotiana benthamiana 叶片组织中。评估的结构参数包括序列重叠长度、突变之间的距离和序列相似性程度。还对观察到的重组体重建频率和重组产物组成的影响进行了特征分析。应用具有不同组成的四个不同类型 X 完整 PVX CP 基因，允许估计精确同源重组的连接点。虽然一个模板转换就足以进行功能重建，但观察到了一个到七个模板转换。使用具有不同长度 CP 缺失的 PVX-GFP 突变体导致重组体重建频率呈线性下降。观察到的同源重组的临界长度为 20-50nt。当使用亲缘关系较远的 PVX 型 Bi CP 基因时，重建频率会降低。最后，CP 和 3'-ntr RNA 二级结构的预测表明，重组连接点主要位于茎环结构区域，允许观察到的重组归类为相似性辅助。
