Human respiratory syncytial virus (RSV) is responsible for a major fraction of severe acute respiratory tract infections, including pediatric, elderly, and immunocompromised individuals, and thus has a global impact on human health. Despite this, there are limited prophylactic and specific treatment options available for RSV infections. RSV nonstructural proteins NS1 and NS2, which are unique to RSV among the pneumoviruses, play multiple major roles that are thought to enhance viral infection and to prevent protection from subsequent RSV reinfection. However, many questions remain that are related to how RSV virally encoded proteins shape the host response. Our unpublished preliminary studies show that NS1 partitions to the nucleus in physiologically relevant cell culture models, binds chromatin at promoters and enhancers of immune response genes, and impacts host gene transcription. In addition, we observe that nuclear NS1 interacts with viral proteins NS2 and matrix (M), an indication of further modulation of host factors in the nucleus by RSV viral proteins. Functional correlates of these observations are poorly understood. Based on these novel findings and to fill this knowledge gap, we propose to determine the molecular mechanisms of NS1 as a major modulator of the epigenome and host responses during RSV infection. Nuclear NS1 may also skew antiviral responses while enhancing immune evasion, thereby promoting viral pathogenesis. Our team, with complementary expertise in biochemistry/structural biology, epigenetics, transcriptional regulation, and pulmonology will define the impact of RSV NS1 on host epigenetic transcriptional control, define and characterize the molecular interactions that contribute to nucleocytoplasmic transport of NS1, and determine the impact of RSV NS2 and M protein interactions on NS1 nuclear functions. To obtain mechanistic insights and to assess the impact of these observations during viral infections, we will use an approach that integrates biochemical, structural, genetic, cellular, and virological studies in relevant cell culture models, including primary human lung derived cells. By completing these synergistic Aims, we expect to provide insights into key contributors to disease and define novel targets for antiviral and vaccine development.