Extended Data Fig. 9: Additional analysis of the VF-like characteristics of PRCs and the conversion of a subset of passive vRNPs into PRCs.

a, Representative image series from time-lapse videos showing an infection with PRCs, where the vRNPs initially split, then subsequently fuse and increase in intensity. b, c, d, e, The effect of cyclopamine (CPM) on infecting vRNPs and VFs was assessed. (b) Representative images of P^exo-fluoro/DARPin-P-fluoro cells infected with WT RSV and treated with CPM for 1 h prior to fixation. Images show infecting vRNPs (4 hpi) and VFs (24 hpi). M2-1 antibody staining was used to evaluate CPM effects. Quantification of M2-1 intensities on vRNPs (c) and VFs (d) is shown. (e) Time-lapse imaging following infections initiated by a single vRNP in control and CPM-treated conditions, showing P^exo-fluoro intensities normalized to the initial intensity of the foci. f, g, Infections with passive vRNPs were further characterized. (f) Schematic highlighting the potential outcomes of passive vRNP infections. (g) Representative image series from a time-lapse video showing an infection with multiple passive vRNPs where one vRNP becomes a PRC post cell entry. Yellow arrowheads indicate first appearance of DARPin-P positivity. h, i, j, k, Analysis of the transcriptional activity of passive vRNP infections that either become PRCs or remain passive. Cumulative incidence graphs depicting the start of viral transcription (h) and violin plots showing the transcription rate (i) for the upstream SunTag^eng RSV strain. Cumulative incidence graphs depicting the start of viral transcription (j) and violin plots showing the transcription rate (k) for the downstream SunTag^eng RSV strain. l, Example intensity-time traces of vRNP P^exo-fluoro and DARPin-P-fluoro foci intensities are shown for the following 3 infection scenarios: (1) infections arising from passive⁻ vRNPs that remain passive, (2) infections from passive vRNPs that becomes a PRC during infection and, (3) infections with a PRC. m, Cumulative incidence graphs showing the kinetics of passive vRNPs becoming PRCs during infection in cells expressing only exogenous P or expressing both exogenous P and exogenous N. Note that the data for P^exo cells is replicated from Fig. 6n for comparison purposes. (e, h, j, m) Mean (line) and SE (shaded area) are shown. (c, d, i, k) The median and quartiles are shown (horizontal lines) (c) One-way ANOVA with Tukey’s multiple comparisons test was used for statistical analysis. (d, i, k) Two-tailed unpaired Student’s t-test was used for statistical analysis.(a, b, g) Scale bars, 10 µm. (a, g) Time, h: min. The number of experimental repeats and fluorophores used are listed in Supplementary Table 1.