Pre-assembly of biomolecular condensate seeds drives RSV replication.

During infection, many RNA viruses, including respiratory syncytial virus (RSV), form specialized biomolecular condensates, viral factories (VFs), where viral transcription and replication occur1,2. Paradoxically, high protein concentrations are typically required for condensate nucleation3, yet attaining sufficient protein levels in infection is thought to require VFs for viral transcription and replication. Here, to uncover how viruses solve this paradox to establish VFs, we visualized early infection of RSV in real time with single genomic viral ribonucleoprotein (vRNP) resolution. Our results reveal that VFs are nucleated from infecting vRNPs rather than de novo in the cytoplasm. VF nucleation further requires in-virion pre-assembly of viral protein-protein interaction networks on vRNPs to form 'pre-replication centres' (PRCs). PRCs are potent condensate nucleation seeds due to their efficient recruitment and retention of viral proteins. The high affinity of PRCs also results in increased association of the viral polymerase and its co-factors, allowing efficient viral transcription even in the absence of VFs. Together, these activities create a feed-forward loop that drives rapid VF formation. PRC assembly depends on in-virion viral protein levels and is highly heterogeneous among virions, explaining cell-to-cell heterogeneity in infection progression, and identifying heterogeneous virions as an important origin of infection heterogeneity. Together, our results show that in-virion pre-assembly of PRCs kick-starts viral condensate nucleation upon host-cell entry and explains cell-to-cell heterogeneity in RSV infection.