MECHANISM OF RNA REPLICATION OF NEGATIVE-STRAND VIRUSES

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The objective of this proposal is two fold. First, the mechanism by which the negative-stranded RNA virus vesicular stomatis virus (VSV) replicates its genome RNA will be investigated. Secondly, the mechanism of interference of the replication of wild-type VSV RNA genomes by defective-interfering (DI) particles of VSV will be studied. Both of these objectives will be investigated using an in vitro system which I developed, and which is ideally suited to study these problems. This system is derived from infected cells and has been shown to support replication of VSV genomic RNA, as well as its encapsidation into nucleocapsids. A complex of two VSV proteins (N and NS) has been previously identified as active in supporting replication of the genome RNA in vitro. Attempts will be made to purify this active protein complex, and study how this complex is involved in the control of the switch from transcription to replication of viral RNA. Experiments will also be performed to determine if this complex can be formed in vitro between the separated proteins, and if so, if this complex is able to support RNA replication. The complex will be examined for the presence of host cell components, and if detected, experiments will be performed to determine if these cellular proteins are functional in the complex. I also plan to produce monoclonal antibodies against the NS and L proteins of the virus, and then use these to determine the role of these proteins in viral RNA replication in vitro. Since these proteins may be multifunctional, a collection of monoclocal antibodies against specific sites of each protein may help define their role in replication. The mechanism by which defective-interfering (DI) particles of VSV interfere with the replication of VSV RNA will be investigated by adding purified DI particles to standard VSV RNA replicating reactions and determining the relative sensitivity of plus and minus stranded 42S RNA to inhibition by the DI. Effects will also be assessed on the synthesis if the VSV leader in these reactions. Two temperature-sensitive (ts) mutants of VSV will be employed in the in vitro RNA replicating system. These are a group IV mutant, tsG41, carrying a lesion in the N protein, and a group II mutant, with a lesion in the NS protein. Both of these mutants have non-temperature-sensitive virion-associated transcriptases and produce viral mRNA at the nonpermissive temperature, but each fails to replicate the viral genomic RNA at nonpermissive temperature. Affects of these ts lesions will be assessed in vitro.