RNA Triggers Liquid Phase Separation Of SARS-CoV-2 Nucleocapsid Protein

The 2020 was marked by the sudden onslaught of coronavirus disease 2019(COVID-19)pandemic.By sequencing the samples from the patients,a virus called severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)is designated as a causative agent.SARS-CoV-2 shares ~80% sequences similarity with severe acute respiratory syndrome coronavirus(SARS-Co V)and SARS-CoV-2 mainly induces infection by entering host cells via angiotensin-converting enzyme 2(ACE2).As of March 1st,2021,SARS-CoV-2 has rapidly spread worldwide and delivered far-reaching impacts on people's health.At least 100 million people have been infected,with 2 million deaths in the pandemic according to a report.Currently,lack of knowledge of SARS-CoV-2 has been hampering development of miracle drugs 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 of great significance.SARS-CoV-2 is a single-stranded,positive-sense RNA virus containing a 30 kb genome with 5' cap structure and 3' poly(A)tail.The genome of SARS-CoV-2 codes 29 proteins,including 4 structural proteins,spike(S),membrane(M),envelope(E)and nucleocapsid(N).Our study mainly found that RNA can trigger liquid-liquid phase separation(LLPS)of N protein.In the study,using bioinformatic tools,IUPred2 and ANCHOR2,we analyzed 29 proteins encoded by the SARS-CoV-2 genome.N protein was predicted as a potentially LLPS protein with numerous intrinsically disordered regions(IDRs).N protein is a 46 k Da protein,and it serves two major functions: compaction of the genome RNA(g RNA)into the virion to shield the pattern recognition receptors(PRRs)and regulation of viral replication and transcription.To study the LLPS of N protein,we first purified N protein in prokaryotic expression and purification system and confirmed its RNA-binding capacity with electrophoretic mobility shift assay(EMSA).By performing the time-lapse and fluorescence recovery after photobleaching(FRAP)assays in phase separation system in vitro,we found that virus RNA(v RNA)can induce drop-like condensate formation of N protein and the photo-bleached fluorescence signal of N-v RNA droplets can be recovered within seconds.At the same time,v RNA can induce foci formation in an inducible N-expressing H1299 cell line.All the above results indicated that N protein can undergo phase separation by v RNA.Furthermore,many RNA mimics also can trigger phase separation of N protein other than v RNAs.As protein phase separation is a context-dependent process,we found that the phase separation of N protein is regulated by many factors,including RNA concentration,protein concentration,p H,salt ionic concentration and RNA length.N is a structural protein shared across all coronaviruses.According to the analysis of sequences,the N protein of SARS-CoV-2 and SARS-Co V are ~ 90% identical.SARSCo V-2 N contains two globular domains,the N-and C-terminal domains(NTD and CTD),which are responsible for RNA-binding and dimer formation,respectively.To further explore the key regions in regulating phase separation of N protein,we constructed truncated N variants,we found that full length(FL)can deliver the strongest RNAbinding capacity.We found that deletion of NTD,CTD or IDR disrupted the LLPS in phase separation system in vitro.Since the first case of the COVID-19 pandemic was reported,full genomic sequences of this virus from all over the world were continuously submitted to public databases,such as GISAID.We analyzed 100,849 genome sequences of SARS-CoV-2 from the database.Surprisingly,while many nucleotide mutations were found across the full length of the N-coding sequence,~37% of the mutant genomes focusing on 608-610 th sites(from GGG to AAC)were identified,resulting in the amino acid substitution,Arginine(R)into Lysine(K)in 203 rd site,Glycine(G)into Arginine(R)in 204 th site.We called it as NR203K/G204 R protein.To further evaluate the LLPS of the mutant protein,we purified the protein.When incubating with RNA,interestingly,NR203K/G204 R protein gained greater ability to undergo LLPS compared to NR203/G204(FL)protein.We also analyzed the correlation between mortality and the polymorphism of N protein.Our results showed that this polymorphism has little effect on the death ratio reported.Taken together,our study found that RNA can trigger the LLPS of SARS-CoV-2 N protein,which is tightly regulated by many factors.NTD,CTD domain and IDR are all essential for the LLPS of N protein.Furthermore,NR203K/G204 R protein can gain a greater ability to undergo LLPS.The LLPS of N protein may serve a better understanding of its biological functions.RNA can trigger condensate formation of N protein by LLPS to preserve the stability of the genome when cells are faced with virus infection.Our work not only reveals a new mechanism that regulates the assembly of SARS-CoV-2,but indicates that disrupting the LLPS of N effectively will provide a promising scientific program for developing selective pharmacologic blockades and combat SARS-CoV-2.