Genetically recoding respiratory syncytial virus to visualize nucleoprotein dynamics and virion assembly

RNA viruses possess small genomes encoding a limited repertoire of essential and often multifunctional proteins. Although genetically tagging viral proteins provides a powerful tool for dissecting mechanisms of viral replication and infection, it remains a challenge. Here, we leverage genetic code expansion to develop a recoded strain of respiratory syncytial virus (RSV) in which the multifunctional nucleoprotein is site-specifically modified with an unnatural amino acid. The resulting virus replicates exclusively in cells capable of amber stop codon suppression and is amenable to labeling with tetrazine-modified fluorophores, achieving high signal-to-background. We use this tool to visualize the transfer of nucleoprotein complexes from cytoplasmic condensates directly to budding viral filaments at the cell surface and to cytoplasmic compartments containing viral surface proteins, suggesting multiple pathways for viral assembly.