key: cord-0925790-ymil1enx
authors: Zhao, Ming; Yu, Yu; Sun, Li-Ming; Xing, Jia-Qing; Li, Tingting; Zhu, Yunkai; Wang, Miao; Yu, Yin; Xue, Wen; Xia, Tian; Cai, Hong; Han, Qiu-Ying; Yin, Xiaoyao; Li, Wei-Hua; Li, Ai-Ling; Cui, Jiuwei; Yuan, Zhenghong; Zhang, Rong; Zhou, Tao; Zhang, Xue-Min; Li, Tao
title: GCG inhibits SARS-CoV-2 replication by disrupting the liquid phase condensation of its nucleocapsid protein
date: 2021-04-09
journal: Nat Commun
DOI: 10.1038/s41467-021-22297-8
sha: 6193ab1cd41987f2f204ebadc6b71e5de4c9ead5
doc_id: 925790
cord_uid: ymil1enx

Lack of detailed knowledge of SARS-CoV-2 infection has been hampering the development of treatments for coronavirus disease 2019 (COVID-19). Here, we report that RNA triggers the liquid–liquid phase separation (LLPS) of the SARS-CoV-2 nucleocapsid protein, N. By analyzing all 29 proteins of SARS-CoV-2, we find that only N is predicted as an LLPS protein. We further confirm the LLPS of N during SARS-CoV-2 infection. Among the 100,849 genome variants of SARS-CoV-2 in the GISAID database, we identify that ~37% (36,941) of the genomes contain a specific trio-nucleotide polymorphism (GGG-to-AAC) in the coding sequence of N, which leads to the amino acid substitutions, R203K/G204R. Interestingly, N(R203K/G204R) exhibits a higher propensity to undergo LLPS and a greater effect on IFN inhibition. By screening the chemicals known to interfere with N-RNA binding in other viruses, we find that (-)-gallocatechin gallate (GCG), a polyphenol from green tea, disrupts the LLPS of N and inhibits SARS-CoV-2 replication. Thus, our study reveals that targeting N-RNA condensation with GCG could be a potential treatment for COVID-19.

H uman coronaviruses have caused two epidemics, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), since the 21st century. A recently identified new member of the coronavirus genera, SARS-CoV-2, is responsible for the outbreak of COVID-19 pandemic, from which the world is suffering now 1, 2 . SARS-CoV-2 shares 80% sequence similarity with SARS-CoV and entries host cells via the same receptor, angiotensin-converting enzyme 2 (ACE2) 3, 4 . As a highly infectious virus, SARS-CoV-2 has rapidly spread worldwide and caused a global health crisis 5 . As of December 1st, 2020, over 63 million people have been confirmed infected and more than 1.4 million deaths have been reported (https://covid19.who.int/). The current treatment for COVID-19 is mainly symptomatic care and supportive 6 . To contain the rapid global spreading of SARS-CoV-2, tremendous efforts have been made to look for efficient treatments for COVID-19. Therefore, a detailed understanding of the molecular events and the underlying mechanisms in the life cycle of SARS-CoV-2, including the viral replication and assembly, is urgently needed.

SARS-CoV-2 is an enveloped, positive-sense RNA virus containing a non-segmented single-stranded RNA genome of 30,000 nucleotides (nt) 1 . The determination of the full-length genome sequence of SARS-CoV-2 allowed the analysis of the encoded proteins 1,7-9 . 29 proteins were predicted, including 4 structural proteins, spike (S), membrane (M), envelope (E) and nucleocapsid (N) . N protein is a highly conserved factor among coronaviruses, for example, the amino acid sequence shares~90% homology between SARS-CoV-2 and SARS-CoV 10, 11 . Similar to N protein of SARS-CoV, the N SARS-CoV-2 is a 46 kDa protein with two domains, NH 2 -terminal RNA-binding domain (NTD) and COOH-terminal dimerization domain (CTD) 11, 12 . Previous studies of coronaviruses suggested that N protein is an RNA-binding factor that plays a critical role in viral genome packaging and virion assembly [13] [14] [15] .

