Mise K, Tsuge S, Nagao K, Okuno T, Furusawa I
Laboratory of Plant Pathology, Faculty of Agriculture, Kyoto University, Japan.
J Gen Virol. 1992 Oct;73 ( Pt 10):2543-51. doi: 10.1099/0022-1317-73-10-2543.
The ATCC66 strain of brome mosaic virus (BMV) (propagated at Kyoto University) contained two types of coat protein in its virion whereas the Russian strain of BMV has been known to contain a single coat protein; both strains have two initiation codons for coat protein in the same reading frame at the 5'-proximal end of the gene in RNA 4. Comparative studies on the nucleotide sequences of the ATCC66 and Russian strains of BMV demonstrated that in the ATCC66 strain, two adjacent adenine residues were absent from RNA 3 in the leader sequences of the coat protein gene just a few nucleotides 5' to the first initiation codon of the coat protein gene. Using biologically active cDNA clones of BMV RNA of the ATCC66 strain, we inserted two adjacent adenine residues into the cDNA of RNA 3 to obtain an RNA 3 transcript which has the same nucleotide sequence as the Russian strain in the non-coding leader sequence of the coat protein gene. Barley protoplasts inoculated with this RNA 3 transcript together with RNA 1 and 2 produced a single coat protein. To obtain further insight into the mechanism of translation of the BMV coat protein, we constructed several types of RNA 4 by changing the sequence surrounding the first AUG codon in the coat protein gene and analysed the in vitro translation products of the mutant RNA 4. The results confirmed that the absence of the two adjacent adenine residues was responsible for the production of two types of coat protein in the ATCC66 strain. The deletion of the two adjacent adenine residues in ATCC66 resulted in a base substitution of A with U three nucleotides 5' to the first AUG in the coat protein gene. The base substitution reduced translational activity from the first AUG codon and concomitantly increased translational activity from the second AUG codon from which a truncated coat protein was translated.
雀麦花叶病毒（BMV）的ATCC66菌株（在京都大学繁殖）在其病毒粒子中含有两种类型的外壳蛋白，而俄罗斯菌株的BMV已知只含有一种外壳蛋白；两种菌株在RNA 4基因的5'近端末端的同一阅读框中都有两个外壳蛋白起始密码子。对BMV的ATCC66和俄罗斯菌株的核苷酸序列进行比较研究表明，在ATCC66菌株中，外壳蛋白基因前导序列的RNA 3中，在外壳蛋白基因第一个起始密码子5'端仅几个核苷酸处缺少两个相邻的腺嘌呤残基。利用ATCC66菌株BMV RNA的生物活性cDNA克隆，我们将两个相邻的腺嘌呤残基插入RNA 3的cDNA中，以获得一种RNA 3转录本，该转录本在外壳蛋白基因的非编码前导序列中具有与俄罗斯菌株相同的核苷酸序列。用这种RNA 3转录本与RNA 1和2一起接种大麦原生质体，产生了一种单一的外壳蛋白。为了进一步深入了解BMV外壳蛋白的翻译机制，我们通过改变外壳蛋白基因中第一个AUG密码子周围的序列构建了几种类型的RNA 4，并分析了突变RNA 4的体外翻译产物。结果证实，缺少两个相邻的腺嘌呤残基是ATCC66菌株产生两种类型外壳蛋白的原因。ATCC66中两个相邻腺嘌呤残基的缺失导致外壳蛋白基因中第一个AUG密码子5'端三个核苷酸处的A被U碱基替换。碱基替换降低了第一个AUG密码子的翻译活性，并同时增加了第二个AUG密码子的翻译活性，从该密码子翻译出截短的外壳蛋白。
