RNA REPLICATION MECHANISM OF DIANTHOVIRUS

石竹病毒的RNA复制机制

• 批准号: 13306005
• 负责人: OKUNO Tetsuro
• 金额: $ 29.04万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13306005/
• 关键词: red clover necrotic mosaic virus; RNA virus; dianthovirus; RNA replication; cap-independent translation; 3'-UTR; RNA silencing suppressor; RNA structure; RNAサイレンシング; RNA構造; 宿主因子; 温度感受性; 3'非翻訳領域

Red clover necrotic mosaic virus genomic RNA1 and RNA2 lacked a 5' cap structure, and uncapped in vitro transcripts of RCNMV RNA1 replicated to a level comparable to capped transcripts in cowpea protoplasts. By using a luciferase reporter assay system in vivo, we showed that the 3'-untranslated region (3'-UTR) of RNA1 alone significantly enhanced translation of the luciferase reporter gene in the absence of the 5' cap structure. Deletion studies revealed that a stem-loop structure predicted in the 3'-UTR plays an important role in cap-independent translation. The cap-independent translational activity is required for RCNMV RNA1 replication.Translation of RNA2 was associated with RNA2 replication and differed from that of RNA1. Requirement of the formation of RNA replication complex for the translation of RNA2 suggested involvement of replication factors in the RNA2 translation.The stem-loop structure that functions as a trans-activator for the RNA-mediated CP expression is critically required for RNA2 replication itself. However, the RNA-RNA interaction is not required for RNA2 replication and these functions were independent. Nucleotide sequences in the stem-loop of RNA2 also are important for RNA2 replication, suggesting their role on minus-strand RNA possibly in plus-strand RNA synthesis.RCNMV RNA1 had RNA silencing suppressor activity. The activity required RCNMV replicase component proteins and viral RNA which could form viral RNA replication complex with the replicase component proteins. This suggests the involvement of RNA replication factors in RNA silencing suppressing mechanism of RCNMV and also the involvement of RNA silencing factors in RNA replicase complex of RCNMV.

红三叶草坏死花叶病毒基因组RNA 1和RNA 2缺少5'帽结构，并且RCNMV RNA 1的未加帽体外转录物复制到与豇豆原生质体中加帽转录物相当的水平。通过在体内使用荧光素酶报告基因测定系统，我们表明在5'帽结构不存在的情况下，单独的RNA 1的3'-非翻译区（3 '-UTR）显著增强荧光素酶报告基因的翻译。缺失研究表明，在3 '-UTR中预测的茎环结构在帽非依赖性翻译中起重要作用。RCNMV RNA 1的复制需要不依赖于帽的翻译活性，RNA 2的翻译与RNA 2的复制相关，但与RNA 1的翻译不同。RNA 2的翻译需要RNA复制复合物的形成，这表明RNA 2的翻译过程中有复制因子的参与，RNA 2的复制本身也需要茎-环结构，这种茎-环结构是RNA介导CP表达的反式激活因子。然而，RNA-RNA相互作用不是RNA 2复制所必需的，并且这些功能是独立的。RNA 2茎环区的核苷酸序列对RNA 2的复制也有重要作用，提示其在负链RNA合成中可能起正链RNA合成的作用。RCNMV的复制酶活性需要复制酶组分蛋白和与复制酶组分蛋白形成病毒RNA复制复合物的病毒RNA。这表明RNA复制因子参与了RCNMV的RNA沉默抑制机制，并且RNA沉默因子也参与了RCNMV的RNA复制酶复合体。

项目成果

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