Many RNA-binding proteins, especially those with high percentage of intrinsically disordered region (IDR), were found to be involved in liquid-liquid phase separation (LLPS) process [16] [17] [18] [19] . Protein LLPS is a physicochemical event and was recently emerged as a critical mechanism in organizing macromolecules, such as proteins and nucleic acids, into membrane-less organelles 16, 20 . These membrane-less cellular compartments were dynamically assembled via LLPS, and conferred important capacities for the cells to initiate biological functions or reactions in response to a number of stresses [20] [21] [22] [23] [24] [25] . Upon RNA virus infection, LLPS mediates the formation of stress granules (SGs) and P-bodies (PBs), which are critical for antiviral immunity by inhibiting viral mRNA translation and promoting RNA decay [16] [17] [18] 26, 27 . Interestingly, LLPS was also thought to be critical in viral assembly, including respiratory syncytial viral (RSV) 28 , measles virus (MeV) 29 and vesicular stomatitis virus (VSV) 30 . A key step during the replication of coronavirus is the association of N protein with viral genomic RNA and the subsequent condensation into higher-order RNA-protein complexes, which initiates the assembly of virions 13, 31 . In the current study, by revealing the RNA-triggered LLPS of N protein, we have been able to find the natural chemical, GCG, can disrupt the LLPS of N protein and inhibit the replication of SARS-CoV-2. Our findings not only provide molecular details in SARS-CoV-2 infection, but also present GCG as a lead compound for the development of drug to treat COVID-19.

RNA triggers the LLPS of N protein. As protein LLPS has been implicated to play important role in viral assembly 29 , we sought to study the SARS-CoV-2 proteins for their ability to undergo LLPS. Using bioinformatic tools, IUPred2, ANCHOR2, PSPredictor, catGranule, P-Score, and PLACC 32-36 , we analyzed the LLPS ability of each of the 29 proteins encoded by SARS-CoV-2 genome. Only N protein was predicted as an LLPS protein ( Fig. 1a, b, Supplementary Fig. 1, 2a and Supplementary Data 1). The known LLPS protein, RNA-binding protein fused in sarcoma (FUS) 19 , and a highly structured, non-LLPS protein, mono-EGFP (mEGFP) 37 , were respectively served as positive and negative controls for the analysis. To further understand the LLPS pattern of N, we analyzed the amino acids and charge distribution using R + Y and DDX4-like predictors 38, 39 . We found that N protein exhibited the similar pattern of charged residues as DDX4-like proteins ( Supplementary Fig. 2b, c) .

To further study the LLPS of N protein, we first purified the mEGFP-tagged recombinant N protein and confirmed its RNAbinding capacity with electrophoretic mobility shift assay (EMSA) ( Supplementary Fig. 3a, b) . When N was incubated with different RNAs, including fragments of SARS-CoV-2 genomic RNAs [a 229-nt 3′ untranslated region (UTR), 229-bp double-stranded RNA (dsRNA) of the 3′ UTR, a 55-nt RNA segment from 5′ UTR or a 60-nt RNA segment from the Nsp1 coding sequence] and the synthetic analog of dsRNAs, polyinosinic-polycytidylic acid [poly (I:C)] and 5′ppp-dsRNA. We found that RNAs triggered the robust LLPS of N protein both in vitro and in vivo (Supplementary Fig. 3c, d) . Using time-lapse microscopy, we observed the dynamic process of RNA-triggered LLPS of N. RNAs formed liquid condensates with N quickly ( Fig. 1c and Supplementary Movie 1) and the smaller N-RNA droplets can fuse into bigger ones ( Fig. 1d and Supplementary Movie 2), which is a hallmark of protein LLPS 40 . The N-RNA condensation was formed in a concentration-dependent manner ( Fig. 4j, k) . With fluorescence recovery after photobleaching (FRAP) experiments, we showed that the photobleached fluorescence signal of N-RNA droplets can be recovered within seconds (Fig. 1h , i and Supplementary Movie 3). This result suggested that the condensates dynamically and rapidly exchange molecules with the environment, which is another feature of protein LLPS 20 . Collectively, these data confirmed that RNA induces the LLPS of N protein.

N undergoes LLPS in vivo. We next investigated the LLPS of N in vivo. We constructed a Doxycycline hyclate (Dox)-inducible N-expressing H1299 cell line (Fig. 2a) . Transfection of Nexpressing cells with poly(I:C) or the vRNA (3′ UTR), which is shared by all the sub-genome mRNAs 8 , resulted in the formation of N protein condensates (Fig. 2b) . Using a Cyanine 5 (Cy5)labeled vRNA (3′ UTR), we confirmed that the transfected RNA formed condensations with N in cells ( Fig. 2c and Supplementary Movie 4). Importantly, the fusion of N-RNA condensates in cells were also observed ( Fig. 2d and Supplementary Movie 5). We further performed the FRAP experiment in cells and showed the active molecule-exchanging process of the N-RNA condensates in vivo (Fig. 2e , f and Supplementary Movie 6). These data indicated that the N-RNA condensates in cells were formed via LLPS.

