Cheng Jai-Hong, Peng Chi-Weng, Hsu Yau-Heiu, Tsai Ching-Hsiu
Graduate Institute of Agricultural Biotechnology, National Chung Hsing University, Taichung 402, Taiwan.
J Virol. 2002 Jun;76(12):6114-20. doi: 10.1128/jvi.76.12.6114-6120.2002.
The 3' terminus of the bamboo mosaic potexvirus (BaMV) contains a poly(A) tail, the 5' portion of which participates in the formation of an RNA pseudoknot required for BaMV RNA replication. Recombinant RNA-dependent RNA polymerase (RdRp) of BaMV binds to the pseudoknot poly(A) tail in gel mobility shift assays (C.-Y. Huang, Y.-L. Huang, M. Meng, Y.-H. Hsu, and C.-H. Tsai, J. Virol. 75:2818-2824, 2001). Approximately 20 nucleotides of the poly(A) tail adjacent to the 3' untranslated region (UTR) are protected from diethylpyrocarbonate modification, suggesting that this region may be used to initiate minus-strand RNA synthesis. The 5' terminus of the minus-strand RNA synthesized by the RdRp in vitro was examined using 5' rapid amplification of cDNA ends (RACE) and DNA sequencing. Minus-strand RNA synthesis was found to initiate from several positions within the poly(A) tail, with the highest frequency of initiation being from the 7th to the 10th adenylates counted from the 5'-most adenylate of the poly(A) tail. Sequence analyses of BaMV progeny RNAs recovered from Nicotiana benthamiana protoplasts which were inoculated with mutants containing a mutation at the 1st, 4th, 7th, or 16th position of the poly(A) tail suggested the existence of variable initiation sites, similar to those found in 5' RACE experiments. We deduce that the initiation site for minus-strand RNA synthesis is not fixed at one position but resides opposite one of the 15 adenylates of the poly(A) tail immediately downstream of the 3' UTR of BaMV genomic RNA.
竹花叶病毒（BaMV）的3'末端含有一个聚腺苷酸（poly(A)）尾巴，其5'部分参与形成BaMV RNA复制所需的RNA假结。在凝胶迁移率变动分析中，BaMV的重组RNA依赖性RNA聚合酶（RdRp）与假结聚腺苷酸尾巴结合（C.-Y. Huang、Y.-L. Huang、M. Meng、Y.-H. Hsu和C.-H. Tsai，《病毒学杂志》75:2818 - 2824，2001年）。靠近3'非翻译区（UTR）的聚腺苷酸尾巴的大约20个核苷酸免受焦碳酸二乙酯修饰，这表明该区域可能用于启动负链RNA合成。使用5' cDNA末端快速扩增（RACE）和DNA测序检查了RdRp在体外合成的负链RNA的5'末端。发现负链RNA合成从聚腺苷酸尾巴内的几个位置开始，起始频率最高的是从聚腺苷酸尾巴5'最末端腺苷酸起计数的第7至第10个腺苷酸。对从用在聚腺苷酸尾巴第1、4、7或16位含有突变的突变体接种的本氏烟草原生质体中回收的BaMV子代RNA进行的序列分析表明存在可变起始位点，类似于在5' RACE实验中发现的那些位点。我们推断负链RNA合成的起始位点不是固定在一个位置，而是位于BaMV基因组RNA 3' UTR下游紧挨着的聚腺苷酸尾巴的15个腺苷酸之一的对面。
