Cap-independent translation mechanism of Red clover necrotic mosaic virus RNA2 differs from that of RNA1 and is linked to RNA replication
Cap-independent translation mechanism of Red clover necrotic mosaic virus RNA2 differs from that of RNA1 and is linked to RNA replication
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DOI：
10.1128/jvi.80.8.3781-3791.2006
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发表时间：
2006-04-01
影响因子：
5.4
通讯作者：
Okuno, T
中科院分区：
文献类型：
--
作者：
Mizumoto, H;Iwakawa, HO;Okuno, T
红三叶坏死花叶病毒（Red三叶草necrotic mosaic virus，RCNMV）是石竹科花叶病毒属（Dianthovirus）的成员，其基因组分为RNA 1和RNA 2两个RNA分子，RNA 1和RNA 2的5'端无帽结构，3'端无poly（A）尾。RCNMV RNA 1的3'非翻译区（3' UTR）含有一个必需的RNA元件（3 'TE-DR 1），它是帽非依赖性翻译所必需的。在这项研究中，我们研究了一个帽独立的翻译机制的RNA 2使用萤火虫荧光素酶（Luc）基因表达测定系统在豇豆原生质体和无细胞裂解液（BYL）从evacuolated烟草BY2原生质体制备。我们不能在RNA 2中检测到可以取代帽结构的功能的顺式作用RNA序列，例如RNA 1的3 'TE-DR 1。然而，未加帽的报告基因RNA 2，RNA 2-Luc，其中Luc开放阅读框（ORF）插入在5' UTR和运动蛋白ORF之间，在p27和p88的存在下，在其中复制RNA 2-Luc的原生质体中，原生质体中的时间过程实验表明，RNA 2-Luc的翻译活性并不反映RNA 2的量。RNA 2的顺式作用RNA复制元件的突变消除了RNA 2-Luc的帽非依赖性翻译活性，表明RNA 2-Luc的翻译活性与RNA复制偶联。我们的研究结果表明，翻译机制不同的两个分段的基因组RNA的RCNMV。我们提出了一个模型，其中只有RNA 2是从头产生的病毒RNA复制机制作为mRNA的翻译功能。
The genome of Red clover necrotic mosaic virus (RCNMV) in the genus Dianthovirus is divided into two RNA molecules of RNA1 and RNA2, which have no cap structure at the 5' end and no poly(A) tail at the 3' end. The 3' untranslated region (3' UTR) of RCNMV RNA1 contains an essential RNA element (3'TE-DR1), which is required for cap-independent translation. In this study, we investigated a cap-independent translational mechanism of RNA2 using a firefly luciferase (Luc) gene expression assay system in cowpea protoplasts and a cell-free lysate (BYL) prepared from evacuolated tobacco BY2 protoplasts. We were unable to detect cis-acting RNA sequences in RNA2 that can replace the function of a cap structure, such as the 3'TE-DR1 of RNA1 However, the uncapped reporter RNA2, RNA2-Luc, in which the Luc open reading frame (ORF) was inserted between the 5' UTR and the movement protein ORF, was effectively translated in the presence of p27 and p88 in protoplasts in which RNA2-Luc was replicated. Time course experiments in protoplasts showed that the translational activity of RNA2-Luc did not reflect the amount of RNA2. Mutations in cis-acting RNA replication elements of RNA2 abolished the cap-independent translational activity of RNA2-Luc, suggesting that the translational activity of RNA2-Luc is coupled to RNA replication. Our results show that the translational mechanism differs between two segmented genomic RNAs of RCNMV. We present a model in which only RNA2 that is generated de novo through the viral RNA replication machinery functions as mRNA for translation.