The LLPS of different N variants. By performing the sequence analysis, we found that similar to SARS-CoV, the N protein of SARS-CoV-2 contains two domains, NTD and CTD (Supplementary Fig. 5a, b) . The domain definition was also reported recently 11 . To understand whether these structured domains contribute to the LLPS ability, we constructed truncated N variants and purified the recombinant proteins (Fig. 3a, b) . Using EMSA, we found that the deletion of any of these domains disrupted the RNA-binding ability of N protein (Fig. 3c) . By incubating these variants with the 60-nt viral genomic RNA, we found that none of the truncated N variants can undergo LLPS To further determine the contribution of IDRs in N for LLPS, another variant of only CTD and NTD (connected by a 'SGGS' linker) was constructed and prepared ( Supplementary Fig. 5c, d) . We found that this variant lost the LLPS ability ( Supplementary Fig. 5e, f) . These b IDR scores of 29 proteins encoded by SARS-CoV-2 genome. FUS and mEGFP are positive and negative controls, respectively. IUPred2 and ANCHOR2 were used as prediction tools. c Time-lapse imaging of N-mEGFP protein (20 μM) in the presence of Cy5-labeled 60-nt vRNA (100 ng/μl), scale bar, 10 μm. d Representative fluorescent images of N-mEGFP-vRNA (60 nt) condensates fusion from a time-lapse movie, scale bar, 3 μm. e-g LLPS of N-mEGFP protein (20 μM) in the presence of indicated concentrations of 60-nt vRNA, scale bar, 10 μm (e). The partition coefficient of fluorescence intensity per droplet (f) and the partition coefficient of total fluorescence intensity in each view (g) were calculated. From left to right, n = 209, 1170, 1026, 1170 droplets (f) from 10 randomly selected views (g). h, i FRAP analysis of vRNAinduced liquid droplets of N-mEGFP protein, scale bar, 2 μm (h), and quantification of fluorescence intensity recovery of a photobleached N-mEGFP protein, n = 3 biologically independent experiments (i). The white dotted circle in h indicated the region of photobleaching. 20 μM N-mEGFP protein and 100 ng/μl 60-nt vRNA were used. Error bars, mean with s.d. (f, g, i). Two-tailed unpaired Student's t-test (f, g), ****P < 0.0001. Data are representative of at least three independent experiments. Source data are provided as a Source Data file. data showed that NTD, CTD, and IDRs are all important for the N-RNA binding and the LLPS of N. N R203K/G204R gained greater ability to undergo RNA-induced LLPS. Since the first identification of the genome sequence of SARS-CoV-2 1 , full genomic sequences of this virus from all over the world were continuously submitted to public databases, such as GISAID (https://www.gisaid.org). We analyzed 100,849 genome sequences of SARS-CoV-2 from GISAID with the attempt to examine the variability of N-coding sequences. Surprisingly, while many nucleotide polymorphisms were found across the full length of the N-coding sequence, a high-frequency trio-nucleotide polymorphism (GGG-to-AAC) was identified in~37% (36,941) of the genomes (Fig. 4a, Supplementary Fig. 5g and Supplementary Data 2). This GGG-to-AAC variation resulted in the amino acid substitutions, R203K/G204R, in N protein. To examine the effect of this high-frequency variation on the LLPS of N, we prepared the recombinant proteins of these variants, N R203/G204 , N R203K , N G204R , and N R203K/G204R (Fig. 4b ). When incubated with viral RNA, we found that, interestingly, N R203K/G204R gained greater ability to undergo LLPS ( Fig. 4c -g and Supplementary Movie 12, 13) . We also analyzed the correlation between the mortality and R203K/G204R polymorphism of N. Our results showed that this polymorphism has little effect on the death ratio reported (Supplementary Fig. 5h ). In the future, analysis of patient clinical outcomes and the coupled SARS-CoV-2 genome sequences will provide important evidences regarding the effect of N R203K/G204R polymorphism on the biology of SARS-CoV-2.

N inhibits RNA-induced IFN expression. According to a previous study of SARS-CoV, N protein inhibits the virus infectioninduced production of interferon (IFN) by interfering with the detection of viral RNA by cellular RNA sensors 41 . To determine the role of SARS-CoV-2 N protein in the RNA-induced expression of IFN, we transfected vRNA (3′UTR) or poly(I:C) into the N-expressing and control cells. Our data showed that the expression of N attenuated the intracellular RNA-triggered expression of IFN (Fig. 5a, b) . We next examined the inhibitory effect of N proteins (both N R203K/G204R and N R203/G204 ) on the RNA-induced expression of IFN. We found that the polymorphism of N R203K/G204R , which exhibited a higher propensity to undergo LLPS in the presence of RNAs, showed a greater effect on the inhibition of IFN expression (Fig. 5c-i) . These data indicated that the RNA-triggered phase separation procedure of N protein may shield viral RNAs from host RNA sensors to avoid immune surveillance. Thus, in addition to mediating the package of viral genomic RNA, N may also affect the host antiviral responses. Our data suggested that the inhibitory effect of N is linked with its ability of LLPS.

