The actin cytoskeleton controls key cellular processes, including cell morphology, motility, and intercellular communication. Many viral pathogens exploit the actin cytoskeletal machinery by a variety of unique mechanisms to facilitate viral infection, replication, and egress. Currently, the molecular underpinnings of these viral mechanisms remain poorly defined for a majority of viruses, including members of the Pneumoviridae family. Human respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are non-segmented negative strand RNA viruses that are members of the recently created Pneumoviridae family. RSV and HMPV cause significant disease, including bronchiolitis and lower respiratory tract infection in the pediatric population, elderly, and immunocompromised individuals. Recent studies from our groups and others have implicated interactions between actin or actin-interacting proteins and specific pneumovirus proteins in replication and viral spread; however, the mechanisms and cellular targets required for facilitating actin rearrangements remain unknown. Our overall hypothesis is that a cascade of specific interactions between pneumovirus proteins and the actin cytoskeleton regulates key steps in viral infection, including formation of novel structures critical for virus cell-to-cell spread. Our collaborative team, with expertise in virology, cell biology, biochemistry, and structural biology, will test our hypothesis through three specific aims. First, we will dissect the interactions between RSV and HMPV matrix (M) proteins and phosphoproteins (P) that regulate the actin cytoskeleton within an infected cell. Second, we will determine the biochemical and structural basis for HMPV phosphoprotein/matrix/actin complex and RSV P/M/actin complex. Finally, we will determine the mechanisms of intercellular extension formation, stabilization, and utilization in viral spread. At the completion of these studies, we expect to define molecular mechanisms by which RSV and HMPV interact with and modulate the host actin cytoskeleton during virus replication, define key elements needed for this modulation, and understand the molecular details of cell-to-cell spread through intercellular extensions, thus providing critical new insights that may be applicable in multiple viral systems.