ANALYSIS OF RNA REPLICASE COMPONENTS OF DIANTHOVIRUS

石竹病毒RNA复制酶成分分析

• 批准号: 11660049
• 负责人: OKUNO Tetsuro
• 金额: $ 2.3万
• 依托单位: KYOTO UNIVERSITY (2000)Kochi University (1999)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11660049/
• 关键词: red clover necrotic mosaic virus; temperature sensitivity; dianthovirus; RNA replication; cis-acting sequence; 3'UTR; 3'非翻訳領域; シス因子; RNA; レプリカーゼ; RNA合成; RNAウィルス; 抗体; RNA依存RNA合成酵素

Infectious cDNA clones of genomic RNAs 1 and 2 of Red clover necrotic mosaic virus Canadian strain (RCNMV-Can) with temperature sensitive (ts) phenotype were constructed and the nucleotide sequences were determined. Experiments with reassortants and chimeric RNA1s between ts-insensitive RCNMV-Aus and RCNMV-Can indicated that the temperature sensitivity of RCNMV-Can is in an early stage of RNA replication and that viral determinants for the ts phenotype are located in the 3'-untranslated region of RCNMV-Can RNA1.RCNMV infection-specific RNA dependent RNA polymerase (RdRp) was effectively prepared from virus-infected protoplasts. Results from in vitro RNA synthesis using the membrane bound RdRp and its solubilized fraction suggested that the ts phenotype of RCNMV-Can was not due to defect in the elongation steps of RNA synthesis but was due to initiation steps including negative-strand RNA.Polyclonal antibody that specifically reacts with both 27 kD and 88 kD replicase component proteins of RCNMV was obtained by using a Histidine tag-fusion polypeptides of 27 kD produced in E.coli. We failed to produce other antibodies reacting with other regions of 88 kD replicase component because of unstable expression of virus fusion proteins in E.coli.

构建了具有温度敏感（ts）表型的红三叶草坏死花叶病毒加拿大株系（RCNMV-Can）基因组RNA 1和RNA 2的感染性cDNA克隆，并测定了其核苷酸序列。利用RCNMV-Can与ts不敏感RCNMV-Aus的嵌合体和RCNMV-Can的重组体进行的实验表明，RCNMV-Can的温度敏感性处于RNA复制的早期阶段，ts表型的病毒决定簇位于RCNMV-Can RNA 1的3 '非翻译区。利用膜结合的RdRp及其可溶性组分进行体外RNA合成的结果表明，RCNMV-Can的ts表型不是由于RNA合成的延伸步骤缺陷，而是由于包括负链RNA的起始步骤。在大肠杆菌中产生的27 kD的融合多肽。由于病毒融合蛋白在大肠杆菌中的表达不稳定，我们未能产生与88 kD复制酶组分的其他区域反应的其他抗体。

项目成果

島田賢治 ら: "レッドクローバーネクロティックモザイクウイルス感染特異的RNA依存RNA複製酵素の抽出とin vitroでの温度感受性"日本ウイルス学会(要旨). 48. 235 (2000)

Kenji Shimada 等：“红三叶草坏死花叶病毒感染特异性 RNA 依赖性 RNA 复制酶的提取和体外温度敏感性”日本病毒学会（摘要）48. 235（2000）。

Mizumoto,H. et al.: "Temperature sensitivity of red clover necrotic mosaic virus-Canadian strain"Abstract in the International Congress of Virology. 11. 351 (1999)

水本，H.

Mizumoto, H.et al.: "Temperature sensitivity of of red clover necrotic mosaic virus Canadian strains"Abstract in 11^<th> International Congress of Viroloty, Sydney. 351 (1999)

Mizumoto, H.等人：“红三叶草坏死花叶病毒加拿大株的温度敏感性”摘要，第 11 届国际 Viroloty 大会，悉尼。

水本裕之 ら: "レッドクローバーネクロティックモザイクウイルスカナダ株とオーストラリア株間でのリアソータントウイルスの温度感受性"日本植物病理学会報. 66. 299 (2000)

Hiroyuki Mizumoto 等：“加拿大和澳大利亚红三叶草坏死花叶病毒之间重配病毒的温度敏感性”日本植物病理学会杂志 66. 299 (2000)。

Shimada, K.et al.: "Extraction of red clover necrotic mosaic virus infection-specific RNA polymerase and its temperature sensitivity in vitro"Congress of Japanese Virology. 48. 235 (2000)

Shimada，K.等人：“红三叶草坏死花叶病毒感染特异性RNA聚合酶的提取及其体外温度敏感性”日本病毒学大会。