Given that the N-mediated genome organization process is a key step for viral assembly 13,14 , our findings, therefore, provided a potential target for the development of means to combat SARS-CoV-2. With this in mind, we listed several chemicals/drugs that were previously reported to interfere with the N-RNA binding or the self-aggregation of N protein of viruses [42] [43] [44] [45] [46] . We also included the chemicals/drugs suggested by a recent report of the proteomics study on SARS-CoV-2 9 (Supplementary Fig. 6 ). Next, we transfected poly(I:C) into the N-expressing cells following the pre-treatment of the above chemicals/drugs. GCG blocked the RNA-triggered LLPS of N, while other drugs did not show detectable effect (Fig. 6a) . Data from multiple views were calculated and analyzed statistically (Fig. 6b) . Using a Cy5-labeled vRNA, we obtained the consistent data ( Fig. 6c and Supplementary Fig. 7a ). The possibility that GCG affected the transfection efficiency was ruled out (Supplementary Fig. 7b) .

To test the cytotoxicity of GCG, different dosages of GCG were used to treat cells, cell viability were measured 48 h after the treatment. Our data showed that the doses of GCG used in our study did not cause an obvious cell death, and the 50% cytotoxicity concentration (CC 50 ) was calculated ( Supplementary  Fig. 7c ). We then examined the LLPS of N protein with the application of increasing concentrations of GCG, the results showed that 12.5 μM was sufficient to block the N protein LLPS ( Supplementary Fig. 7d, e) . We further titrated the concentrations of GCG below 10 μM and found that 6-8 μM were the starting concentrations for GCG to inhibit LLPS of N protein (Fig. 6d, e) . By using EMSA, we showed that the presence of GCG significantly impaired the RNA-binding of N protein (Fig. 6f ). In addition, by incubating N with GCG, we showed the direct binding of GCG and N protein (Fig. 6g) . We further used GCGbeads to pull-down proteins in cells expressing N, and found that GCG selectively bound to N (Fig. 6h) . Previously, our group reported that epigallocatechin gallate (EGCG), a structural isomer of GCG inhibited interferon production by disrupting the interaction between GTPase-activating protein-(SH3 domain)binding protein 1 (G3BP1) and Cyclic GMP-AMP synthase (cGAS) 47 . We then tested the effect of EGCG on blocking the RNA-triggered LLPS of N protein. Interestingly, although these two molecules are isomers, EGCG had much weaker effect on the inhibition of N-RNA condensation ( Supplementary Fig. 7f, g) . Taken together, GCG directly bound N protein and disrupted N LLPS.

GCG suppresses SARS-CoV-2 replication. We next examined whether GCG could inhibit N protein LLPS in the context of SARS-CoV-2 infection. To do so, we obtained the antibody against SARS-CoV-2 N protein, and the specificity of the antibody was verified ( Supplementary Fig. 7h, i) . We then observed the N LLPS upon SARS-CoV-2 infection, robust formation of N condensates was observed in infected cells (Fig. 7a-c) . These data indicated that N protein indeed underwent LLPS during the SARS-CoV-2 infection. By applying GCG treatment on SARS-CoV-2 infected cells, we found that the viral titers were dramatically inhibited (Fig. 7d) , and the 50% inhibitory concentration (IC 50 ) was calculated (Fig. 7e) . The selective index (ratio of CC 50 to IC 50 ) was 3.5. Importantly, the administration of GCG significantly impaired the LLPS of N protein during SARS-CoV-2 infection (Fig. 7f, g) . To rule out the possibility that GCG restrict SARS-CoV-2 at the entry step, cells were infected with SARS-CoV-2 for 1 h and then treated with GCG for 24 h. The viral titers were measured, and the results showed that GCG still significantly inhibited the viral replication (Fig. 7h) . Together, our data suggested that GCG effectively inhibited SARS-CoV-2 replication and most likely through the disruption of LLPS of N.

