ANALYSIS OF RNA REPLICASE COMPONENTS OF DIANTHOVIRUS

石竹病毒RNA复制酶成分分析

基本信息

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

项目摘要

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.

构建了带有温度敏感（TS）表型的红色三叶草坏死性马赛亚菌株（RCNMV-CAN）的基因组RNA 1和2的传染性cDNA克隆，并确定了核苷酸序列。在TS不敏感的RCNMV-AUS和RCNMV-CAN之间进行了重新成分和嵌合RNA1的实验表明，RCNMV-CAN的温度敏感性处于RNA复制的早期阶段，并且对TS表型的病毒决定因素位于3'-Untranslanslatanslatanslated RCNMV-CAN RNA1.RCNMV感染特异性RNA依赖性RNA聚合酶（RDRP）的区域有效地从病毒感染的原生质体中制备。使用膜结合的RDRP及其溶解的馏分是由体外RNA合成的结果表明，RCNMV-CAN的TS表型不是由于RNA合成的伸长步骤中的缺陷而导致的，而是由于启动步骤，包括负链RNA。链链RNA.polyclonal.polyclonal抗体。通过使用E.Coli中产生的27 kD的组氨酸标签 - 融合多肽获得了RCNMV的27 kD和88 kD复制酶成分蛋白的特异性反应。由于病毒融合蛋白在大肠杆菌中的不稳定表达，我们未能产生其他与88 kD复制酶成分反应的其他抗体。