Extended Data Fig. 7: Validation of the SunTag^eng RSV strains that allow real-time visualization of viral mRNA expression.

a, Schematic illustrating the upstream and downstream insertion locations of the ‘additional SunTag genes’ in the two SunTag^eng RSV strains. b, c, Viral growth analysis based on plaque assays were carried out to determine the fitness of the engineered RSV strains compared to the WT. (b) Representative images of observed plaques 6 days post viral inoculation. For each viral strain, 2 repeats, each with 2 inoculum dilutions are shown. (c) Quantification of growth assay. Line connects mean of experimental repeats (dots). d, Representative images of time-lapse videos of cells expressing STAb-fluoro after infection with upstream SunTag^eng RSV prior to (top panel) and following (bottom panel) puromycin administration. e, f, STAb-fluoro foci observed with the SunTag^eng RSV strains were assessed by smFISH. (e) Representative images of smFISH for SunTag mRNA foci and STAb-fluoro foci in the same infected cell for the upstream strain is shown. (f) Scatter dot plot quantifies the number of SunTag mRNAs (via smFISH) and translation sites (via STAb) in the same cells. Each dot indicates a single cell and dashed line indicates linear correlation with Pearson R² (top left). g, The number of translating SunTag mRNAs over time was quantified for both the upstream and downstream SunTag^eng strains. Overall, both strains show an increase in the number of translating SunTag mRNAs over time. Mean (line) and SE (shaded area) are shown. h, i, j, Downstream SunTag^eng RSV viral mRNA expression dynamics were assessed for infections originating from passive vRNPs and PRCs in cell lines labelling all vRNPs (P^exo-fluoro) and DARPin-P⁺ vRNPs (DARPin-P-fluoro). (h) Two representative image series from time-lapse videos of SunTag mRNAs in cells infected with a PRC are shown. (i) Cumulative incidence graphs depict the start of viral transcription for infections with passive vRNPs and PRCs. Mean (line) and SE (shaded area) are shown. (j) The transcription rate was calculated and plotted as violin plots with the median and quartiles shown (horizontal lines). Two-tailed unpaired Student’s t-test was used for statistical analysis. (d, e, h) Scale bar, 10 µm. (h) Time, h: min. The number of experimental repeats and fluorophores used are listed in Supplementary Table 1.