Thivierge Karine, Cotton Sophie, Dufresne Philippe J, Mathieu Isabelle, Beauchemin Chantal, Ide Christine, Fortin Marc G, Laliberté Jean-François
Department of Plant Science, McGill University, 21,111 Lakeshore, Ste-Anne-de-Bellevue, Quebec, Canada H9X 3V9.
Virology. 2008 Jul 20;377(1):216-25. doi: 10.1016/j.virol.2008.04.015. Epub 2008 May 23.
Eukaryotic elongation factor 1-alpha (eEF1A) was identified as an interactor of Turnip mosaic virus (TuMV) RNA-dependent RNA polymerase (RdRp) and VPg-protease (VPg-Pro) using tandem affinity purification and/or in vitro assays. Subcellular fractionation experiments revealed that the level of eEF1A substantially increased in membrane fractions upon TuMV infection. Replication of TuMV occurs in cytoplasmic membrane vesicles, which are induced by 6K-VPg-Pro. Confocal microscopy indicated that eEF1A was included in these vesicles. To confirm that eEF1A was found in replication vesicles, we constructed an infectious recombinant TuMV that contains an additional copy of the 6K protein fused to the green fluorescent protein (GFP). In cells infected with this recombinant TuMV, fluorescence emitted by 6KGFP was associated with cytoplasmic membrane vesicles that contained VPg-Pro, the eukaryotic initiation factor (iso) 4E, the poly(A)-binding protein, the heat shock cognate 70-3 protein, and eEF1A. These results suggest that TuMV-induced membrane vesicles host at least three plant translation factors in addition to the viral replication proteins.
利用串联亲和纯化和/或体外实验，真核延伸因子1-α（eEF1A）被鉴定为芜菁花叶病毒（TuMV）RNA依赖性RNA聚合酶（RdRp）和VPg蛋白酶（VPg-Pro）的相互作用因子。亚细胞分级分离实验表明，TuMV感染后，膜分级中eEF1A的水平显著增加。TuMV在由6K-VPg-Pro诱导产生的细胞质膜泡中进行复制。共聚焦显微镜显示eEF1A包含在这些膜泡中。为了证实eEF1A存在于复制膜泡中，我们构建了一种感染性重组TuMV，其包含一个与绿色荧光蛋白（GFP）融合的6K蛋白的额外拷贝。在用这种重组TuMV感染的细胞中，6KGFP发出的荧光与包含VPg-Pro、真核起始因子（iso）4E、聚腺苷酸结合蛋白、热休克同源蛋白70-3和eEF1A的细胞质膜泡相关。这些结果表明，除了病毒复制蛋白外，TuMV诱导的膜泡还容纳至少三种植物翻译因子。
