Preliminary Study On The Mechanism Of Selective Genome Encapsulation By SARS-CoV-2N Protein

Background and ObjectiveThe coronavirus disease 2019(COVID-19)pandemic,caused by severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)infection,is still spreading worldwide.According to the epidemic report released by the World Health Organization(WHO),the number of confirmed cases of new coronary pneumonia in223 countries around the world has exceeded 460,280,168.The virus poses a huge threat to human life and health.SARS-CoV-2 is a single-stranded positive-sense RNA(+ss RNA)virus belonging to the family Betacoronavirus of the family Coronaviridae,family B.Its genome RNA is about 30 kb in length and encodes at least 20 proteins.Including 4structural proteins:spike protein(S),envelope protein(E),membrane protein(M)and nucleocapsid protein(N).Among them,the nucleocapsid protein(N)is the main structural component of the virus particle.It combines with the viral RNA to form a helical nucleocapsid structure,which protects the genomic RNA from being degraded by nucleases in the host,and plays a key role in the virus life cycle.The precise packaging of the N protein and viral genomic RNA is a key factor for the correct assembly and budding of the virus to produce progeny viruses.Under the interference of numerous nucleic acids in the cytoplasm,the mechanism of how the N protein correctly wraps the viral genomic RNA is still unclear.Focusing on the scientific issue of precise packaging of viral genomic RNA,this study focused on the N protein of SARS-CoV-2 and the SUD domain of the non-structural protein Nsp3 to preliminarily study the mechanism of how viral genomic RNA is selectively packaged.This study provides a theoretical and practical basis for understanding the SARS-CoV-2 genome-specific packaging mechanism and establishing preventive methods for targeted viral packaging.Methods1.Construction of protein expression vectorUsing molecular cloning technology,the sequence of the target protein and the tag sequence required for purification were inserted into the prokaryotic expression vector p ET32a,p GEX-6P-1,and the eukaryotic expression vector pc DNA3.1,respectively.2.Expression and purification of N protein of SARS-CoV-2 in prokaryotic systemThe constructed prokaryotic expression plasmid was transformed into BL21(DE3)E.coli,induced by IPTG at 16°C overnight for 16 h,the cells were collected,and the expression of N protein was detected by Coomassie blue staining and Western blot.We used His-tag affinity chromatography,desalting column,heparin column and size exclusion chromatography to obtain high-purity N protein.3.Expression and purification of N protein of SARS-CoV-2 and its glycosylation and phosphorylation mutants in eukaryotic systemThe constructed eukaryotic expression plasmid was transfected into the mammalian expression cell HEK293F.After 36 hours,samples were taken for WB to verify the expression of the N protein.The pure modified N protein was expressed and purified by the same method in order to explore the functional effects of post-translational modifications on the N protein.4.Measurement of protein particle Size by Dynamic Light ScatteringThe particle sizes of prokaryotic N proteins,eukaryotic N proteins,and glycosylation and phosphorylation mutant N proteins were measured by Dynamic Light Scattering(DLS)method,and we compared their differences.5.Measuring protein stability by Micro Differential Fluorescence ScanningThe thermal stability of prokaryotic system N protein,eukaryotic system N protein and glycosylated and phosphorylated mutant proteins were measured by differential scanning fluorescence scanning(DSF),and we compared their differences.6.Expression and purification of the SUD domain of Nsp3 protein in prokaryotic systemThe constructed prokaryotic expression plasmid was transformed into BL21(DE3)E.coli,induced by IPTG at 16°C overnight for 16 h,the cells were collected,and the expression of SUD protein was detected by Coomassie blue staining and Western blot.High purity SUD domains were isolated by GST-tag affinity chromatography and size exclusion chromatography.7.In vitro transcription of RNAThe two RNA fragments on the SARS-CoV-2 genome were cloned into the p Bluescript II-SK(+)vector with the T7 promoter,and then the 5’end RNA and packaging signal(PS)of the SARS-CoV-2 genome were obtained by in vitro transcription.8.Detection of the role of SUD domain and RNA by isothermal titration calorimetryIsothermal Titration Calorimetry(ITC)is a label-free technique for studying intermolecular interactions.Computational qualitative and quantitative information on intermolecular interactions,we used this technique to accurately measure the binding affinity between the SUD domain and two RNA fragments from the SARS-CoV-2 genome.9.Study the interaction between SUD domain and N protein by GST pull-down methodGST-pulldown is a classic method to study protein-protein interactions,The purified prokaryotic system N protein,eukaryotic system N protein and mutants were subjected to pull down experiments with GST-tagged SUD domains respectively,and the interaction was verified by Western blot.Results1.Design of fusion protein and successful construction of expression vectorWe inserted a His tag at the N-terminus of the N protein of SARS-CoV-2 to construct a prokaryotic expression vector,named p ET32a-His-n CoVN,and inserted a flag tag into the N-terminus of the N protein to construct a eukaryotic expression vector,Named pc DNA3.1-flag-n CoVN,and glycosylation mutants pc DNA3.1-flag-n CoVN47Q,pc DNA3.1-flag-n CoVN269Q,phosphorylated mutants pc DNA3.1-flag-n CoVN-S188A-S206A.We inserted a GST tag into the N-terminus of the SUD domain of the SARS-CoV-2 Nsp3 protein to construct a prokaryotic expression vector,named p GEX-6P-1-GST-SUD,they were successfully expressed by Western blot.2.The N protein expressed in prokaryotic system can bind nucleic acid nonspecificallyThe N protein of SARS-CoV-2 was finally obtained in E.coli BL21(DE3)through the His-tag.After desalting,heparin column,and size exclusion chromatography(FPLC)purification,we