Identification and characterization of a cis-acting replication element (cre) adjacent to the internal ribosome entry site of foot-and-mouth disease virus
Identification and characterization of a cis-acting replication element (cre) adjacent to the internal ribosome entry site of foot-and-mouth disease virus
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DOI：
10.1128/jvi.76.19.9686-9694.2002
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发表时间：
2002-10-01
影响因子：
5.4
通讯作者：
Henry, TM
中科院分区：
文献类型：
--
作者：
Mason, PW;Bezborodova, SV;Henry, TM
在过去的几年中，在几种小核糖核酸病毒的蛋白质编码区发现了一种被称为顺式作用复制元件（ere）的基本RNA结构。ere是一个包含保守的AAACA基序的茎环结构，其功能是将U残基添加到蛋白质引物3B上的模板。通过对几种口蹄疫病毒（FMDV）血清型代表的基因组进行调查，我们在基因组的5'非翻译区（与内核糖体进入位点[IRES]相邻）发现了一个假定的位点。为了证实这一假设在复制中的作用，我们测试了AAACA基序和碱基配对在FMDV基因组复制中的重要性。为此，这些突变被克隆到FMDV复制子和合成病毒基因组中。对这些复制子和基因组特性的分析揭示了以下几点。(i) AAACA基序的突变严重减少了复制，从含有突变AAACA序列的基因组中恢复的所有病毒都恢复到野生型序列。（ii）茎区的突变表明，形成这种碱基配对结构的能力对复制很重要。虽然ere与IRES相邻，但我们创建的突变在体内并没有显著降低IRES介导的翻译。最后，研究表明，在基因组的5‘端，如果在3D（pol）编码区之后的基因组中添加一个野生型ere，则可以获得缺乏5’ ere的功能性复制子和病毒，因此，该位置对复制并不是至关重要。综上所述，这些结果支持了ere在复制中的重要性，并证明了这一基本元件的活性不需要定位在基因组的多蛋白编码区。
Over the last few years, an essential RNA structure known as the cis-acting replicative element (ere) has been identified within the protein-coding region of several picornaviruses. The ere, a stem-loop structure containing a conserved AAACA motif, functions as a template for addition of U residues to the protein primer 3B. By surveying the genomes of representatives of several serotypes of foot-and-mouth disease virus (FMDV), we discovered a putative ere in the 5' untranslated region of the genome (contiguous with the internal ribosome entry site [IRES]). To confirm the role of this putative ere in replication, we tested the importance of the AAACA motif and base pairing in the stem in FMDV genome replication. To this end, ere mutations were cloned into an FMDV replicon and into synthetic viral genomes. Analyses of the properties of these replicons and genomes revealed the following. (i) Mutations in the AAACA motif severely reduced replication, and all viruses recovered from genomes containing mutated AAACA sequences had reverted to the wild-type sequence. (ii) Mutations in the stem region showed that the ability to form this base-paired structure was important for replication. Although the ere was contiguous with the IRES, the mutations we created did not significantly reduce IRES-mediated translation in vivo. Finally, the position of the ere at the 5' end of the genome was shown not to be critical for replication, since functional replicons and viruses lacking the 5' ere could be obtained if a wild-type ere was added to the genome following the 3D(pol) coding region. Taken together, these results support the importance of the ere in replication and demonstrate that the activity of this essential element does not require localization within the polyprotein-encoding region of the genome.