Extended Data Fig. 3: Validation of the P^exo-fluoro cell line and RSV polymerase inhibitor, PC786.

a, The P^exo-fluoro cell line expresses low levels of RSV P-SunTag and fluorescently-tagged STAb. Representative images show the levels of P mRNA (via smFISH using a probe set for P) and protein (anti-RSV P antibody) in the cell line. b, c, RSV infection in the P^exo-fluoro cell line was compared to that in WT A549 cells at 2 and 6 h post viral inoculum addition by antibody staining for vRNPs (anti-RSV N antibody). Representative images (b) and quantification of the number of vRNPs per cell (c) is shown. d, e, f, RSV infection in the P^exo-fluoro cell line, WT A549 cells and in the presence of the RSV polymerase inhibitor, PC786, was assessed at 2 and 6 h post viral inoculum addition by smFISH staining towards the RSV genome and N transcripts. (d) Representative images are shown. Quantification of the (-)vRNA (e) and N mRNA (f) levels. g, Characterization of RSV vRNP separation after host cell entry observed for cells infected with virions containing 1-4 vRNPs. PC786 was added to all cells to ensure increases in vRNP number are caused by splitting of incoming vRNPs and not a consequence of genome replication. Foci intensities of P^exo-fluoro for the foci before splitting (red) and individual foci intensities after reaching the maximum foci number after splitting (grey). Each data point is an individual foci. h, High-time resolution imaging (10 sec interval) captured transient cytoplasmic co-localization of incoming vRNPs from the same virion in P^exo-fluoro cells. Cell were supplemented with PC786 to prevent infection progression. For virions carrying multiple vRNPs, the time interval from entry to initial vRNP separation was quantified. See also Supplementary Video 2. (c, e, f, g) Two-way ANOVA with Tukey’s multiple comparisons test used for statistical analysis. (a, b, d) Scale bar, 10 µm. The number of experimental repeats and fluorophores used are listed in Supplementary Table 1.