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. 2021 Aug 17;7:69. doi: 10.1038/s41421-021-00306-w

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Schematic diagram of SARS-CoV-2 N protein highlights an ITFG motif located in the N-IDR. IDR intrinsically disordered region. b GST–G3BP1^NTF2 pull-down of purified SARS-CoV-2 N^1–246 and ITFG mutants. c SARS-CoV-2 N^1–174, but not N^1–30 or N^30–174, is sufficient to inhibit phase separation of G3BP1 in the presence of RNA. Equimolar N fragment was mixed with MBP-G3BP1. TEV was added in solution to cleave the MBP fusion, and OD_575nm was recorded over time. Data represent mean±s.d. from 3 independent experiments. d Mutations in the ITFG motif of N protein impaired its ability to inhibit G3BP1-mediated phase separation. Experiments were performed similar to (c). e Summary of the phase separation behaviors of purified recombinant G3BP1 and RNA, in the absence of N protein or the presence of various N mutants. Red: no droplet; green: droplet. f Mutation of three basic amino acids (R92E/R107E/R149E, 3E) in the RRM domain of SARS-CoV-2 N protein significantly impaired the inhibitory effect of N protein on G3BP1 phase separation. g, h Overexpression of SARS-CoV-2 N protein inhibited the formation of SGs in cells, and mutation of the ITFG motif or RRM significantly abrogated the inhibition. Data shown in (h) represent mean ± s.d. from three independent experiments. Results were evaluated by one-way ANOVA test (**P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant). Scale bar: 10 μm. i Turbidity of G3BP1 with Caprin-1 or its mutants over time in the presence of RNA. Data represent mean ± s.d. from three independent experiments. j Turbidity of G3BP1 with Caprin-1 or N protein over time in the presence of RNA. Data represent mean ± s.d. from three independent experiments. k, l Caco-2 cells expressing N wild-type or mutants were infected with SARS-CoV-2 GFP/ΔN trVLP, and cell culture supernatant was collected to infect Caco-2-N wild-type cells. GFP signal was observed using microscopy, and cellular RNA was extracted for RT-qPCR analysis to determine viral subgenomic RNA levels. Data represent mean ± s.d. from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Significance assessed by one-way ANOVA. Scale bar: 100 μm.