Genetically\nRecoding Respiratory Syncytial Virus to\nVisualize Nucleoprotein Dynamics and Virion Assembly

RNA viruses possess small genomes encoding a limited\nrepertoire\nof essential and often multifunctional proteins. Although genetically\ntagging viral proteins provides a powerful tool for dissecting mechanisms\nof viral replication and infection, it remains a challenge. Here,\nwe leverage genetic code expansion to develop a recoded strain of\nrespiratory syncytial virus (RSV) in which the multifunctional nucleoprotein\nis site-specifically modified with a noncanonical amino acid. The\nresulting virus replicates exclusively in cells capable of amber stop\ncodon suppression and is amenable to labeling with tetrazine-modified\nfluorophores, achieving high signal to background. Virus with labeled\nnucleoprotein remains functional, retaining ∼70% infectivity\nrelative to unlabeled controls. We leverage this tool to visualize\nRSV assembly, capturing the transfer of nucleoprotein complexes from\ncytoplasmic condensates directly to budding viral filaments at the\ncell surface and to cytoplasmic compartments containing viral surface\nproteins. Collectively, these results suggest multiple pathways for\nRSV assembly and establish a framework that may be extended to other\nviral nucleoproteins.