Belli B A, Samuel C E
Department of Biological Sciences, University of California, Santa Barbara 93106.
Virology. 1993 Mar;193(1):16-27. doi: 10.1006/viro.1993.1099.
The reovirus S1 gene cDNA was systematically altered by site-directed mutagenesis in an attempt to identify regions important in determining the relative efficiency of translation of the two open reading frames of the bicistronic S1 mRNA. The synthesis of the minor capsid protein sigma 1 encoded by ORF1, extending from AUG14 to UGA1424, and the synthesis of the nonstructural protein sigma 1NS encoded by ORF2, extending from AUG75 to UAG432, were examined in transfected COS cells. Deletion of the 5'-untranslated region upstream of ORF1 AUG14 did not significantly affect either the relative amount, or the ratio, of sigma 1 and sigma 1NS synthesized. Creation of a strong context ORF1 initiation site by substitution of 5'-untranslated region nucleotides flanking AUG14 likewise did not affect sigma 1 synthesis, but sigma 1NS synthesis from ORF2 was decreased about twofold relative to wild-type S1 mRNA. The amount of sigma 1NS synthesis was increased less than twofold either by elimination of the ORF1 AUG14 initiation codon or by termination of sigma 1 synthesis shortly after initiation from AUG14. No sigma 1 synthesis was detected when the ORF1 AUG14 was mutated to a UUG14 codon. When ribosomes which initiated translation at AUG14 were frameshifted at the next codon after AUG14, elongation occurred with comparable efficiency in the sigma 1NS ORF2 and in sigma 1 ORF1. No sigma 1NS synthesis was detected when the ORF2 AUG75 was mutated to an CUG75 codon, and this mutation did not affect the amount of sigma 1 synthesis. sigma 1NS synthesis was not affected by truncation of the 3'-untranslated region or premature termination of sigma 1 synthesis shortly after the ORF2 UAG432. However, truncation of the ORF2 5'-untranslated region at either 6 or 36 nt following the ORF1 AUG14 significantly increased the efficiency of sigma 1NS synthesis. These results indicate that the region of the S1 mRNA immediately downstream of the sigma 1 ORF1 initiation codon AUG14 but well upstream of the sigma 1NS ORF2 initiation codon AUG75 plays a major role in determining the relative efficiency of synthesis of sigma 1 and sigma 1NS from the reovirus bicistronic s1 mRNA.
呼肠孤病毒S1基因cDNA通过定点诱变进行系统改变，旨在确定对双顺反子S1 mRNA两个开放阅读框翻译相对效率起重要作用的区域。在转染的COS细胞中检测了由ORF1编码的小衣壳蛋白σ1（从AUG14延伸至UGA1424）和由ORF2编码的非结构蛋白σ1NS（从AUG75延伸至UAG432）的合成。删除ORF1 AUG14上游的5'-非翻译区对合成的σ1和σ1NS的相对量或比例均无显著影响。通过替换AUG14侧翼的5'-非翻译区核苷酸来创建一个强上下文的ORF1起始位点同样不影响σ1的合成，但相对于野生型S1 mRNA，来自ORF2的σ1NS合成减少了约两倍。通过消除ORF1 AUG14起始密码子或在从AUG14起始后不久终止σ1合成，σ1NS合成量增加不到两倍。当ORF1 AUG14突变为UUG14密码子时，未检测到σ1合成。当在AUG14起始翻译的核糖体在AUG14后的下一个密码子处发生移码时，在σlNS ORF2和σ1 ORF1中延伸效率相当。当ORF2 AUG75突变为CUG75密码子时，未检测到σ1NS合成，且该突变不影响σ1合成量。σ1NS合成不受3'-非翻译区截短或在ORF2 UAG432后不久σ1合成提前终止的影响。然而，在ORF1 AUG14之后6或36个核苷酸处截短ORF2的5'-非翻译区显著提高了σ1NS合成效率。这些结果表明，S1 mRNA中紧接在σ1 ORF1起始密码子AUGl4下游但远在σ1NS ORF2起始密码子AUG75上游的区域在决定呼肠孤病毒双顺反子s1 mRNA中σ1和σ1NS合成的相对效率方面起主要作用。
