Pelet T, Delenda C, Gubbay O, Garcin D, Kolakofsky D
Department of Genetics and Microbiology, University of Geneva School of Medicine, CMU, Switzerland.
Virology. 1996 Oct 15;224(2):405-14. doi: 10.1006/viro.1996.0547.
We have used a cDNA copy of a natural, internally deleted, Sendai virus defective interfering genome to study the effect of insertions and deletions (which maintain the hexamer genome length) on the ability of viral genomes to be amplified in a transfected cell system. The insertion of 18 nt at nt72 (In the 5' untranslated region of the N gene, just downstream of the le+ region) was found to be lethal, whereas similar insertions further from the genome ends were well tolerated. Curiously, the insertion of 6 nt on either side of the le+/N junction (at nt47 and nt87) was well tolerated, but the insertion of 12 nt at either site, or of 6 nt at both sites, largely ablated genome amplification. These results suggest that an element of this replication promoter is located downstream of nt87, in the 5' untranslated region of the first gene. Remarkably, the addition of 6 nt by the insertion of 2, 3, or 4 nt at nt47 plus the insertion of 4, 3, or 2 nt, respectively, at nt87 was poorly tolerated, presumably because the hexamer phase of the intervening sequence was altered with respect to the N subunits of the template. These results suggest that the rule of six operates, at least in part, at the level of the initiation of antigenome synthesis.
我们使用了一种天然的、内部缺失的仙台病毒缺陷干扰基因组的cDNA拷贝，来研究插入和缺失（保持六聚体基因组长度）对病毒基因组在转染细胞系统中扩增能力的影响。发现在核苷酸72处（在N基因的5'非翻译区，就在le+区域下游）插入18个核苷酸是致死性的，而在离基因组末端更远的位置进行类似插入则能被很好地耐受。奇怪的是，在le+/N连接处两侧（核苷酸47和87处）插入6个核苷酸能被很好地耐受，但在任一位置插入12个核苷酸，或在两个位置都插入6个核苷酸，在很大程度上消除了基因组的扩增。这些结果表明，这个复制启动子的一个元件位于第一个基因的5'非翻译区中核苷酸87的下游。值得注意的是，在核苷酸47处分别插入2、3或4个核苷酸，同时在核苷酸87处分别插入4、3或2个核苷酸，从而总共插入6个核苷酸，这种情况耐受性很差，推测是因为中间序列的六聚体相位相对于模板的N亚基发生了改变。这些结果表明，六碱基规则至少在部分程度上在反基因组合成起始水平起作用。
