Respiratory syncytial (RS) virus contains a single-stranded RNA genome that is devoid of any messenger RNA (mRNA) activity, and formation of mRNA is therefore the first biosynthetic step carried out under the direction of the virus in infected cells. It is our long-term objective to examine the enzymatic steps involved in RS virus mRNA and genomic RNA synthesis in greater detail in order to obtain definitive information about the replication of this important human pathogen. The goal of this proposal is to elucidate the in-intro capability of purified virus to synthesize mRNAs in a cell-free transcrition system. The structural and functional characteristics of in-vitro synthesized RNAs will be compared to those of mRNAs isolated from infected cells by several techniques, including annealing, sedimentation and electrophoretic analyses as well as by translation in cell-free systems, to establish the similarity of the in-vitro product to mRNAs. Experiments on the synthetic capabilities of the system to determine whether the genome is completely transcribed and reinitiation occurs are planned. Finally, transcriptional complexes will be isolated from virions to investigate the proteins required for transcription and whether complexes retain the complete synthetic capabilities of unfractionated virions.
