The rhabdovirus vesicular stomatitis virus (VSV) is a model system for the study of the group of viruses possessing non-segmented single- stranded RNA genomes of the negative strand sense. It includes such human pathogens as measles, mumps, rabies, respiratory syncytial virus, human parainfluenza viruses, and many viruses that infect economically important animals. A central question concerning the growth of these viruses is: by what mechanism do they control the expression of their genetic information through transcription and replication of the genome. The goals of the research in this application are two fold. First, we will continue our study of the form and function of the proteins involved in the assembly of newly replicating nucleocapsids. We will attempt to clarify the relationship between the soluble forms of the N protein in the cell and the various RNA-binding activities of the molecule (encapsidation of genome-length RNA, encapsidation of free leader RNA, and binding to mRNAs). Secondly, we will perform a molecular genetic analysis of cis-acting regulatory sequences in controlling the processes of transcription, replication, and nucleocapsid assembly. We will do this by producing a full-length cDNA copy of the genome RNA of a defective-interfering (DI) particle of the virus, and placing this cDNA under the control of a bacterial promoter in the plasmid pPMI. In this way, we will be able to produce RNA of identical sequence to the DI genome in vitro. We have developed a system to assemble the RNA into functional nucleocapsids in vitro, and will use this system in conjunction with site-directed mutagenesis of the cloned DI genome to assess the role of specific RNA sequences in nucleocapsid assembly, initiation and termination of transcription, intragenic pausing of the virion polymerase, and to address questions relating to the mechanism of interference of standard virus by DI particles. We will also investigate the possible construction and use of chimeric DI nucleocapsids as vectors for gene expression in mammalian cells.
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