We plan a systematic, detailed comparative study of the mechanism of replication of the RNA genomes of two different negative strand viruses, vesicular stomatitis virus (VSV), a rhabdovirus and Sendai virus, a paramyxovirus. We will carry out these studies with the use of an in vitro reconstitution assay for RNA genome replication that we have recently developed, utilizing nucleocapsids derived from either the defective interfering particles of each virus or the wild type viruses as templates. We shall determine if the requirements for initiation are the same as those for elongation during RNA replication. For each virus we shall identify and purify the individual viral proteins required for the various steps of RNA replication and encapsidation. We also propose to develop an assay by which we can identify and purify any host cell factors which may be involved specifically in the replication of the RNA genomes of these viruses. We plan to select hybridomas secreting monoclonal antibodies to the VSV and Sendai nucleocapsid proteins, and also to isolate and use amber mutants in these proteins, as approaches to understanding the specific functions of the individual viral nucleocapsid proteins in RNA replication.
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