This work is directed towards understanding the molecular processes of viral replication by the myxo-paramyxo-rhabdovirus group. Using vesicular stomatitis virus as the model system, we will study the interpedendence of transcription and replication with emphasis on the processes that regulate the transition between them. This proposal is based upon the hypothesis that the nucleocapsid protein plays a pivotal role in the transition from transcription to replication by formation of a sequence specific nucleation complex near the 5' end of plus sense RNA when free N protein reaches a critical concentration. The growth of nucleocapsides from that point inhibits processing into mRNA at intercistronic junctions. In the proposed work, we will determine the nucleotide sequence at the junction between the leader RNA and the first gene (N protein gene) by synthesizing cDNA from the 3' end of VSV RNA. This region on VSV plus strand RNA might be near the N protein nucleation site. Furthermore, nascent RNPs will be isolated or reconstituted in order to localize and sequence the initial nucleoprotein binding site on VSV RNA. The structure of mature ribonucleoproteins of VSV and DI particles will be examined by dissection with ribonucleases of various specificities and by placing radiolabels at accessible regions of RNA within RNP. We further propose that small RNAs synthesized from the 3' end of the RNAs of defective interfering particles are products of abortive replication. Therefore, small RNA products from the 3' termini of both VSV RNA and DIP RNA will be isolated from infected cells in order to determine whether they are present as free RNAs or as ribonucleoproteins. In addition, a unique defective interfering particle which is capable of transcription in vitro and in vivo, will be analyzed to determine its ability to interfere with the replication of VSV RNA. Also, we will test the ability of exogenously added VSV RNAs and RNA fragments to interfere with VSV replication and will measure the target size of the interfering components of a number of structurally dissimilar DI particles.
NIH ExPorter
NIH ExPorter