Extended Data Fig. 5: DARPin-P⁺ vRNPs give rise to successful infections.

a, b, WT RSV infections in P^exo-fluoro cells were further characterized. The P^exo foci area (a) and intensity (b) of the successful and stalled infections shown in Fig. 3a,b and Supplementary Video 3 were quantified. Foci were assessed post splitting from 3 hpi onwards, every hour, till 15 hpi. c, Following VF formation in RSV infected P^exo-fluoro cells, there is a rapid increase in foci. These are vRNP progeny that stain positive for RSV genomic RNA ((-)vRNA) by smFISH. d, e, Cells where a successful RSV infection was established were assessed for viral mRNA expression (d) and viral protein levels (e) in P^exo-fluoro cells at 24 hpi. f, g, h, M2-1^exo-fluoro allowed visualization of Inclusion Body-Associated Granules (IBAGs), internal substructures present in a subset of RSV VFs. (f) Schematic representation of the M2-1^exo-fluoro system. Here exogenous, fluorescently-tagged M2-1 is expressed in cells. Exogenous M2-1 encodes a SunTag peptide and the cells additionally co-express fluorescently-tagged STAb allowing its visualization. Combining with P^exo-fluoro (tagged with the ALFA-Tag, visualized by nanobody-ALFA-fluoro) and DARPin-P-fluoro allows visualization of IBAG formation during RSV infection. Two representative example image series (g) and quantification (h) assess the moment of IBAG formation in relation to RSV replication. i, The number of VFs present at 20 h post RSV infection was quantified. j, k, RSV infected cells that were positive for extracellular viral G protein were assessed for their viral RNA composition by smFISH. These cells always displayed viral antigenomes (j) as well as high levels of viral mRNA transcripts (RSV whole transcriptome smFISH probe set) (k). l, m, n, o, p, Cell culture supplemented fluorescently-conjugated RSV G antibody (AbG-fluoro) identifies successful RSV infections (as defined by observing VFs and vRNP replication). (l) Schematic of the AbG-fluoro labeling approach. (m) P^exo-fluoro cells were infected with viral inoculum supplemented with increasing concentrations of AbG-fluoro and infection assessed at 6 hpi. Quantification shows no inhibitory effect on viral entry at concentrations up to 1:50 of the antibody compared to no antibody control. Each data point represents an imaged FOV. A dilution of 1:1000 was used for all experiments. (n) Representative images of extracellular AbG-fluoro accumulation on infected cells relative to the moment of vRNP fusion. (o) The timing of AbG-fluoro labeling relative to vRNP fusion is displayed by a cumulative incidence graph. Only infections with >1 vRNP were included to analyze vRNP fusion. (p) For infections with >1 vRNP, AbG-fluoro positivity is exclusively observed in infections where vRNPs have fused. All cells were imaged for at least 20 hpi. q, r, The potential variations in infection by virions carrying DARPin-P⁺ and DARPin-P⁻ vRNPs were assessed. (q) Cumulative incidence graphs showing the time from inoculum addition to vRNP entry for both DARPin-P⁺ and DARPin-P⁻ virions. (r) The number of vRNPs per infected cell for DARPin-P⁺ and DARPin-P⁻ virions was quantified. Comparing DARPin-P⁺ and DARPin-P⁻ virions show no significant difference across all foci numbers. s, t, Infection success for DARPin-P⁻ (s) and DARPin-P⁺ (t) infections were assessed in relation to the number of vRNPs and displayed as cumulative incidence graphs. u, DARPin-P state of successfully infected cells was assessed. All VFs in these infections are DARPin-P positive. (o, q, s, t) Lines and shaded areas indicate mean and SE, respectively. (m) Ordinary one-way ANOVA with Dunnett’s multiple comparisons test used for statistical analysis. (r) Two-way ANOVA with Tukey’s multiple comparisons test used for statistical analysis. (c, d, e, j, k, n, u) Scale bar, 10 µm. (g) Scale bar, 2 µm. (n) Time, h: min. The number of experimental repeats and fluorophores used are listed in Supplementary Table 1.