The purpose of this research is to study the mechanism of replication of the negative-stranded RNA viruses using vesicular stomatitis virus (VSV) as a prototype. The unifying aim of this proposal is to identify factors relevant to the control of expression of information in the genome of the negative-strand virus. In particular the questions we propose to ask are designed to evaluate the role of several factors in controlling whether the VSV negative strand template will be expressed by transcription to yield discrete mRNAs or by replication to produce the genome-sized virion complementary (VC) RNA.
Our specific aims are as follows: 1. Evaluation of the relation of the discrete secondary structures which we have identified in the VSV genome-size RNAs, but not the mRNAs, to possible biological function. To do this the location of these structures on the genome will be mapped and then the size, number, sequence relatedness and primary sequence in the structures will be determined. This information will be related to the location and known sequence of the virion RNA termini, intercistronic and leader RNA junctions. 2. Analysis of the virion RNA and RNP in situ for interacting structures. This will test whether or not the secondary structures observed in VSV RNAs are due to interactions of contiguous sequences or distance sequences in the genome. 3. Analysis of the architecture of VSV RNA synthetic structures by electron microscopy. 4. Study of the relation of the VSV NS protein structure, conformation and levels of phosphorylation to RNA transcription and replication. 5. Characterization of an in vitro translation assisted RNA synthesis system. 6. Characterization of RNAs synthesized by respiratory syncytial (RS) virus in vivo and in vitro. Identification of polyadenylated mRNA species and assignment of mRNA coding capacity by in vitro translation of separated messages.
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