Fig 2: Only hardened inclusions emerge in nucleozin-treated Rab11a-DN cell line.(A–B) A549 cells constitutively expressing GFP-Rab11a-WT and GFP-Rab11a-DN were infected for 10 hr with PR8 at an MOI of 3 and treated with 5 µM nucleozin (Ncz) (n=10) or dimethyl sulfoxide (DMSO) (n=10) before fixing for analysis by immunofluorescence. The data are a representative analysis from biological duplicates. (A) Representative images of cells analysed by immunofluorescence staining using antibodies against viral protein nucleoprotein (NP) (magenta), host Rab11 (green), and ER (cyan). Nuclei and cell periphery delimited by yellow and white dashed line respectively, and white boxes are insets showing presence or absence of viral inclusions. Scale bar = 10 µm. (B) Scatter plot of circularity versus roundness of viral inclusions. (C–E) A549 cells constitutively expressing GFP-Rab11a-DN were transfected with mcherry-NP and co-infected with PR8 virus at an MOI of 3. At 12 hr post-infection (hpi), the cells were treated with 5 µM Ncz or DMSO for 10 min before imaging. (C) Schematic depicting the possible outcomes when Rab11a-DN cell lines are treated with Ncz. (D) Representative time lapse pseudo-colour images show fusion of IAV inclusions in a coarsening assay of PR8 infected Rab11a-DN cell line treated with Ncz or DMSO (extracted from Figure 8—videos 1–6). (E) Plot depicting the aspect ratio of fusing inclusions over time in infected Rab11a-DN cell line treated with Ncz.

Fig 3: Changes in concentration of viral ribonucleoproteins (vRNPs) and Ras-related in brain 11a (Rab11a) modestly alter the material properties of viral inclusions.(A–H) A549 cells stably expressing GFP, or GFP-Rab11a-WT were infected at an MOI of 3 with PR8 virus and, at the indicated timepoints, were fixed, and analysed by immunofluorescence using an antibody against nucleoprotein (NP) (as a proxy for vRNPs). (Number of cells (n)=14–18 for GFP and 19–25 for GFP-Rab11-WT). (C–H) Each dot is the average value of measured parameters per cell. Above each boxplot, same letters indicate no significant difference between them, while different letters indicate a statistical significance at a=0.05 using one-way ANOVA, followed by Tukey multiple comparisons of means for parametric analysis, or Kruskal Wallis Bonferroni treatment for non-parametric analysis. All thermodynamic related values are displayed in Supplementary file 1 (Sheets 2 and 3). Abbreviations: AU, arbitrary unit. (A) Representative depiction of the experimental analysis workflow. (B) Immunofluorescence images of infected cells at different hours post-infection (hpi) in cells overexpressing GFP (left) or GFP-Rab11 (right) (both in green); NP (red, as a proxy of vRNPs), and nucleus (blue). Scale bar = 10 µm. (C) Boxplot depicting the fold change in the ratio of cytoplasmic to nuclear vRNPs concentration at different times of infection, with endogenous or overexpressed Rab11a; p<0.001; Kruskal Wallis Bonferroni treatment. (D) Boxplot of mean inclusion area (µm2) per cell; p<0.001 by one-way ANOVA, followed by Tukey multiple comparisons of means. (E) Scatter plot showing nucleation density (?, µm–2) versus degree of supersaturation (S). (F) Boxplot of Cdense (AU); p<0.001 by Kruskal Wallis Bonferroni treatment. (G) Boxplot of Cdilute (AU); p<0.001 by Kruskal Wallis Bonferroni treatment. (H) Boxplot of ??G (J/mol); p<0.001 by Kruskal Wallis Bonferroni treatment. Conditions were normalised to an infection state without IAV inclusions (3 hpi) that is indicated by the dashed black line.

