Structural mechanisms of respiratory syncytial virus and human metapneumovirus assembly

About This Grant

PROJECT SUMMARY/ABSTRACT Respiratory Syncytial Virus (RSV) and Human Metapneumovirus (HMPV) are leading causes of severe respiratory illness in infants, the elderly, and immunocompromised individuals, yet effective treatments and vaccines remain elusive. This critical unmet medical need stems from a limited understanding of the structural and molecular mechanisms governing how these viruses assemble and spread within host cells. This proposal will leverage cutting-edge cryo-electron tomography (cryo-ET), cryo-correlative light and electron microscopy (cryo-CLEM), and cryo-focused ion beam (cryo-FIB) milling, complemented by molecular biology and biochemical approaches, to provide unprecedented in situ structural insights into these processes. We hypothesize that distinct, yet conserved, molecular interactions between viral proteins and host factors orchestrate the precise formation of infectious virions, and disrupting these interactions represents a viable therapeutic strategy. Aim 1 will elucidate the high-resolution structural architecture of RSV and HMPV virion assembly directly within infected cells. We will employ cryo-FIB milling, cryo-ET, volume segmentation, and sub-tomogram averaging to determine the precise spatial organization and structures of viral components, identify structural intermediates at the plasma membrane, and investigate the involvement of host cellular machinery and membrane microdomains. Aim 2 will define the molecular mechanisms governing the critical nucleocapsid-matrix-envelope-glycoprotein linkages essential for RSV and HMPV assembly. This involves mapping protein-protein interfaces and characterizing conformational changes using cryo-EM/ET and biochemical assays, followed by functional validation of targeted mutations to assess their impact on virion formation and infectivity. Aim 3 will characterize the structural basis and functional differences between RSV and HMPV F proteins and their impact on virion morphogenesis. We will determine native high-resolution cryo-ET structures of F proteins, including HMPV F in complex with host receptors. Cryo-CLEM will visualize the localization and clustering of F protein during assembly and assess how mutations affect attachment, fusion, and virion infectivity. Collectively, this research will provide fundamental mechanistic understanding of pneumovirus assembly and dissemination in situ, directly informing the rational design of next-generation antivirals and other broadly protective therapeutics to combat these significant global health threats.