van der Vossen E A, Notenboom T, Bol J F
Institute of Molecular Plant Sciences, Gorlaeus Laboratories, Leiden University, The Netherlands.
Virology. 1995 Oct 1;212(2):663-72. doi: 10.1006/viro.1995.1524.
RNA 3 of alfalfa mosaic virus encodes the movement protein P3 and the viral coat protein (CP). CP is translated from a subgenomic (sg) messenger, RNA 4. To characterize the sg promoter that is responsible for RNA 4 synthesis in vivo, putative sg promoter sequences were inserted in a unique Xhol site located between the initiation codon of the P3 gene and a second in-frame ATG codon in an infectious cDNA clone of RNA 3. Mutants with an active sg promoter insert expressed an N-terminally truncated P3 protein and were able to accumulate in plants. In addition, sg promoter activity was analyzed in protoplasts. When the transcription start site is taken as +1, the sequence of nucleotides -26/+1 was found to have a basal level of sg promoter activity. This activity was increased to near maximum levels when the sg promoter sequence was extended to -136/+12. The upstream positive regulatory element was mapped to nucleotides -136/-94. Engineering of point mutations and small deletions in RNA 3 around the transcription start site for RNA 4 synthesis revealed elements important for sg promoter activity with similarity to sequences conserved in sg promoters of alpha-like viruses. Some of these elements appeared to be required in cis for RNA 3 accumulation. A deletion of the C-terminal three amino acids of the P3 protein rendered this protein nonfunctional in cell-to-cell movement.
苜蓿花叶病毒的RNA 3编码运动蛋白P3和病毒外壳蛋白（CP）。CP由亚基因组（sg）信使RNA 4翻译而来。为了表征在体内负责RNA 4合成的sg启动子，将推定的sg启动子序列插入到RNA 3感染性cDNA克隆中P3基因起始密码子与第二个框内ATG密码子之间的一个独特Xhol位点。带有活性sg启动子插入片段的突变体表达N端截短的P3蛋白，并能够在植物中积累。此外，还在原生质体中分析了sg启动子活性。以转录起始位点为+1时，发现核苷酸序列-26/+1具有sg启动子活性的基础水平。当sg启动子序列延伸至-136/+12时，该活性增加到接近最大水平。上游正调控元件定位于核苷酸-136/-94。对RNA 3中围绕RNA 4合成转录起始位点的点突变和小缺失进行工程改造，揭示了对sg启动子活性重要的元件，这些元件与α样病毒sg启动子中保守的序列相似。其中一些元件似乎在顺式作用中是RNA 3积累所必需的。P3蛋白C端三个氨基酸的缺失使其在细胞间运动中无功能。
