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. 2020 Jul 1;11:3281. doi: 10.1038/s41467-020-17101-y

Fig. 3. NSs fibrilization relies on specific cysteine residues.

Fig. 3

a Vero cells were infected with RVFV and four mutant viruses each coding for NSs with cysteine substitutions, namely C39S/C40S, C149S, C178S, and C194S, for 8 h. Infected cells were analyzed by confocal microscopy after staining of nuclei with Hoechst (blue) and Abs against intracellular N (red) and NSs (green). 3D-modeling of NSs fibrillary and globular aggregates were achieved with the IMARIS software and are shown underneath each respective virus strain. Images are representative of three independent experiments. Scale bars, 5 µm. b, d, f Confocal Z-stack obtained in a were classified, segmented, and analyzed with the software ilastik as described in Supplementary Fig. 2. The number of NSs aggregates is given per cell as follow: b nuclear globular aggregates, d nuclear fibrillary aggregates, and f cytosolic globular aggregates. n = 29, 33, 25, 35, and 31 cells exposed to the wt virus and the mutants C39S/C40S, C149S, C178S, and C194S were respectively examined. Unpaired t-test, two-tailed, with Welch’s correction [parametric, not equal standard deviations (SDs)] was used in b while multiple comparisons, one-way ANOVA tests with Tukey corrections was employed in f. *p = 0.0288; ****p < 0.0001; ns non-significant. Same as in b, d, f but showing the volume of NSs globular c and fibrillary e aggregates. In c, n = 26 and 23 cells were examined for nuclear NSs globular aggregates (black) made from the NSs mutants C39S/C40S and C149S while n = 24, 32, 25, 28, and 30 cells were analyzed for cytosolic globular aggregates (gray) of NSs wt, C39S/C40S, C149S, C178S, and C194S, respectively. In e, n = 51, 50, and 66 nuclear NSs fibrillary aggregates built from NSs wt, C178S, and C194S were respectively examined. Unpaired t-test, two-tailed, with Welch’s correction (parametric, not equal SDs) was applied in c. ***p = 0.0002. b–f Center line, median; box limits, upper and lower quartiles; whiskers, 1.5× interquartile range; points, outliers. g Vero cells were exposed to RVFV and the two mutant viruses NSs C39S/C40S and C149S (MOI ~5) for 16 h. Electron micrographs show the ultrastructure of wt NSs fiber-bundles (upper left panel) and globular aggregates made of the NSs mutants C39S/C40S (upper right panel) and C149S (lower panel) after immunogold labeling with Abs against NSs (black spots). Experiments were repeated independently thrice with similar results. Scale bars, 500 nm.