Eggen R, Verver J, Wellink J, De Jong A, Goldbach R, van Kammen A
Department of Molecular Biology, Agricultural University, Dreijentaan, Wageningen, The Netherlands.
Virology. 1989 Dec;173(2):447-55. doi: 10.1016/0042-6822(89)90557-6.
Full-length DNA copies of both B- and M-RNA of cowpea mosaic virus (CPMV) were constructed downstream from a T7 promoter. By removal of nucleotides from the promoter sequence, B- and M-RNA-like transcripts with varying numbers of additional nonviral sequences at the 5' end were obtained upon transcription with T7 RNA polymerase. The infectivity of the transcripts in cowpea protoplasts was greatly affected by only a few extra nonviral nucleotides at the 5' end. The addition of about 400 nonviral nucleotides at the 3' end did not have any effect. Using the most infectious transcripts, in 40% of the cowpea protoplasts replication and expression of B-RNA like transcripts were observed and in 10% of the protoplasts both B- and M-RNA-like transcripts multiplied. Moreover, cowpea plants could also be infected with these transcripts. Sequence analysis showed that the 5' terminus of the M-RNA transcripts and the 3' terminus of the B-RNA transcripts were completely restored during replication in plants, including a poly(A) tail of variable length. Swapping experiments have been used to identify an influential point mutation in the coding region for the viral polymerase of a noninfectious B transcript. This experiment demonstrates the potential of the optimized infection system for future analysis of virus-encoded functions.
豇豆花叶病毒（CPMV）的B-RNA和M-RNA的全长DNA拷贝构建于T7启动子下游。通过去除启动子序列中的核苷酸，用T7 RNA聚合酶转录后可获得5'端带有不同数量额外非病毒序列的B-RNA样和M-RNA样转录本。转录本在豇豆原生质体中的感染性仅受到5'端少数几个额外非病毒核苷酸的极大影响。在3'端添加约400个非病毒核苷酸没有任何作用。使用感染性最强的转录本，在40%的豇豆原生质体中观察到了B-RNA样转录本的复制和表达，在10%的原生质体中B-RNA样和M-RNA样转录本都进行了增殖。此外，豇豆植株也能被这些转录本感染。序列分析表明，M-RNA转录本的5'末端和B-RNA转录本的3'末端在植物复制过程中完全恢复，包括可变长度的聚（A）尾。交换实验已被用于鉴定一种非感染性B转录本的病毒聚合酶编码区中的一个有影响的点突变。该实验证明了优化感染系统在未来病毒编码功能分析中的潜力。
