Soluble, template-dependent extracts from Nicotiana benthamiana plants infected with potato virus X transcribe both plus- and minus-strand RNA templates.
Soluble, template-dependent extracts from Nicotiana benthamiana plants infected with potato virus X transcribe both plus- and minus-strand RNA templates.
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DOI：
10.1006/viro.2000.0512
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发表时间：
2000-09
期刊：
影响因子：
3.7
通讯作者：
C. Plante;K. H. Kim;N. Pillai-Nair;T. Osman;K. Buck;C. Hemenway
中科院分区：
文献类型：
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作者：
C. Plante;K. H. Kim;N. Pillai-Nair;T. Osman;K. Buck;C. Hemenway
我们开发了一种方法，将从感染了马铃薯病毒X （PVX）的benthamiana植物中分离的膜结合复制复合物转化为可溶的模板依赖系统，用于分析RNA合成。内源性模板提取物的膜结合RNA依赖RNA聚合酶活性分析表明，三种主要产物对应于PVX基因组RNA的双链版本和两种主要的亚基因组RNA。在Nonidet P-40 （NP-40）存在的情况下，用BAL 31核酸酶处理内源性模板从膜结合复合物中去除。在添加全长正链或负链PVX转录本后，检测相应大小的产物。当加入红三叶草坏死花叶dianthovirus (RCNMV) RNA 2模板时，未观察到合成，表明模板特异性。缺少3‘末端区域的正链PVX模板没有被复制，这表明3’区域的元件是RNA合成起始所必需的。支持内源性模板RNA合成的提取物也可以用牛磺酸去氧胆酸钠溶解，然后通过BAL 31核酸酶/NP-40处理使其具有模板依赖性。溶解的制剂复制PVX的正链和负链转录本，但不支持从RCNMV RNA 2合成。这些膜结合和可溶性模板依赖系统将有助于PVX RNA合成所需的病毒和宿主成分的分析。
We have developed a method to convert membrane-bound replication complexes isolated from Nicotiana benthamiana plants infected with potato virus X (PVX) to a soluble, template-dependent system for analysis of RNA synthesis. Analysis of RNA-dependent RNA polymerase activity in the membrane-bound, endogenous template extracts indicated three major products, which corresponded to double-stranded versions of PVX genomic RNA and the two predominant subgenomic RNAs. The endogenous templates were removed from the membrane-bound complex by treatment with BAL 31 nuclease in the presence of Nonidet P-40 (NP-40). Upon the addition of full-length plus- or minus- strand PVX transcripts, the corresponding-size products were detected. Synthesis was not observed when red clover necrotic mosaic dianthovirus (RCNMV) RNA 2 templates were added, indicating template specificity for PVX transcripts. Plus-strand PVX templates lacking the 3' terminal region were not copied, suggesting that elements in the 3' region were required for initiation of RNA synthesis. Extracts that supported RNA synthesis from endogenous templates could also be solublized using sodium taurodeoxycholate and then rendered template-dependent by BAL 31 nuclease/NP-40 treatment. The solubilized preparations copied both plus- and minus-strand PVX transcripts, but did not support synthesis from RCNMV RNA 2. These membrane-bound and soluble template-dependent systems will facilitate analyses of viral and host components required for PVX RNA synthesis.