Ding B, Li Q, Nguyen L, Palukaitis P, Lucas W J
Section of Plant Biology, University of California, Davis 95616.
Virology. 1995 Mar 10;207(2):345-53. doi: 10.1006/viro.1995.1093.
Contrary to a previous report, electron microscopic studies on the Fny strain of cucumber mosaic virus (CMV)-infected tobacco tissues revealed that plasmodesmata were not structurally modified during CMV infection, nor were virions ever observed in plasmodesmata connecting infected cells. To further explore the basis of CMV infection, experiments were performed on the CMV 3a ORF. The 3a protein of CMV was expressed in and purified from Escherichia coli. The purified protein was labeled with fluorescein isothiocyanate (FITC) and subsequently microinjected into mesophyll cells of mature leaves of Nicotiana tabacum cv. Turkish Samsun NN. Within a brief period (as little as 1 sec), the microinjected FITC-labeled CMV 3a protein moved into neighboring cells. Co-injection of unlabeled CMV 3a protein with 9.4-kDa fluorescein-conjugated dextran (F-dextran) resulted in extensive cell-to-cell movement (diffusion) of the F-dextran, indicating that the 3a protein can interact with and dilate plasmodesmata. Furthermore, co-injection of unlabeled 3a protein with fluorescently labeled infectious CMV RNA molecules resulted in rapid and extensive cell-to-cell transport. In contrast, a mutant form of the 3a protein was unable to traffic from cell to cell, to increase the size exclusion limit of plasmodesmata, or to potentiate cell-to-cell trafficking of CMV RNA molecules. Microinjection studies performed on transgenic tobacco plants expressing the CMV 3a protein indicated that fluorescently labeled CMV RNA moved out of the target cell into the surrounding mesophyll tissue. In addition, expression of the CMV 3a protein also potentiated the cell-to-cell movement of 9.4-kDa F-dextran. Collectively, these results provide direct experimental evidence that the CMV 3a protein functions as the movement protein of CMV. These findings are consistent with the hypothesis that CMV moves from cell-to-cell in the form of a ribonucleoprotein complex.
与之前的一份报告相反，对感染黄瓜花叶病毒（CMV）Fny株系的烟草组织进行的电子显微镜研究显示，在CMV感染期间胞间连丝的结构并未改变，在连接受感染细胞的胞间连丝中也从未观察到病毒粒子。为了进一步探究CMV感染的基础，对CMV 3a开放阅读框进行了实验。CMV的3a蛋白在大肠杆菌中表达并纯化。纯化后的蛋白用异硫氰酸荧光素（FITC）标记，随后显微注射到烟草品种土耳其萨姆松NN成熟叶片的叶肉细胞中。在短时间内（短至1秒），显微注射的FITC标记的CMV 3a蛋白就进入了相邻细胞。将未标记的CMV 3a蛋白与9.4 kDa荧光素偶联葡聚糖（F-葡聚糖）共同注射，导致F-葡聚糖在细胞间广泛移动（扩散），表明3a蛋白能够与胞间连丝相互作用并使其扩张。此外，将未标记的3a蛋白与荧光标记的感染性CMV RNA分子共同注射，导致病毒在细胞间快速且广泛地运输。相比之下，3a蛋白的突变形式无法在细胞间移动、增加胞间连丝的大小排阻极限或促进CMV RNA分子在细胞间的运输。对表达CMV 3a蛋白的转基因烟草植株进行的显微注射研究表明，荧光标记的CMV RNA从靶细胞移出并进入周围的叶肉组织。此外，CMV 3a蛋白的表达还增强了9.4 kDa F-葡聚糖在细胞间的移动。总的来说，这些结果提供了直接的实验证据，证明CMV 3a蛋白作为CMV的运动蛋白发挥作用。这些发现与CMV以核糖核蛋白复合物的形式在细胞间移动的假说一致。
