Analysis on mechanisms of brome mosaic virus RNA replication

参与病毒RNA复制的细胞结构或微细胞器一直存在争议。从BMV感染的大麦植物中提取对病毒感染特异的RNA依赖性RNA聚合酶（RdRp）。RdRp与感染细胞的膜结构和RNA模板相关。使用BMV RNA特异性探针对BMV感染特异性膜结构的超微结构研究尚未成功分配病毒RNA复制位点的更详细结构。目前，BMV复制酶组分、大肠杆菌中产生的1a和2a蛋白可用于获得单克隆抗体，并可用于进行蛋白质的表位定位。用抗BMV 1a蛋白单克隆抗体对BMV感染的细胞进行免疫电镜观察，结果表明，BMV感染细胞的膜结构特异性发育，并含有1a蛋白特异性的金颗粒。细胞膜结构与健康大麦细胞中常见的微细胞器不同。侵染特异性结构随侵染期的延长而发育。使用BMV感染大麦植物的膜部分进行的类似研究表明，BMV 1a蛋白特异性的金颗粒与健康细胞中存在的任何微细胞器无关。纯化的BMV RdRp级分用于类似的研究。经SDS处理的RdRp组分固定在筛网上，与1a单克隆抗体反应，可观察到1a蛋白特异性的单或双金颗粒，而未经SDS处理的样品则观察到4-7个金颗粒的团簇，这表明BMV 1a蛋白以寡聚体形式存在，BMV复制酶可能由1a寡聚体组成。用11种抗BMV 1a蛋白的单克隆抗体对BMV 1a蛋白进行了表位定位。

Cellular structures or micro-organella involved in virus RNA replication have been debated. RNA dependent RNA polymerase (RdRp) specific to virus infection is extracted from BMV infected barley plants. The RdRp was associated with membrane structure and RNA templates in the infected cells. Ultrastructural study of the BMV-infection specific membrane structure using BMV RNA specific probe has not been successful to assign more detailed structure for sites of virus RNA replication. Now, BMV replicase components, 1a and 2a proteins produced in E.coli are available to obtain monoclonal antibody which can be used to make epitope mapping of the proteins. Immunoelectron microscopic study of BMV-infected cells using monoclonal antibody against BMV 1a protein indicated that mebrane structures specifically developed to virus infection and the structures contained 1a protein specific gold particles. The membrane structures were distinct from micro-organella usually observed in healthy barley cells. The infection specific structures developed with infection stage. Similar study using membrane fraction of BMV infected barley plants showed that gold particles specific to BMV 1a protein was not associated with any micro-organella present in healthy cells. Purified BMV RdRp fraction was used for similar study. When RdRp fraction treated with ionic detergent (SDS) was fixed on a mesh and reacted with 1a monoclonal antibody, 1a protein specific gold particles were observed in single or double, while gold particles were observed in a cluster containing 4-7 particles in samples untreated with SDS.This suggests that BMV 1a protein exists as oligomer and that BMV replicase may consist of the 1a oligomer. Epitope mapping of BMV 1a protein was made using 11 monoclonal anitodies against 1a protein.