Haldeman-Cahill R, Daròs J A, Carrington J C
Institute of Biological Chemistry, Washington State University, Pullman 99164-6340, USA.
J Virol. 1998 May;72(5):4072-9. doi: 10.1128/JVI.72.5.4072-4079.1998.
The 3'-terminal 350 nucleotides of the tobacco etch potyvirus (TEV) genome span the end of the capsid protein (CP)-coding sequence and the 3' nontranslated region (NTR). The CP-coding sequence within this region contains a 105-nucleotide cis-active element required for genome replication (S. Mahajan, V. V. Dolja, and J. C. Carrington, J. Virol. 70:4370-4379, 1996). To investigate the sequence and secondary structure requirements within the CP cis-active region and the 3' NTR, a systematic linker-scanning mutagenesis analysis was done. Forty-six mutations, each with two to six nucleotide substitutions, were introduced at consecutive hexanucleotide positions in the genome of a recombinant TEV strain expressing a reporter protein (beta-glucuronidase). Genome amplification activity of each mutant in the protoplast cell culture system was measured. Mutations that severely debilitated genome amplification were identified throughout the CP-coding cis-active sequence and at several distinct locations within the 3' NTR. However, based on a computer model of RNA folding, mutations that had the most severe effects mapped to regions that were predicted to form base-paired secondary structures. Linker-scanning mutations predicted to affect either strand of a base-paired structure within the CP-coding cis-active sequence, a base-paired structure between two segments of the CP-coding cis-active sequence and a contiguous 14-nucleotide segment of the 3' NTR, and a base-paired structure near the 3' terminus of the 3' NTR inactivated genome amplification. Compensatory mutations that restored base pair interactions in each of these regions restored amplification activity, although to differing levels depending on the structure restored. These data reveal that the 3' terminus of the TEV genome consists of a series of functionally discrete sequences and secondary structures and that the CP-coding sequence and 3' NTR are coadapted for genome amplification function through a requirement for base pair interactions.
烟草蚀纹马铃薯Y病毒（TEV）基因组的3'末端350个核苷酸跨越衣壳蛋白（CP）编码序列的末端和3'非翻译区（NTR）。该区域内的CP编码序列包含一个基因组复制所需的105个核苷酸的顺式作用元件（S. 马哈詹、V. V. 多尔贾和J. C. 卡林顿，《病毒学杂志》70:4370 - 4379, 1996）。为了研究CP顺式作用区域和3' NTR内的序列及二级结构要求，进行了系统的接头扫描诱变分析。在表达报告蛋白（β - 葡萄糖醛酸酶）的重组TEV株基因组中，在连续的六核苷酸位置引入了46个突变，每个突变有两到六个核苷酸替换。在原生质体细胞培养系统中测量了每个突变体的基因组扩增活性。在整个CP编码顺式作用序列以及3' NTR内的几个不同位置都鉴定到了严重削弱基因组扩增的突变。然而，基于RNA折叠的计算机模型，影响最严重的突变映射到预计形成碱基配对二级结构的区域。预计影响CP编码顺式作用序列内碱基配对结构的任一条链、CP编码顺式作用序列的两个片段与3' NTR的一个连续14核苷酸片段之间的碱基配对结构以及3' NTR 3'末端附近的碱基配对结构的接头扫描突变使基因组扩增失活。恢复这些区域中碱基对相互作用的补偿性突变恢复了扩增活性，尽管根据恢复的结构不同，恢复程度也不同。这些数据表明，TEV基因组的3'末端由一系列功能上离散的序列和二级结构组成，并且CP编码序列和3' NTR通过对碱基对相互作用的需求共同适应基因组扩增功能。
