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      Liquid–liquid phase separation by SARS-CoV-2 nucleocapsid protein and RNA

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          Abstract

          Dear Editor, The COVID-19 pandemic worldwide is caused by a novel coronavirus SARS-CoV-2 (the severe acute respiratory syndrome coronavirus 2). 1 After viral invasion into the host cells, the ~30 kb viral genome RNA injected is translated into structural and non-structural proteins to replicate viral genome and assemble more viral particles. Many copies of nucleocapsid (N) protein can bind to viral genome RNA and pack it into ~100 nm particles, assisting membrane (M) and envelope (E) proteins to efficiently assemble the viral envelope. 2 The exact molecular mechanism by which N protein packs up the viral genome still remains elusive. An N protein of SARS-CoV-2 consists of an N-terminal RNA-binding domain (NTD) and a C-terminal dimerization domain (CTD) and shares ~90% sequence identity with N protein of SARS-CoV (Supplementary information, Fig. S1a). The regions located between the N-terminus and NTD, between NTD and CTD, and between CTD and the C-terminus of the N protein of SARS-CoV-2 (thereafter referred to as N protein) are predicted to be intrinsically disordered (Supplementary information, Fig. S1b, c). At neutral pH, the N protein is positively charged (+24 e), consistent with its strong binding affinity with negatively charged RNA, and this has also been validated by the nucleotide contaminant in N protein purification (Supplementary information, Fig. S2a, b). The gel filtration and dynamic light scattering results further suggested the oligmerization of N protein (Supplementary information, Fig. S2c–f). Altogether, the sequence and structure features of N protein are similar to those of other proteins that have been reported to undergo liquid–liquid phase separation (LLPS) with nucleic acids. 3 Thus, we hypothesized that N protein may also undergo LLPS with viral genome RNA and potentially facilitate viral assembly. To test this hypothesis, we directly mixed the Alexa-488-labeled recombinant full-length N protein (488-N) with 20-nt single strand RNA (ssRNA; A20) (Supplementary information, Table S1). We observed formation of liquid-like droplets when N protein or RNA concentration was higher than certain thresholds (Supplementary information, Fig. S3a). The shapes of LLPS droplets were determined by the ratio of N protein and ssRNA concentrations. At fixed N protein concentration, typical liquid-like droplets were formed at higher RNA concentrations, whereas denser, solid-like sediments were observed at lower RNA concentrations. To further quantify the properties of N protein/RNA LLPS, we established the phase diagram of Alexa-647-labeled N protein (647-N) with another 20-nt ssRNA labeled with HEX (H20) (Fig. 1a; Supplementary information, Fig. S3b and Table S1). We found that H20 and 647-N co-localized well within the phase-separated droplets (Fig. 1b), further confirming that the observed phase-separated droplets were indeed formed by N protein and ssRNA. Moreover, the observed LLPS is the intrinsic biochemical property of N protein and ssRNA, as it was still present even with no or low percentage of crowding polymer PEG8000 (Supplementary information, Fig. S3c). Upon mixing, 647-N and H20 formed micrometer-sized droplets rapidly within minutes, and the small droplets further fused into larger ones (Supplementary information, Fig. S4a–c). Even after 0.5–1 h, we still could observe ongoing droplet fusion (Fig. 1b). Fig. 1 N protein of SARS-CoV-2 undergoes LLPS with ssRNA in vitro. a Phase diagram of a 20-nt RNA H20 separating with N protein of SARS-CoV-2. The histogram shows the percentage of area occupied by N protein/H20 droplets. b Upper panel, representative fluorescence and DIC images of LLPS droplets formed by H20 RNA (orange) and N protein (red) after 1-h mixing. White arrows indicate ongoing fusion events. Bottom panel, fluorescence intensity profiles of two fluorescence channels (H20 RNA, orange; N protein, red) along the white dashed line in the upper panels. c The length of ssRNA modulates the N protein/RNA LLPS. Representative fluorescence images of HEX-labeled ssRNA (orange, 5 μM) of different lengths forming phase-separated liquid droplets with Alexa-647-labeled N protein (red, 20 μM). d Comparison of the percentage of area occupied by droplets in each field of view in c. e Zn2+ promotes N protein/RNA LLPS. Representative fluorescence images of Alexa-647-labeled N protein (20 μM) mixed with H20 RNA (5 μM) to form phase-separated liquid droplets in the presence of the indicated concentrations of Zn2+. Scale bars, 20 μm. Error bars refer to SD of five independent experiments. When 488-N was injected into the phase-separated solution formed by 647-N and H20, we found that the fluorescence intensity of Alexa-488 in droplets increased gradually along with the decrease of Alexa-647 fluorescence intensity (Supplementary information, Fig. S4d). When H20 was injected into the phase-separated solution that had already been formed by 488-N and A20, HEX fluorescence also appeared gradually inside the original droplets. Both the intensity of HEX fluorescence in each droplet and the number of droplets with HEX fluorescence increased over time (Supplementary information, Fig. S4e). All these results suggest that both N protein and ssRNA are exchangeable between the dense and solute phases. The sequences of H20 and A20 are totally different, and both can phase separate with N protein and exhibit similar phase diagram (Fig. 1a; Supplementary information, Fig. S3a, b), thus the ability of N protein to undergo LLPS with short RNA is independent of short RNA sequence. This is consistent with the previously reported finding that the binding of SARS-CoV’s N protein with RNA was not dependent on RNA sequence 4 and also agrees with the fact that N protein could bind various sites on genome RNAs. 5 It should be noted that these results do not rule out the possibility that N protein may undergo stronger LLPS with longer RNAs containing specific sequences or secondary