SARS-CoV-2 is still raging around the world. The daily confirmed cases are about 491,000 and this number is still increasing. The development of strategies to combat SARS-CoV-2 holds the highest priority. Tremendous efforts have been made to understand the infection of SARS-CoV-2, and the spike-ACE2mediated viral entry was a major target for many studies 3, 4, 48, 49 . In addition to the viral entry process, it is also critical to understand the details of other molecular events in the life cycle of SARS-CoV-2, such as viral assembly and replication. Recently studies revealed that SARS-CoV-2 carries almost the largest genome in RNA virus family and rapidly replicates in cells 8, 50 . The efficient genomic RNA package is therefore important for its replication. Investigation on the mechanisms underlying the assembly of SARS-CoV-2 will be critical in identifying new targets for treating COVID-19. Our work, by unveiling the LLPS of N protein with viral RNA, provided important detailed knowledges of SARS-CoV-2 assembly.

As a physicochemical process, LLPS was more and more realized to be a crucial mechanism that governing the functional organization of macromolecules in numerous biological processes 20, 23 . LLPS is believed to be critical in viral assembly 29 .

A key step during the replication of coronavirus is the association Foci of N R203/G204 -mEGFP and N R203K/G204R -mEGFP proteins per cell were quantified, n = 100 biologically independent cells (e). f qPCR analysis of IFNB expression in H1299 cells stimulated with 500 ng/ml poly(I:C), n = 3 biologically independent samples. g, h Representative fluorescent images of H1299 cells stimulated with 3 μg/ml vRNA (3′ UTR) (g). Foci of N R203/G204 -mEGFP and N R203K/G204R -mEGFP proteins per cell were quantified, n = 100 biologically independent cells (h). i qPCR analysis of IFNB expression in H1299 cells stimulated with 500 ng/ml vRNA (3′ UTR), n = 3 biologically independent samples. Hoechst (blue), nuclear staining (d, g). Scale bars, 10 μm (d, g). Violin plots showing foci of cells from each group, lines within the plots, with 25th, 50th, and 75th percentiles marked (e, h). Error bars, mean with s. d. (a, b, f, i) . Two-tailed unpaired Student's t-test (a, b, e, f, h, i), *P < 0.05, **P < 0.01, ****P < 0.0001. Data are representative of at least three independent experiments. Source data are provided as a Source Data file. of N protein with viral genomic RNA and the subsequent condensation into higher-order RNA-protein complexes, which initiates the assembly of virions 13, 31 . Our data suggested that in addition to virion assembly, the N-RNAs condensation is also important for shielding viral RNAs from host RNA sensors to avoid host immune surveillance. Interestingly, a recent proteome study identified the protein-protein interaction between N and G3BP1 9 . G3BP1 is a core organizer of SGs assembly [16] [17] [18] and SGs play a crucial role in antiviral responses against RNA viruses 51 . Because G3BP1 mediates the formation of SGs through LLPS [16] [17] [18] , N protein may be also involved in SARS-CoV-2 infection-induced formation of SGs through the binding to G3BP1. This involvement could be important for the host to block the translation of SARS-CoV-2 RNAs. On the other hand, N could also hijack G3BP1 or SGs to facilitate virion replication 51, 52 .

By analyzing the reported genome sequences, we found that the N R203K/G204R variant, contained by~37% of the total sequenced SARS-CoV-2 viruses, gained greater ability to undergo RNA-triggered LLPS. Interestingly, N R203K/G204R exhibited a higher propensity to undergo LLPS in the presence of RNAs and showed a greater effect on the inhibition of IFN expression. This finding linked the LLPS ability of N protein with its effect on IFN inhibition. Although our results showed that N R203K/G204R has little effect on the death ratio of COVID-19 patients, future studies with patient clinical outcomes and the coupled SARS-CoV-2 genome sequences will provide important evidences regarding the effect of N R203K/G204R polymorphism on the biology of SARS-CoV-2. In our study, we have also determined that the acidic microenvironment (pH 6.5) is favorable condition for the RNAtriggered LLPS of N. Although this observation needs to be further investigated, this may propose another perspective for the development of antiviral strategies.

During the revision of this manuscript, a few publications also reported the LLPS of N 53-57 . Our work, however, not only revealed the RNA-triggered LLPS of N as an important molecular event during the life cycle of SARS-CoV-2, but also found that GCG can inhibit SARS-CoV-2 replication by disrupting the LLPS of N. Our findings thus present GCG as a lead compound for the design of anti-SARS-CoV-2 drugs. Given that N protein is a highly conserved protein factor shared by the coronavirus family 58 , targeting N protein represents a novel avenue for drug discovery, not only for SARS-CoV-2, but also for the potential new coronavirus in the future.