Fig 4: Changes in concentration of viral ribonucleoproteins (vRNPs) and Ras-related in brain 11a (Rab11a) modestly alter the material properties of viral inclusions.(A–I) A549 cells stably expressing GFP, or Rab11a-WT were transfected with a plasmid encoding mCherry-NP and simultaneously co-infected with PR8 virus at an MOI of 10 and were live imaged at 12–16 hr post-infection (hpi). (Number of cells (n)=10–18 for GFP and GFP-Rab11-WT.) (F–I) Each dot is the average value of measured parameters per cell. Above each boxplot, same letters indicate no significant difference between them, while different letters indicate a statistical significance at a=0.05 using one-way ANOVA, followed by Tukey multiple comparisons of means for parametric analysis, or Kruskal Wallis Bonferroni treatment for non-parametric analysis. All related values are displayed in Supplementary file 1 (Sheet 5). (A) Representative time lapse images of fission (blue arrow) and fusion (yellow arrow) dynamics of viral inclusions in cells with endogenous levels or overexpressing Rab11a (Figure 5—videos 1–4). (B) Schematic depiction of an internal rearrangement of viral inclusion after an ROI within the inclusion is photobleached. (C) At 12 hpi, cells were treated with nocodazole (10 µg/mL) for 2 hr to reduce the highly stochastic motion of liquid influenza A virus (IAV) inclusions in GFP and GFP-Rab11a lines. Small regions inside IAV inclusions were photobleached to assess internal rearrangement of vRNPs (mCherry-NP as proxy). Time lapse pseudo-colour images show internal rearrangements after photobleaching (extracted from Figure 5—videos 5; 6). Scale bar = 2 µm. (D) Schematic of a fluorescence loss after photoactivation (FLAPh) experiment. (E) Time lapse pseudo-colour images showing the fluorescence loss in photoactivated IAV inclusions (photoactivatable paCherry-NP used as proxy) in GFP (n=10) or GFP-Rab11a cell lines (n=10) (extracted from Figure 5—videos 7–12). The analysis is a representative of two biological replicates. Bar = 2 µm. (F) Fluorescence intensity decay of photoactivated (paCherry-NP) normalised to GFP. Coloured lines are single exponential model fitting (y0 = (1-a) + ae-kt) of the data point, dots are the mean of the data per second, and vertical lines denote the standard deviation (SD) per time (s), (Supplementary file 1 (Sheet 5)). (G) Half-life (t1/2 = In(2)k, k=rate constant) of GFP and GFP-Rab11a developed inclusions decay post-activation (s); p=0.0003 by Kruskal Wallis Bonferroni treatment (Supplementary file 1 (Sheet 5)). (H) Boxplot showing the rate constant, k, of liquid inclusions (using paCherry-NP as proxy) arising in GFP and GFP-Rab11a cell lines; p=0.0003 by Kruskal Wallis Bonferroni treatment (Supplementary file 1 (Sheet 5)). (I) Boxplot showing the immobile fractions from FLAPh experiment calculated by the formula y0 = (1-a) + ae-kt, where 1-a is the immobile fraction arising from GFP and GFP-Rab11a cell lines; p=0.898 by Kruskal Wallis Bonferroni treatment (Supplementary file 1 (Sheet 5)).

Fig 5: Hardening of influenza A virus (IAV) inclusions changes its proteome solubility.(A–E) A549 cells constitutively expressing GFP-Rab11a-WT or GFP-Rab11a-DN were infected for 12 hr with PR8 at an MOI of 5 and treated with 5 µM nucleozin (Ncz) or dimethyl sulfoxide (DMSO) for 1 hr. Thereafter, cells were lysed in mild (NP40) or strong detergent (SDS), while NP40 lysate was ultracentrifuged (100,000 × g) to pellet materials in condensates from the soluble fraction in the supernatant. Soluble and total host and viral proteome were identified by LC-MS/MS and solubility was determined as the ratio of soluble NP40- to SDS-derived total proteome abundances at the indicated timepoints. Data were from two biological replicates. (A) Schematic representation of solubility proteome profiling (SPP). (B) Volcano plot representing relative host protein abundance in Rab11a-WT and Rab11a-DN infected cell lines (at 12 hr post-infection [hpi]) after treatment with Ncz or DMSO. Differentially upregulated proteins in these conditions (statistical significance – see Materials and methods) are indicated in blue dots. (C) Bar graphic comparing abundances of viral proteins (in log2 scale) in Rab11a-WT and Rab11a-DN cell lines PR8-infected (12 hpi) and treated with either Ncz or DMSO. (D) Volcano plot representing relative solubility of host and viral proteins in Rab11a-WT and Rab11a-DN infected cell lines (at 12 hpi) after treatment with Ncz. Differentially soluble proteins in these conditions (statistical significance – see Materials and methods) are indicated in pink and green dots. (E) Bar graph comparing solubility (in log2 scale) of viral proteins when PR8 infected (12 hpi) Rab11a-WT and Rab11a-DN cell lines were treated with either Ncz or DMSO.