The long-term goal of this project is to understand the molecular basis of growth and pathogenicity of human respiratory syncytial virus (RSV), the single most important agent of respiratory diseases in the young children throughout the world and a probable agent of sudden infant death syndrome (SIDS). A recurring cause of pneumonia-like disease characterized by rhinitis, pharyngitis, and croup, it accounts for significant morbidity in the first year of human life. It is now clear that a better understanding of the regulation of viral growth at the molecular level is an essential prerequisite to a successful control of this human pathogen, particularly in view of the failure of immune therapy. Although considerable information is available regarding the nucleotide sequence of the viral genome and its constituent genes, our knowledge about the molecular mechanism of expression of the genes remains poor. Recently, we have taken an important step in this direction by establishing an in vitro of transcription system of RSV which efficiently synthesizes all viral mRNAs. The fidelity of the system is underscored by our finding that the mRNAs are capped, methylated and polyadenylated. More importantly, we have demonstrated an essential requirement of cellular protein(s) in viral transcription. Recently, we have dissected the RSV transcription apparatus even further: we have purified viral L protein, phosphoprotein, and the N-RNA template from one another and reconstituted functional transcription using these purified macromolecules. The reconstituted system is now amenable to studies of structure and function of its components. We have recently expressed RSV P protein in E. coli in a phosphate-free form and shown that it can be phosphorylated by cell kinase in vitro. Inhibition of the cell kinase also inhibited RSV transcription suggesting an essential role for P protein phosphorylation in its function. The immediate major goal is to use the reconstituted system to understand the role of P protein phosphorylation in detail, and to characterize the identity of the cellular protein (s) and determine its exact role in RSV transcription. These studies should provide important insights into the functioning of the viral transcription machinery which is an essential component in RSV growth and pathogenesis.
