Analysis on mechanisms of brome mosaic virus RNA replication

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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.