Li W Z, Qu F, Morris T J
School of Biological Sciences, University of Nebraska-Lincoln 68588-0118, USA.
Virology. 1998 May 10;244(2):405-16. doi: 10.1006/viro.1998.9125.
Previous studies on turnip crinkle virus (TCV) have suggested that the two small, centrally located ORFs, conserved in all Carmoviruses, are both required for cell-to-cell movement (Hacker et al., 1992). We now demonstrate that the cell-to-cell movement of TCV is mediated by in trans complementation of the two proteins. First, both of the putative movement proteins (MPs p8 and p9) were shown to be translated in vitro from transcripts representing the 1.7-kb subgenomic RNA. Western blot analysis, using antisera prepared against GST fusion proteins of both genes, was then used to show that the p8 but not the p9 protein accumulated to detectable levels in particulate fractions of infected cells. Cell-to-cell movement of various MP mutants in Arabidopsis was evaluated by in situ hybridization of inoculated leaves. Changes in either of the two MP genes resulted in failure of the mutants to move cell-to-cell. Coat protein was found to be unnecessary for cell-to-cell movement. Complementation of cell-to-cell movement by co-inoculating p8-defective mutants with a p9-defective mutant resulted in delayed systemic infection. In contrast, efficient cell-to-cell movement was achieved when the MP mutants were inoculated into transgenic plants expressing the corresponding functional gene(s). These experiments provide further evidence that both MP genes encoded by Carmoviruses must function in trans in the same cell in order to mediate cell-to-cell movement.
先前对芜菁皱缩病毒（TCV）的研究表明，在所有 Carmoviruses 中保守的两个位于中心的小开放阅读框对于细胞间运动都是必需的（Hacker 等人，1992 年）。我们现在证明 TCV 的细胞间运动是由这两种蛋白质的反式互补介导的。首先，两个假定的运动蛋白（MPs p8 和 p9）都显示可从代表 1.7-kb 亚基因组 RNA 的转录本在体外翻译。然后使用针对这两个基因的 GST 融合蛋白制备的抗血清进行蛋白质印迹分析，结果表明 p8 蛋白而非 p9 蛋白在受感染细胞的颗粒组分中积累到可检测水平。通过对接种叶片进行原位杂交来评估拟南芥中各种 MP 突变体的细胞间运动。两个 MP 基因中任何一个的变化都会导致突变体无法进行细胞间运动。发现外壳蛋白对于细胞间运动不是必需的。将 p8 缺陷型突变体与 p9 缺陷型突变体共接种来互补细胞间运动，会导致系统感染延迟。相反，当将 MP 突变体接种到表达相应功能基因的转基因植物中时，可实现有效的细胞间运动。这些实验提供了进一步的证据，表明 Carmoviruses 编码的两个 MP 基因必须在同一细胞中以反式发挥作用，才能介导细胞间运动。
