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。RNA依赖RNA聚合酶(RdRp)是从BMV侵染的大麦植株中提取的针对病毒感染的RNA依赖RNA聚合酶。RdRp与感染细胞的膜结构和RNA模板有关。用BMV RNA特异性探针对BMV感染的特异膜结构进行超微结构研究，尚不能确定病毒RNA复制部位的更详细结构。目前，在大肠杆菌中生产的BMV复制酶组分、1a和2a蛋白可用于获得可用于蛋白质表位定位的单抗。用抗BMV-1a蛋白的单抗对BMV感染的细胞进行免疫电子显微镜研究，结果表明，BMV-1a蛋白的膜结构是病毒感染所特有的，膜结构中含有1a蛋白特异的金颗粒。这些膜结构不同于健康大麦细胞中常见的微细胞器。感染特异性结构随感染阶段的进展而发展。用BMV感染的大麦植株的膜部分进行的类似研究表明，BMV 1a蛋白的特异性金粒子与健康细胞中存在的任何微细胞器都不相关。纯化的BMV RdRp组分用于类似的研究。用离子洗涤剂(SDS)处理过的RdRp组分与1a单抗反应时，1a蛋白特异性金粒呈单粒或双粒状，而未经离子洗涤剂处理的样品中金粒呈4~7粒簇状分布，提示BMV 1a蛋白以低聚体形式存在，BMV复制酶可能由1a寡聚体组成。用11个抗1a蛋白的单克隆无性体对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.