structures. When mixing N protein with 5 μM HEX-labeled poly-U RNA oligos of lengths ranging from 10 nt to 60 nt (U10, U20, U40 and U60) to induce LLPS in vitro, we found that N protein could form larger droplets with longer ssRNAs (Fig. 1c), indicating the enhancing effect of RNA length on LLPS. The percentage of area occupied by the dense phase relative to the solute phase 6 increased rapidly from ~10% for 10-nt RNA (U10) to ~70% for 20-nt RNA (U20) and reached ~80% for 40-nt and 60-nt RNAs (U40 and U60) (Fig. 1d). When inducing N protein/RNA LLPS by using these poly-U oligos with the same mass and the same concentrations of N protein, we found that the longest ssRNA, U60, could phase separate with N protein to form much larger droplets compared with other shorter oligoes (Supplementary information, Fig. S5a), confirming that the LLPS is indeed dependent on ssRNA length. The genome RNA of SARS-CoV-2 is about 30 kb, much longer than RNA oligoes that we used above. We next tested long RNA in the LLPS. Two longer RNAs, 85-nt (A85) RNA and 1541-nt (N1541, the positive strand RNA of N protein), were tested. Both A85 and N1541 RNAs could form droplets with N protein, and the droplets turned to be solid-like structure instead of droplets as the length of RNA increased (Supplementary information, Fig. S5b, c). We thereby hypothesized that the viral packaging of SARS-CoV-2 might be driven by N protein/RNA LLPS. However, the exact role of N protein/RNA LLPS in viral packaging remains to be further validated. To test whether divalent cation could modulate N protein/RNA LLPS, we performed LLPS experiments in the presence of various cations. We found that Zn2+ had a significant effect on enhancing the LLPS (Fig. 1e; Supplementary information, Fig. S6), while Mg2+, Mn2+ and Ca2+ did not (Supplementary information, Fig. S6). Moreover, Ni2+ and Cu2+ could also modulate the LLPS at a relative lower concentration (10 μM) compared with Zn2+ (20 μM) (Supplementary information, Fig. S6). Zn2+ physiologically regulates cell functions, 7 and its regulation on LLPS has been reported previously. 8,9 We further investigated how the concentration of Zn2+ affected N protein/RNA LLPS. We found that 20 μM or higher concentration of Zn2+ could remarkably enhance the LLPS and turn the LLPS droplets to solid-like condensates, whereas lower concentrations of Zn2+ (< 10 μM) could not (Fig. 1e). When 0.5 mM Zn2+ was added into freshly made N protein/RNA droplets, liquid-like droplets turned to be denser, solid-like structures, which could recover to liquid-like droplets after addition of 50 mM EDTA (Supplementary information, Fig. S7a, b). In addition, negative staining EM (Supplementary information, Fig. S7c) or cryo-EM (Supplementary information, Fig. S7d) imaging of N protein/RNA LLPS in the presence and absence of Zn2+ revealed similar loose filament-like structures as those observed in the RNP particles of another β-coronavirus MHV, 10 supporting the potential role of N protein/RNA LLPS in viral assembly. N protein alone formed small condensates in the presence of 20 μM Zn2+ and became more aggregative when Zn2+ concentration was increased (Supplementary information, Fig. S7e). In contrast, H20 alone did not show significant change upon supplementation with 20 μM Zn2+ (Supplementary information, Fig. S7d). The result indicates that Zn2+ promotes N protein/RNA LLPS via inducing N protein oligomerization. Because the free cytoplasmic Zn2+ concentration is very low, the physiological relevance of Zn2+-promoted N protein/RNA LLPS is still not clear. Nevertheless, A recent study suggests that Chloroquine is a zinc ionophore. 11 Chloroquine has been shown to effectively inhibit the infection of SARS-CoV-2, 12 but the mechanism is still not known. We speculate that Chloroquine may inhibit N protein/RNA LLPS through chelating the free Zn2+ in cytosol. To identify the essential regions of N protein to facilitate LLPS with RNAs, we designed a panel of N protein mutants (Supplementary information, Fig. S8a). Interestingly, the deletion of CTD (dCTD) dramatically attenuated the droplet formation, whereas the deletions of NTD (dNTD), a basic amino acid-rich sequence (dBRS) or both (ddRBD) only mildly attenuated N protein/RNA LLPS (Supplementary information, Fig. S8b). These results are in agreement with previous findings that N protein harbors extra RNA-binding sites other than NTD (NTD, CTD and intrinsic disorder regions are all involved in RNA binding), and that the oligomerization of N protein mediated mainly by the CTD plays an essential role in N protein/RNA LLPS. We further tested the ability of N protein single domain only (NTD or CTD) to undergo phase separation with H20. We found that NTD alone could not induce LLPS, whereas CTD alone had weak ability to induce phase separation with H20 (Supplementary information, Fig. S8b). This result indicates that CTD could also bind RNA without affecting the oligomerization states. Interestingly, droplet formation could be enhanced by the addition of 20 μM Zn2+ for all these N protein mutants (Supplementary information, Fig. S8c), even the NTD domain only mutant, which failed to form phase-separated droplets with H20 in the absence of Zn2+ (Supplementary information, Fig. S8b). This suggests that Zn2+ promotes new interactions between NTDs and RNA, or it triggers N protein dimerization or multimerization via NTD. Zn2+ also significantly modulated the phase separation of CTD/H20 to form smaller droplets (Supplementary information, Fig. S8c), suggesting that the CTD also contributes to Zn2+-enhanced N protein/RNA LLPS. In summary, we revealed that N protein and RNA underwent LLPS. The LLPS is dependent on the length and concentration of ssRNA. N protein forms typical sphere-like droplets with short ssRNAs, but solid-like structures with long ssRNAs. We further identified that the LLPS could be enhanced by Zn2+. Our findings suggest that N protein/RNA LLPS may be essential for SARS-CoV-2 viral assembly, which may shed light on developing intervention strategies to prevent COVID-19 pandemic by disrupting the LLPS and viral assembly. Supplementary information Supplementary information

