Fig. 1: SARS-CoV-2 N protein phase separates into stress granules in cells with G3BP1 and in vitro. | Cell Discovery

Fig. 1: SARS-CoV-2 N protein phase separates into stress granules in cells with G3BP1 and in vitro.

From: SARS-CoV-2 nucleocapsid protein undergoes liquid–liquid phase separation into stress granules through its N-terminal intrinsically disordered region

Fig. 1

a Prion-like domain analysis of SARS-CoV-2 N protein revealed that the N-terminal 1–39 aa was a probable IDR, which inclined to undergo LLPS. b Images of HeLa cells overexpressing GFP-tagged N protein (top) or N protein without the N-terminal 1–39 residues (∆N) (bottom). The left and right panels showed cells in the absence and presence of arsenite at 500 μM for 30 min, respectively. N protein aggregated into spherical droplets of several micrometers in diameter. Scale bar, 10 μm. c, d Fluorescence recovery of N-GFP protein after photobleaching showed that N protein demonstrated exceptional recovery in 60 s. Scale bar, 5 μm. The error bar represents SD (n = 18). e N-GFP protein underwent droplet fusion in a time-lapse movie after arsenite stress. Scale bar, 5 μm. f Fluorescence images of N-GFP condensation with endogenous G3BP1. ∆N-GFP failed to phase separate into G3BP1 SGs. Arsenite was added into medium at 500 μΜ for 30 min. Scale bar, 10 μm. As, arsenite. g Western blotting of HeLa G3BP1 knockdown cell lines showed effective knockdown by #2 and #3 shRNAs. h Statistic analysis showing that HeLa cells formed N-GFP puncta after G3BP1 knockdown with arsenite treatment for 30 min at 500 μM. For each group of cells, more than 50 cells were calculated for each time. i Upper panel, SARS-CoV-2 N proteins were diluted at 2.5, 5, 10, 20, and 40 μM separately in 25 mM Hepes (pH 7.5), 50 mM NaCl, and observed under bright field of a confocal microscope. Lower panel, SARS-CoV-2 N protein were diluted at 2.5, 5, 10, 20, and 40 μM separately in 25 mM Hepes (pH 7.5), 50 mM NaCl with additional 6.25 ng/μL total RNAs from HeLa cells. Droplets were observed under a bright field of a confocal microscope. Scale bar, 10 μm. j N protein at 10 μM in 25 mM Hepes (pH 7.5), 50 mM NaCl were stimulated with increased concentration of total RNAs. Scale bar, 10 μm. k FITC-labeled N protein were observed under 488 laser at different concentrations with or without RNA stimulation. The concentration of added HeLa cell total RNAs was 25 ng/μL. Scale bar, 10 μm. l Left, N proteins at 10 μM were stimulated with 6.25 ng/μL HeLa cell total RNAs in 25 mM Hepes (pH 7.5), 50 mM NaCl; right, the salt concentration of the left protein solution was raised to 500 mM and droplets dissolved. Scale bar, 10 μm.

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