Characterization of the behavior of RABV N in acellular minimal systems.

A) Schematic representation of the organization of RABV N and P constructs used. A StrepTagII was amino-terminally fused to the N protein to purify the N°-P complex from insect cells. Domains delimitations are indicated for both proteins. B) SDS PAGE analysis of N°-P complex. C) Purified N°-P complex labelled with NHS-Ester-Atto-488, was incubated at 2 μM concentration in 50 mM NaCl, 20 mM Tris-HCl pH 7.5 in absence or presence of 5% PEG 8000. Presence of N°-P droplets was assessed by fluorescence microscopy. Scale bar: 10 μm. D) Box plot representation of N°-P droplets aspect ratios (formed at 2μM with 5% PEG 8000 as in C). Sample size: n = 1707. The cross on the boxplot indicates the mean. E) Fluorescent P (resp. fluorescent N°-P) at 25 nM was incubated with 1μM N°-P (resp 5μM P) in 50 mM NaCl, 20 mM Tris-HCl pH 7.5 and 5% PEG 8000. Presence of droplets was assessed by fluorescence microscopy. Scale bar: 10 μm F) Whole droplets formed by N°-P complex at the concentration of 2 μM (25 nM of fluorescent complex) in the presence of 5% PEG 8000 in 50 mM NaCl and 20 mM Tris-HCl pH 7.5 were photobleached. Images were acquired on a Spinning-Disk microscope. Scale bars: 5 μm. For the right plot, FRAP data were corrected for background, normalized to the minimum and maximum intensity. The mean is shown with error bars representing the SD. G) Top: Electron microscopy image of negatively stained viral nucleocapsids (Nuc), purified from RABV-infected BSR cells. Bottom: SDS PAGE analysis of purified nucleocapsids. H) Purified nucleocapsids (500 nM of N protein, 10% fluorescently-labelled), labelled with NHS-Ester-Atto-488 were incubated in absence or presence of increasing concentrations of P (125 mM NaCl, 20 mM Tris-HCl pH 7.5, 5% PEG 8000, 25 nM of fluorescent P). Nucleocapsids and P droplets were observed by fluorescence microscopy. Scale bar: 10 μm. I) Segregation of RNA-Cy5 (40-A nucleotides) at the concentration of 100 nM into non-fluorescent P droplets (P at 2 μM in presence of 5% PEG 8000 in 125 mM NaCl, 20 mM Tris-HCl pH 7.5). Analysis was performed by fluorescence microscopy. Scale bars: 10 μm. J) N°-P droplets (2 μM concentration among which 25 nM of fluorescent complex, 5% PEG 8000, NaCl 50 mM, 20 mM Tris-HCl pH 7.5) in absence of RNA or incubated in presence of 40-A RNA added either 10 min before PEG-induced phase separation, or at the time of PEG 8000 addition, or 10 min after PEG-induced phase separation were observed by fluorescence microscopy. Images were acquired 20, 40, and 60 min after LLPS induction. Scale bars: 10μm. K) Boxplot analysis of N°-P droplets aspect ratios in absence or presence of RNA added 10 min after LLPS. The time indicated on the left corresponds to the time elapsed since LLPS induction. From left to right, the sample size in each category is n = 536, n = 1065, n = 1087, n = 694, n = 962, n = 882. The cross on each boxplot indicates the mean. P-values were calculated using Kolmogorov-Smirnov test. ** p−4, *** p−6.