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          Most cited references6

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          Is Open Access

          Zinc in Cellular Regulation: The Nature and Significance of “Zinc Signals”

          In the last decade, we witnessed discoveries that established Zn2+ as a second major signalling metal ion in the transmission of information within cells and in communication between cells. Together with Ca2+ and Mg2+, Zn2+ covers biological regulation with redox-inert metal ions over many orders of magnitude in concentrations. The regulatory functions of zinc ions, together with their functions as a cofactor in about three thousand zinc metalloproteins, impact virtually all aspects of cell biology. This article attempts to define the regulatory functions of zinc ions, and focuses on the nature of zinc signals and zinc signalling in pathways where zinc ions are either extracellular stimuli or intracellular messengers. These pathways interact with Ca2+, redox, and phosphorylation signalling. The regulatory functions of zinc require a complex system of precise homeostatic control for transients, subcellular distribution and traffic, organellar homeostasis, and vesicular storage and exocytosis of zinc ions.
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            Coronavirus genomic RNA packaging

            RNA viruses carry out selective packaging of their genomes in a variety of ways, many involving a genomic packaging signal. The first coronavirus packaging signal was discovered nearly thirty years ago, but how it functions remains incompletely understood. This review addresses the current state of knowledge of coronavirus genome packaging, which has mainly been studied in two prototype species, mouse hepatitis virus and transmissible gastroenteritis virus. Despite the progress that has been made in the mapping and characterization of some packaging signals, there is conflicting evidence as to whether the viral nucleocapsid protein or the membrane protein plays the primary role in packaging signal recognition. The different models for the mechanism of genomic RNA packaging that have been prompted by these competing views are described. Also discussed is the recent exciting discovery that selective coronavirus genome packaging is critical for in vivo evasion of the host innate immune response.
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              Multiple nucleic acid binding sites and intrinsic disorder of severe acute respiratory syndrome coronavirus nucleocapsid protein: implications for ribonucleocapsid protein packaging.