obtained N protein samples with a small amount of nucleic acid and heterogeneous properties.The measured ratio of A260 to A280 of the N protein was 1.84.Since the ratio was much greater than 1,it indicated that the N protein bound a large number of bacterial nucleic acids and was a non-specifically binding nucleic acid protein.3.The N protein expressed in eukaryotic systems is essentially unbound to nucleic acidsThe SARS-CoV-2 N protein was finally obtained by transient transfection in mammalian expression cells HEK293F.After purification by flag affinity chromatography and size exclusion chromatography(FPLC),we obtained relatively homogeneous N protein samples.The results showed that the ratio of A260 to A280of N protein purified by eukaryotic system was 0.61,which was very different from that of prokaryotic system,and basically did not bind nucleic acid.4.The difference in the nucleic acid binding ability of N proteins expressed in eukaryotic and prokaryotic systems may be due to glycosylation and phosphorylation modificationsThe mass spectrometry results of post-translational modification of N protein in eukaryotic system show that N protein has many glycosylation and phosphorylation modifications,and the measured phosphorylation sites are S51,T54,T57,T76,S78,S79,S105,T135,T141,T148,S176,T282,T325,S327,T362,T391,T393,of which the phosphorylation level at site 176 is the highest,and the measured O-glycosylation sites are T115,T165,T332,T334,S412,S416,N-glycosylation sites are N47,N269.5.Effects of N-protein glycosylation mutants and phosphorylation mutants on its function in eukaryotic systemsIn order to explore the functional effects of post-translational modifications on N protein,N protein glycosylation and phosphorylation mutants were expressed and purified in a eukaryotic system.The results showed that glycosylation mutant N47Q and N269Q proteins have nucleic acids similar to wild-type N protein.Binding characteristics,the phosphorylated mutant N-S188A-S206A protein exists mainly in the unbound nucleic acid state.6.Particle size measurement of prokaryotic system N proteins and eukaryotic system N proteins and mutantsThe results of dynamic light scattering showed that the solution of N protein purified by the prokaryotic system was very heterogeneous,including monomers,dimers,and possibly multimers.The eukaryotic system N protein and its glycosylated and phosphorylated mutants exist mainly in dimer form.7.Stability measurement of prokaryotic system N protein and eukaryotic system N protein and mutantsThe measured Tm values of prokaryotic system N protein,eukaryotic system N protein,eukaryotic system glycosylation mutant N47Q,N269Q protein,and phosphorylated mutant N-S188A-S206A protein were 45.9℃,51.6℃,48.9℃,respectively.47.9°C,49.5°C.The higher the Tm value,the more stable the protein is,and the degree of stability is obtained.Eukaryotic system N protein>eukaryotic system N-S188A-S206A>eukaryotic system N47Q protein>eukaryotic system N269Q protein>prokaryotic system N protein,it can be speculated that after translation the post-modified N protein may have a more stable structure.8.The SUD domain can specifically bind to viral packaging signalWe used the T7 promoter to transcribe the 5’end RNA and packaging signal RNA on the SARS-CoV-2 genome in vitro,and obtained RNA with higher purity by concentration measurement and electrophoresis identification.The binding affinity between the SUD domain and these two RNA fragments was measured by isothermal calorimetry,and the results showed that the K_D value of the SUD domain for the reaction with 5’RNA was 1.47e-06 M,and the K_D value for the reaction with PS RNA was 1.05e-09M.The smaller the K_D value,the stronger the binding ability between protein and nucleic acid.The ability of the SUD domain to bind to putative packaging signals is nearly a thousand-fold stronger than that of 5’genomic RNA,reaching the n M level.This indicates that the SUD domain is highly selective for the recognition of packaging signal.9.Phosphorylation modification of N protein affects the binding to SUD domainThe purified prokaryotic N protein,eukaryotic N protein and mutants were subjected to pull down with the GST-tagged SUD domain,and Western blot was used to verify the situation.We found that the prokaryotic N protein interacts with the SUD domain.The eukaryotic N protein cannot interact with the SUD domain,indicating that post-translational modification affects the binding of the N protein to the SUD domain.The eukaryotic glycosylation mutant N protein does not interact with the SUD domain,while the eukaryotic phosphorylation The mutant N protein can interact with the SUD domain,indicating that the phosphorylation modification affects the interaction between the N protein and the SUD domain.Conclusions1.Post-translational modification of N protein is related to RNA binding ability(1)The N protein expressed in the prokaryotic system lacks post-translational modifications,is easily degraded,and binds non-specifically to nucleic acids.(2)The N protein expressed in the eukaryotic system is post-translationally modified,so it is not easy to non-specifically bind to nucleic acids,and its properties are relatively stable.2.The N protein cooperates with the nsp3 protein in the DMV membrane pore complex to complete the selective packaging of viral genomic RNA(1)The single SUD domain of nsp3 can selectively bind to RNAs containing packaging signals.(2)The N protein has a direct interaction with the SUD domain of the nsp3protein and is regulated by the post-translational modification of the N protein in eukaryotic cells.After the N protein is phosphorylated in the SR region in eukaryotic cells,its binding to the SUD domain is weakened.(3)Combined with previous findings in various coronaviruses,the N protein may have complex and diverse interactions with multiple domains at the N-terminus of Nsp3.It is speculated that Nsp3 entraps PS-bearing viral genomic RNA and delivers RNA by altering the role of N protein modifications,assisting in nucleocapsid assembly,and release from the vicinity of membrane pores.