Mutational analysis of upstream AUG codons of poliovirus RNA.
Mutational analysis of upstream AUG codons of poliovirus RNA.
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脊髓灰质炎病毒 RNA 上游 AUG 密码子的突变分析。
DOI：
10.1128/jvi.62.12.4486-4492.1988
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发表时间：
1988
影响因子：
5.4
通讯作者：
Sonenberg,N
中科院分区：
文献类型：
--
作者：
Pelletier,J;Flynn,ME;Kaplan,G;Racaniello,V;Sonenberg,N
脊髓灰质炎病毒2型Lansing RNA的5'非翻译区由744个核苷酸组成，其中包含7个AUG密码子，这些密码子后面是帧内终止密码子，从而形成短的开放阅读帧（orf）。为了确定这些小orf的生物学意义，我们将所有上游的AUG密码子突变为UUG。将点突变引入传染性脊髓灰质炎病毒cDNA克隆，并将改变后的cDNA体外转录的RNA转染到HeLa细胞中以恢复病毒。AUG 7的突变产生一种具有小斑块表型的病毒（称为R2-5NC-14），而其他6个AUG密码子的突变产生具有野生型斑块形态的病毒。为了确定R2-5NC-14的小斑块表型是否与病毒mRNA的翻译效率改变有关，我们构建了含有脊髓灰质炎病毒mRNA的5'非编码区与氯霉素乙酰转移酶（CAT）编码序列融合的嵌合mRNA。含有突变的AUG 7密码子的mRNA在体外的翻译效率下降。结果表明，脊髓灰质炎病毒RNA的上游orf不是病毒复制所必需的，也不作为脊髓灰质炎病毒mRNA翻译的障碍。AUG 7及其侧翼序列可能在脊髓灰质炎病毒RNA翻译中发挥积极作用。
The 5' untranslated region of poliovirus type 2 Lansing RNA consists of 744 nucleotides containing seven AUG codons which are followed by in-frame termination codons, thus forming short open reading frames (ORFs). To determine the biological significance of these small ORFs, all of the upstream AUG codons were mutated to UUG. The point mutations were introduced into an infectious poliovirus cDNA clone, and RNA transcribed in vitro from the altered cDNA was transfected into HeLa cells to recover the virus. Mutation of AUG 7 resulted in a virus (called R2-5NC-14) with a small-plaque phenotype, whereas mutation of the other six AUG codons produced virus with a wild-type plaque morphology. To determine whether the small-plaque phenotype of R2-5NC-14 was due to altered translational efficiency of the viral mRNA, we constructed chimeric mRNAs containing the 5' noncoding region of poliovirus mRNA fused to the chloramphenicol acetyltransferase (CAT) coding sequence. mRNA containing a mutated AUG 7 codon showed decreased translational efficiency in vitro. The results indicate that the upstream ORFs of poliovirus RNA are not essential for viral replication and do not act as barriers to the translation of poliovirus mRNA. AUG 7 and flanking sequences may play a positive acting role in poliovirus RNA translation.