Abstract
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 a noncanonical 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. Virus with labeled nucleoprotein remains functional, retaining ∼70% infectivity relative to unlabeled controls. We leverage this tool to visualize RSV assembly, capturing 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. Collectively, these results suggest multiple pathways for RSV assembly and establish a framework that may be extended to other viral nucleoproteins.