              The nucleocapsid protein (N) of the severe acute respiratory syndrome coronavirus (SARS-CoV) packages the viral genomic RNA and is crucial for viability. However, the RNA-binding mechanism is poorly understood. We have shown previously that the N protein contains two structural domains--the N-terminal domain (NTD; residues 45 to 181) and the C-terminal dimerization domain (CTD; residues 248 to 365)--flanked by long stretches of disordered regions accounting for almost half of the entire sequence. Small-angle X-ray scattering data show that the protein is in an extended conformation and that the two structural domains of the SARS-CoV N protein are far apart. Both the NTD and the CTD have been shown to bind RNA. Here we show that all disordered regions are also capable of binding to RNA. Constructs containing multiple RNA-binding regions showed Hill coefficients greater than 1, suggesting that the N protein binds to RNA cooperatively. The effect can be explained by the "coupled-allostery" model, devised to explain the allosteric effect in a multidomain regulatory system. Although the N proteins of different coronaviruses share very low sequence homology, the physicochemical features described above may be conserved across different groups of Coronaviridae. The current results underscore the important roles of multisite nucleic acid binding and intrinsic disorder in N protein function and RNP packaging.
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                Author and article information

                Contributors
                jackweichen@zju.edu.cn
                jlou@ibp.ac.cn
                Journal
                Cell Res
                Cell Res
                Cell Research
                Springer Singapore (Singapore )
                1001-0602
                1748-7838
                8 September 2020
                : 1-3
                Affiliations
                [1 ]GRID grid.258164.c, ISNI 0000 0004 1790 3548, Shenzhen Baoan Women’s and Children’s Hospital, , Jinan University, ; Shenzhen, Guangdong 518102 China
                [2 ]GRID grid.9227.e, ISNI 0000000119573309, Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, , Chinese Academy of Sciences, ; Beijing, 100101 China
                [3 ]GRID grid.410726.6, ISNI 0000 0004 1797 8419, University of Chinese Academy of Sciences, ; Beijing, 100084 China
                [4 ]GRID grid.13402.34, ISNI 0000 0004 1759 700X, Department of Cell Biology and Department of Cardiology of the Second Affiliated Hospital, , Zhejiang University School of Medicine, ; Hangzhou, Zhejiang 310058 China
                [5 ]GRID grid.13402.34, ISNI 0000 0004 1759 700X, Key Laboratory for Biomedical Engineering of Ministry of Education, State Key Laboratory for Modern Optical Instrumentation, and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, , Zhejiang University, ; Hangzhou, Zhejiang 310058 China
                [6 ]GRID grid.27255.37, ISNI 0000 0004 1761 1174, Advanced Medical Research Institute, Cheeloo College of Medicine, , Shandong University, ; Jinan, Shandong 250012 China
                [7 ]Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou, Guangdong 510320 China
                Author information
                http://orcid.org/0000-0001-6853-2423
                http://orcid.org/0000-0001-5366-7253
                http://orcid.org/0000-0003-4031-6125
                Article
                408
                10.1038/s41422-020-00408-2
                7477871
                32901111
                00e17882-1c7b-437e-bf4e-30417ff6b5f8
                © Center for Excellence in Molecular Cell Science, CAS 2020

                This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

                History
                : 23 June 2020
                : 19 August 2020
                Funding
                Funded by: FundRef https://doi.org/10.13039/501100004751, Chinese Ministry of Science and Technology | Department of S and T for Social Development (Department of S&T for Social Development);
                Award ID: 2019YFA0707001
                Award ID: 2017ZX10203205
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/501100001809, National Natural Science Foundation of China (National Science Foundation of China);
                Award ID: 11672317
                Award ID: 31971237
                Award Recipient :
                Funded by: the COVID-19 emergency tackling research project of Shandong University (2020XGB03)
                Funded by: Chinese Ministry of Science and Technology | Department of S and T for Social Development (Department of S&T for Social Development)
                Funded by: Zhejiang University special scientific research fund for COVID-19 prevention and control (2020XGZX077)
                Categories
                Letter to the Editor

                Cell biology
                molecular biology,cell biology
                Cell biology
                molecular biology, cell biology

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