Our long range goal is to understand the molecular mechanism of transcription and replication of the RNA genomes of the negative strand viruses. For VSV, we will determine the transcriptive properties of a synthetic virion (-) strand nucleocapsid which can be assembled from synthetic RNA and purified N protein in vivo. We will test the ability of this nucleocapsid to bind the individual VSV polymerase subunits derived from purified virus or synthesized de novo from an expression vector. Productive binding will be measured by synthesis of (+) strand leader RNA from the nucleocapsid. By mutating the RNA of the nucleocapsid, we can identify the cis sequences in the RNA which regulate transcription and RNA processing. We plan to extend our studies on the sequence requirements for the specific in vitro encapsidation of synthetic VSV RNAs by purified wild type and mutant N proteins. Ultimately we would like to assemble defective interfering nucleocapsids in order to identify the minimal sequences which are required for replication and interference in vitro. We also plan to characterize a newly discovered cellular factor which confers specificity for VSV RNA on the encapsidation process. Using Sendai virus, a system very amenable for in vitro replication studies, we hope to understand the functional domains of the viral proteins required for the various steps of RNA replication. We will synthesize wild type and mutant NP (nucleocapsid) proteins via an expression vector containing the NP cDNA for use in replication reconstitution experiments. We shall identify and characterize a second viral protein which is required for the initiation step of SV RNA encapsidation. We shall characterize the nature of the interaction between and the roles of the Sendai virus M protein and cellular actin which act to inhibit viral RNA synthesis and promote morphogenesis, respectively, utilizing purified and/or genetically engineered proteins. For both viruses we shall continue to characterize the role of tubulin and MAPs in RNA synthesis by studying how these proteins interact with the RNA polymerase subunits and the RNA-N template and affect transcription.
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