Antibodies and reagents. Anti-Flag M2 (F3165, 1:5000) was from Sigma-Aldrich; anti-N (40143-R019, 1:5000) was from Sino-Biological; anti-N (ARG66782, 1:1000) was from Arigo Biolaboratories; anti-β-Actin (20536-1-AP, 1:2000) was from Proteintech Group. Anti-human GAPDH (1:5000) was prepared in our laboratory and generated by immunizing rabbits with human GAPDH protein. Cell culture and transfection. H1299 (ATCC #CRL-5803) cells were cultured in RPMI-1640 medium containing 10% FBS, 2 mM L-glutamine, 100 U ml −1 penicillin, and 100 mg ml −1 streptomycin. A549 (ATCC #CCL-185) and A549-hACE2-Flag cells (this paper) were cultured in MCCOY'S 5A containing 10% FBS, 1.5 mM L-glutamine, 100 U ml −1 penicillin, and 100 mg ml −1 streptomycin. All the cell lines were tested routinely and confirmed to be free of mycoplasma contamination. Transfection of RNAs and ssDNA were performed with Lipofectamine 2000 (Invitrogen). Lenti-virus for the preparation of N-expressing cells were produced in HEK293T (ATCC #CRL-3216) cells.

Plasmids. cDNA encoding N protein of SARS-CoV-2 was from Sango Biotech. We subcloned the coding sequence of N protein into pcDNA3.0-Flag vector for transient expression, and into pET28a(+) vector linked with C-terminal mEGFP for recombinant protein purification. mEGFP, N-mEGFP, and N R203K/G204R -mEGFP were subcloned into pCDX-Tet-On vector with an N-terminal Flag tag and fused with an mEGFP tag at C-terminus for the inducible expression in cells. Five truncations (N NTD , N CTD , N ▵NTD , N ▵CTD , and N NTD-CTD ) and three mutations (N R203K , N G204R , and N R203K/G204R ) were generated from full-length N-mEGFP and subcloned into pET28a(+) vector.

Cell viability assay. A549-hACE2-Flag cells were seeded into 96-well plates at a density of 10,000 cells per well and incubated with GCG at the indicated concentrations for 48 h. The cell viability was analyzed with CellTiter One Solution Cell Proliferation Assay (MTS) (G3580, Promega) according to the manufacturer's instruction. 50% cytotoxicity concentration (CC 50 ) was calculated by non-linear regression analysis.

N gene variant identification. Complete SARS-CoV-2 genome sequences (100,849) updated on September 18th, 2020 were downloaded from GISAID database (https://www.gisaid.org). To extract all N gene sequences, "Exonerate 2.2.0" software 59 was used to align N protein-coding sequences to the SARS-CoV-2 genome sequences (-model protein2 genome: bestfit -score 5 -g y). The gene sequences of N protein were aligned with MUSCLE 3.8.31 60 and the annotations and visualizations of mutation sites were processed within R 3.6.0 (https://cran.rproject.org).

The correlation analysis between mutation frequencies and death ratio. The frequencies of R203K/G204R polymorphism of N protein were calculated with each country and the death ratio information of indicated countries were obtained from WHO website (https://covid19.who.int/). The correlation between the mortality and R203K/G204R polymorphism of N protein was calculated with a linear regression model within R 3.6.0. The subgroup analysis was performed stratified by different continents.

Sequence alignment analysis. The sequence alignment of SARS-CoV-2 N protein and SARS-CoV N protein (GenBank: AY278741.1) was analyzed and visualized through the msa package within R 3.6.0 61 .

Phase separation prediction analysis of SARS-CoV-2 proteins. IDR scores of all SARS-CoV-2 proteins were calculated with an IUPred2A python script 3.7.3 32 for each amino acid. A score greater than 0.5 was regarded as intrinsically disordered and the percentage of amino acids with scores greater than 0.5 for each protein was calculated. Modular domains were predicted with InterProScan 5.31-70.0 62 and we used the predicted results of pfam and SMART for further analysis. Prion-like domains were identified with PLACC 36 , foci-formation propensity was calculated with catGranule 34 , Pi-Pi interactions were analyzed with P-Score 35 , and LLPS ability was predicted with an extra machine learning prediction tool PSPredictor 33 . The charges of N protein were analyzed according to DDX4-like predictor 39 and nuclear staining (a, c, d) . Violin plots showing foci of cells from each group, lines within the plots, with 25th, 50th, and 75th percentiles marked (b, e). Scale bars, 10 μm (a, c, d). Two-tailed unpaired Student's t-test (b, e), **P < 0.01, ****P < 0.0001. Data are representative of at least three independent experiments. Source data are provided as a Source Data file. cells) from each group, lines within the plots, with 25th, 50th, and 75th percentiles marked (g). h Cells were infected with SARS-CoV-2 for 1 h followed by 24-h GCG treatment, n = 3 biologically independent samples. Representative images were shown. SARS-CoV-2 was used at an MOI of 1. Hoechst (blue), nuclear staining (a, c, f). Error bars, mean with s. d. (b, d, e, g, h) . Two-tailed unpaired Student's t-test, *P < 0.05, ****P < 0.0001. Data are representative of at least three independent experiments. Source data are provided as a Source Data file.

the amino acid frequencies of N protein were analyzed according to R + Y predictor 38 within R 3.6.0.

