PROJECT ABSTRACT The long-term goal of our project is to elucidate the molecular basis of the poorly understood mechanism of the RNA synthesis machinery of NNS RNA viruses and to determine the structures of key protein complexes involved in the RSV RNA synthesis, facilitating the development of antiviral drugs. Despite several decades of research, the molecular mechanism of RNA synthesis by NNS RNA viruses remains elusive. The catalytic core of the RNA synthesis machine of NNS RNA viruses is the RNA-dependent RNA polymerase (RdRP) that comprises a large 250 kDa protein (L) and a cofactor phosphoprotein (P). The L protein catalyzes three enzymatic activities: nucleotide polymerization, cap addition, and cap methylation. P is essential for the full activity of L to act on the viral genome. In some cases, additional viral proteins (VP30 in Ebola and M2-1 in RSV) are necessary for full RdRP processivity. Several important and long-standing questions for RNA synthesis by NNS RNA viruses remain unanswered - First, how does L carry out the catalytic reactions of the phosphodiester bond formation of the first two nucleotides (de novo RNA synthesis), RNA elongation (RNA extension), and RNA polyadenylation (shutter mechanism)? Second, how does a single protein L coordinate three distinct enzymatic activities (makes, caps, and methylates RNA), and what determines the switch to the next process? Third, how do the cofactor P and transcription factor M2-1 regulate the activities of the L protein? The lack of clear answers to these critical questions represents a major knowledge gap in our understanding of the mechanism of NNS RNA synthesis. We propose to tackle these challenging questions with a novel hybrid approach that integrates biochemistry, enzymology, mutagenesis, virology, genetics, crystallography, and cryo- EM methods. Guided by our preliminary data, we hypothesize that the catalytic activities of RNA polymerization, cap addition, and cap methylation reside within RSV L and that L requires a dynamic assembly with P and M2- 1 to coordinate these activities during RNA synthesis. To test this hypothesis, we will investigate the functional interplay between the multi-functional enzyme L, cofactor P, and transcription factor M2-1. We will seek to provide clear answers to the long-standing questions regarding the mechanisms governing RNA synthesis of RSV and other NNS RNA viruses. The proposed research is significant and groundbreaking because the novel knowledge and structures obtained from this proposed research will significantly advance our understanding of RSV and NNS RNA synthesis. Ultimately, such knowledge will provide a framework for developing novel antivirals to treat RSV infections.