Ivanov P A, Karpova O V, Skulachev M V, Tomashevskaya O L, Rodionova N P, Atabekov J G
Department of Virology and A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.
Virology. 1997 May 26;232(1):32-43. doi: 10.1006/viro.1997.8525.
Most eukaryotic mRNAs are translated by a "scanning ribosome" mechanism. We have found that unlike the type member of the genus Tobamovirus, translation of the 3'-proximal coat protein (CP) gene of a crucifer infecting tobamovirus (crTMV) (Dorokhov et al., 1993; 1994) occurred in vitro by an internal ribosome entry mechanism. Three types of synthetic dicistronic RNA transcripts were constructed and translated in vitro: (i) "MP-CP-3'NTR" transcripts contained movement protein (MP) gene, CP gene and the 3'-nontranslated region of crTMV RNA. These constructs were structurally equivalent to dicistronic subgenomic RNAs produced by tobamoviruses in vivo. (ii) "deltaNPT-CP" transcripts contained partially truncated neomycin phosphotransferase I gene and CP gene. (iii) "CP-GUS" transcripts contained the first CP gene and the gene of Escherichia coli beta-glucuronidase (GUS) at the 3'-proximal position. The results indicated that the 148-nt region upstream of the CP gene of crTMV RNA contained an internal ribosome entry site (IRES(CP)) promoting internal initiation of translation in vitro. Dicistronic IRES(CP), containing chimeric mRNAs with the 5'-terminal stem-loop structure preventing translation of the first gene (MP, deltaNPT, or CP), expressed the CP or GUS genes despite their 3'-proximal localization. The capacity of crTMV IRES(CP) for mediating internal translation distinguishes this CP tobamovirus from the well-known-type member of the genus, TMV UI. The equivalent 148-nt sequence from TMV RNA was incapable of mediating internal translation. Two mutants were used to study structural elements of IRES(CP). It was concluded that integrity of IRES(CP) was essential for internal initiation. The crTMV provides a new example of internal initiation of translation, which is markedly distinct from IRESs shown for picornaviruses and other viral and eukaryotic mRNAs.
大多数真核生物的信使核糖核酸（mRNA）是通过“扫描核糖体”机制进行翻译的。我们发现，与烟草花叶病毒属的典型成员不同，十字花科侵染性烟草花叶病毒（crTMV）（多罗霍夫等人，1993年；1994年）3'-近端衣壳蛋白（CP）基因的翻译在体外是通过内部核糖体进入机制发生的。构建了三种类型的合成双顺反子RNA转录本并在体外进行翻译：（i）“MP-CP-3'NTR”转录本包含运动蛋白（MP）基因、CP基因和crTMV RNA的3'-非翻译区。这些构建体在结构上等同于烟草花叶病毒在体内产生的双顺反子亚基因组RNA。（ii）“deltaNPT-CP”转录本包含部分截短的新霉素磷酸转移酶I基因和CP基因。（iii）“CP-GUS”转录本在3'-近端位置包含第一个CP基因和大肠杆菌β-葡萄糖醛酸酶（GUS）基因。结果表明，crTMV RNA中CP基因上游148个核苷酸的区域包含一个内部核糖体进入位点（IRES(CP)），可促进体外翻译的内部起始。双顺反子IRES(CP)包含嵌合mRNA，其5'-末端茎环结构阻止第一个基因（MP、deltaNPT或CP）的翻译，尽管CP或GUS基因位于3'-近端，但仍能表达。crTMV IRES(CP)介导内部翻译的能力使其与烟草花叶病毒属的著名典型成员TMV UI区分开来。来自烟草花叶病毒RNA的等效148个核苷酸序列无法介导内部翻译。使用两个突变体研究IRES(CP)的结构元件。得出的结论是，IRES(CP)的完整性对于内部起始至关重要。crTMV提供了一个翻译内部起始的新例子，它与微小核糖核酸病毒以及其他病毒和真核生物mRNA所显示的内部核糖体进入位点（IRES）明显不同。