GCG pull-down assay. Pull-down assays were previously described 47 . Briefly, GCG was conjugated with cyanogen bromide (CNBr)-activated agarose beads (C500099, Sangon Biotech). The recombinant N protein (40588-V08B) was from Sino-Biological. A549-hACE2-Flag cells were transfected with pcDNA3.0-Flag-N for 24 h and then lysed with lysis buffer (20 mM Tris-HCl, pH 7.5; 0.5% Nonidet P-40; 250 mM NaCl; 3 mM EDTA and 3 mM EGTA) containing complete protease inhibitor cocktail (04693132001, Roche), followed by centrifugation at 20,000 × g for 20 min at 4°C. The recombinant N protein and the supernatants from cell lysates were incubated with GCG conjugated beads at 4°C for 6 h. The beads were then washed five times with lysis buffer. The proteins pulled down were examined by 10% SDS-PAGE followed by immunoblotting with indicated antibodies.

Electrophoretic mobility shift assay (EMSA). The EMSA was performed to determine RNA-binding capacity of N protein. Recombinant full-length and truncated N-mEGFP proteins were incubated with 55-nt Cy3-labeled vRNA. The mixtures were then applied to an 8% Native-PAGE and the electrophoresis was performed in 0.5 × TBE (Tris-Borate-EDTA) buffer for 1 h at 200 V. The gels were analyzed by ChemiScope 6100 Touch Chemiluminescence imaging system (CLiNX) and ChemiDoc MP Imaging System (Bio-Rad).

RNA isolation and quantitative PCR (qPCR). Cells were collected and total RNAs were isolated using TRI reagent (93289, Sigma-Aldrich). Total RNAs (500 ng) were reversed-transcribed to cDNA using PrimeScript RT Master Mix (RR036A, TaKaRa). qPCR was performed with PowerUp SYBR Green Master Mix (A25778, Applied Biosystems), using StepOnePlus Real-Time PCR System (Applied Biosystems) according to the manufacturer's instructions. Data were analyzed with StepOnePlus v2.2 software. Primers used are as follows: hIFNB-Fwd: 5′-AGGAC AGGATGAACTTTGAC-3′; hIFNB-Rev: 5′-TGATAGACATTAGCCAGGAG-3′; hGAPDH-Fwd: 5′-GAGTCAACGGATTTGGTCGT-3′ and hGAPDH-Rev: 5′-TTGATTTTGGAGGGATCTCG-3′. GAPDH was used for normalization.

In vitro phase separation assay. Recombinant N-mEGFP proteins were diluted in phase separation buffer (10 mM Na 3 PO 4 , 150 mM NaCl, pH 6.5), and RNAs were added and mixed in glass-bottom cell culture dishes (801002, NEST) for microscopic observation and image acquirement.

Fluorescence recovery after photobleaching (FRAP). Recombinant mEGFPtagged N proteins were used to performed FRAP assays in vitro. Selected regions were bleached with a 488-nm laser pulse. The fluorescence intensity was collected every 1 s and normalized to the intensity before bleaching. For in vivo FRAP assays, H1299 cells were seeded on the glass bottom cell culture dishes and treated with 100 ng ml −1 Dox for the inducible expression of N-mEGFP. After 12-h Dox treatment, the cells were transfected with 1 μg ml −1 poly(I:C) for another 6 h. FRAP assays were performed with 488-nm laser pulse and the fluorescence intensity was collected every 0.5 s in vivo and normalized to the intensity before bleaching.

Protein expression and purification. Constructs for recombinant protein purification were transformed into E. coli BL21 (DE3) strain (S106-02, GenStar), and 0.6 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) (VA20321, GenStar) was used to induce the expression of recombinant proteins. Cells were collected and resuspended in lysis buffer (20 mM Na 3 PO 4 , 1.5 M NaCl, 20 mM imidazole, pH 7.5). Following the sonication and centrifugation, the cleared supernatants were purified with Nickle-coupled agarose beads (G106-01, GenStar) according to the manufacturer's instructions.

