Extended Data Fig. 2: DARPin-P labels a subset of vRNPs in infected cells.

a, Quantification of the false-positive foci detection for indicated antibodies, recombinant DARPin-P protein and RSV genome smFISH probe set used to detect RSV vRNPs and virions. Data are shown as a box and whiskers plots, with the box extending from the 25th to 75th percentiles, the center line indicating the median, and whiskers spanning the 1st to 99th percentiles representing the minima and maxima. b, c, RSV N antibody staining identifies RSV vRNPs. N antibody foci in infected cells colocalize with RSV genomic RNA ((-)vRNA). Representative images (b) and quantification of colocalization (c). d, Schematic highlighting dual antibody staining-based approach for distinguishing RSV vRNPs in virions (N⁺/F⁺) and in the cytoplasm of infected cells (N⁺/F⁻). e, Representative images of RSV infected WT A549 cells stained for RSV F, N and recombinant DARPin-P. The green insert highlights a virion attached to the cell while the orange and purple inserts highlight DARPin-P⁻ and DARPin-P⁺ vRNPs in the infected host cell cytoplasm, respectively. f, For cytoplasmic vRNPs (F⁻), the DARPin-P state was determined and quantified. Note that the analysis was performed at 4 h post RSV inoculum addition to ensure mostly single vRNPs were analyzed (see Fig. 2j). g, h, The effect of paraformaldehyde fixation on DARPin-P-fluoro foci observed following RSV infection was assessed. (g) Representative images of the same cells imaged live and after fixation. (h) Heat map visualizes the frequency of the number of DARPin-P-fluoro foci observed by live imaging and imaging following fixation, in the same cells. i, Quantification of the colocalization between DARPin-P signal from genetically-encoded DARPin-P-fluoro and recombinant DARPin-P signal of RSV vRNPs in infected DARPin-P-fluoro cells (related to Fig. 1f). j, k, l, RSV infection in the DARPin-P-fluoro cell line was comparable to that in WT A549 cells. (j) Quantification of the fraction of infected cells at 6 h post viral inoculum addition for DARPin-P-fluoro and WT A549 cells. Representative images (k) and quantification (I) of viral mRNA transcripts (using a smFISH probe set for all RSV mRNAs) in infected DARPin-P-fluoro and WT A549 cells at 10 h post viral inoculum addition. m, Flow cytometry-based quantification of the percentage of infected cells expressing viral G protein (a read-out of successful infections, see Extended Data Fig. 5j–p and Supplementary Fig. 1) at 24 and 48 h post viral inoculum addition for DARPin-P-fluoro and WT A549 cells. (a, m) Two-way ANOVA with Tukey’s multiple comparisons test used for statistical analysis. (j, l) Two-tailed unpaired Student’s t-test was used for statistical analysis. (b, e, g, k) Scale bar, 10 µm. The number of experimental repeats and fluorophores used are listed in Supplementary Table 1.