Fig. 6: PRCs are biomolecular condensate seeds.

a–c, Visualization of L protein levels in virions and on vRNPs after host cell. Representative image series showing L protein levels in virions before entry and on vRNPs after entry for passive vRNPs and PRCs (a). Quantification of L levels in virions (b) and on vRNPs over time (c). Minpi, minutes post infection. d–g, Visualization of endogenous P (P^endo) on vRNPs using the P-ALFA-Tag^eng RSV strain. A schematic of P^endo visualization in cells expressing Nb-ALFA-fluoro (d); a representative image series of the vRNP P^endo signal over time on passive vRNPs and PRCs (e); quantification of P^endo levels on the infecting vRNP (f) and the relative P^endo levels on vRNPs over a 1-h period post-entry (g) are shown. h–j, Viral protein association kinetics with passive vRNPs and PRCs, assessed using the P^exo-fluoro/DARPin-P-fluoro system. Infections using the P-ALFA-Tag^eng RSV strain in cells additionally expressing Nb-ALFA-fluoro, allowed vRNP identification independent of the P^exo-fluoro signal. A schematic showing visualization of exogenous viral P protein association with cytoplasmic vRNPs (h); a representative image series of the vRNP P^exo-fluoro signal over time (i); and quantification of the P^exo-fluoro levels on vRNPs over 30 min (j). k,l, Quantification of vRNP separation kinetics (k) and vRNP fusion kinetics (l) as cumulative incidence graphs. Data are median (k). m–o, Analysis of passive vRNPs transitioning to PRCs. A representative image series illustrating such transitions (m; see also Supplementary Video 6), cumulative incidence graphs showing the kinetics of passive vRNPs becoming PRCs (n) and their infection success rate (o) are shown. p, Schematic showing the mechanism by which a subset of infecting vRNPs seed VFs. For simplicity, single vRNP virions are shown. The lines are means and the shaded regions are s.e. (c,g,j,l,n,o). A two-tailed unpaired Student’s t-test was used for statistical analysis (b,f,k). Scale bars, 1 µm (a,e,i,m). Experimental repeats and fluorophores are listed in Supplementary Table 1.