Formation of N condensates in vivo. H1299 cells were seeded in 24-well plates and treated with 100 ng ml −1 Dox for 12 h to induce the expression of N-mEGFP. Then the cells were treated with different chemicals as indicated concentrations, followed by transfection with different RNAs. Cells were fixed with 4% paraformaldehyde for 10-15 min at room temperature, and the nuclei were stained with Hoechst for 10 min. Images were acquired using Zeiss LSM 880 confocal microscope or DeltaVision Deconvolution microscope.

Virus RNA detection. A549-hACE2-Flag cells were pre-treated with GCG for 1 h, and then infected with SARS-CoV-2 nCoV-SH01 at an MOI of 1 for 24 h, or cells were infected with SARS-CoV-2 for 1 h followed by 24-h GCG treatment. Total RNAs were extracted from cells and viral RNAs were determined using the Taq-Path 1-Step RT-qPCR Master Mix (A15299, Thermo Fisher Scientific). Primers and probes used are as follows: SARS-CoV-2-N-Fwd: 5′-GACCCCAAAATCAG CGAAAT-3′; SARS-CoV-2-N-Rev: 5′-TCTGGTTACTGCCAGTTGAATCTG-3′ and SARS-CoV-2-N-Probe: 5′-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1-3′. 50% inhibitory concentration (IC 50 ) was calculated by non-linear regression analysis.

Statistical analysis. To determine the partition coefficient of indicated groups, 8 or 10 microscopy images were randomly selected, and the fluorescence intensity was acquired with Volocity 6.1.1 63 . Partition coefficient of total fluorescence intensity was calculated as the total fluorescence intensity of droplets divided by the bulk fluorescence intensity of background. Partition coefficient of fluorescence intensity per droplets was calculated as average fluorescence intensity of droplets divide by the bulk fluorescence intensity per pixel of background.

GraphPad Prism 8.0 was used to perform the statistical analysis. Statistical data are presented as mean with s.d. or s.e.m. as indicated in figure legends. The fluorescence intensity was calculated by Volocity 6.1.1. A standard two-tailed unpaired Student's t-test was used for statistical analysis of two groups.

Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Complete SARS-CoV-2 genome sequences (100,849) updated on September 18th, 2020 were downloaded from GISAID database (https://www.gisaid.org). The death ratio information updated on September 18th, 2020 was obtained from WHO website (https:// covid19.who.int/). The annotations and visualizations of mutation sites, the correlation analysis between mutation frequencies and death ratio, the sequence alignment of SARS-CoV-2 N protein and SARS-CoV N protein, and the amino acid frequencies of N protein were analyzed within R 3.6.0 (https://cran.r-project.org). The full-length genome sequence of SARS-CoV-2 nCoV-SH01 strain (accession no. MT121215) and the sequence of SARS-CoV (accession no. AY278741.1) are downloaded from GenBank. Other data related to this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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The coronavirus nucleocapsid is a multifunctional protein

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G3BP1 is a tunable switch that triggers phase separation to assemble stress granules

RNA-induced conformational switching and clustering of G3BP drive stress granule assembly by condensation

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Protein phase separation: a new phase in cell biology

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P-body purification reveals the condensation of repressed mRNA regulons

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Respiratory syncytial virus sequesters NF-κB subunit p65 to cytoplasmic inclusion bodies to inhibit innate immune signaling

Measles virus nucleo-and phosphoproteins form liquid-like phase-separated compartments that promote nucleocapsid assembly

Phase transitions drive the formation of vesicular stomatitis virus replication compartments

Coronavirus nucleocapsid protein is an RNA chaperone

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Prediction of liquid-liquid phase separation proteins using machine learning

A concentration-dependent liquid phase separation can cause toxicity upon increased protein expression

Pi-Pi contacts are an overlooked protein feature relevant to phase separation

PLAAC: a web and command-line application to identify proteins with prion-like amino acid composition

Coactivator condensation at super-enhancers links phase separation and gene control

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SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with human hnRNPs

protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates

Genomic RNA elements drive phase separation of the SARS-CoV-2 nucleocapsid

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InterProScan: protein domains identifier

We provided the code that we programmed and used in this study on GitHub at https:// github.com/TintingLi/Nprotein_LLPS_analysis.

We thank Pei-Hui Wang (Shandong University) for providing materials. 

The authors declare no competing interests.

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-021-22297-8.Correspondence and requests for materials should be addressed to T.Z., X.-M.Z. or T.L.Peer review information Nature Communications thanks Jonathon A Ditlev and other, anonymous, reviewers for their contributions to the peer review of this work. Peer review reports are available.

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