key: cord-0956121-4o6hx5mb authors: Wu, Chao; Qavi, Abraham J.; Hachim, Asmaa; Kavian, Niloufar; Cole, Aidan R.; Moyle, Austin B.; Wagner, Nicole D.; Sweeney-Gibbons, Joyce; Rohrs, Henry W.; Gross, Michael L.; Peiris, J. S. Malik; Basler, Christopher F.; Farnsworth, Christopher W.; Valkenburg, Sophie A.; Amarasinghe, Gaya K.; Leung, Daisy W. title: Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain date: 2021-06-01 journal: iScience DOI: 10.1016/j.isci.2021.102681 sha: 3f48aa311fb74e31f46ccc7441715e75a44948dd doc_id: 956121 cord_uid: 4o6hx5mb Nucleocapsid (N) encoded by SARS-CoV-2 plays key roles in the replication cycle and is a critical serological marker. Here we characterize essential biochemical properties of N and describe the utility of these insights in serological studies. We define N domains important for oligomerization and RNA binding and show that N oligomerization provides a high affinity RNA binding platform. We also map the RNA binding interface, showing protection in the N-terminal domain and linker region. In addition, phosphorylation causes reduction of RNA binding and redistribution of N from liquid droplets to loose-coils, showing how N/RNA accessibility and assembly may be regulated by phosphorylation. Finally, we find that the C-terminal domain of N is the most immunogenic, based upon antibody binding to patient samples. Together, we provide a biochemical description of SARS-CoV-2 N and highlight the value of using N domains as highly specific and sensitive diagnostic markers. connects the NTD and CTD (Figure 1A and Supp. Figure 1A ) . 73 Phosphorylation of residues in the serine-arginine of LKR is believed to regulate 74 J o u r n a l P r e -p r o o f 4 discontinuous transcription, particularly for shorter subgenomic mRNA closer to the 3' 75 end during early stages of replication (Wu et al., 2014; Wu et al., 2009 ). The LKR along 76 with residues at the extreme N and C termini (Narm: amino acid residues 1-43 and 77 Carm: amino acid residues 370-419) are intrinsically disordered 78 Cubuk et al., 2021). However, relative to the Narm and Carm, the LKR is more 79 conserved (Figure 1B and Supp. Figure 1B) . 80 Given its abundant expression and conservation within the genome, N has been used (Figure 2A) . From this assay, we find that 140 N WT binds the 20-nt ssRNA with high affinity (K D = 0.007 ± 0.001 μM). Removal of the 141 Narm and Carm do not impact ssRNA binding (K D = 0.006 ± 0.002 and 0.006 ± 0.002 142 μM for N NTD-LKR-CTD-Carm and N NTD-LKR-CTD , respectively) ( Figure 2B ). In contrast, the 143 J o u r n a l P r e -p r o o f 7 isolated N NTD and N CTD have low affinity binding (K D = 20 ± 10 and 13 ± 5 μM, 144 respectively). However, inclusion of the LKR region increased RNA binding affinity 145 significantly (0.50 ± 0.08 and 0.35 ± 0.04 μM for N NTD-LKR and N LKR-CTD ) (Figure 2B-2D) . 146 Addition of CTD onto NTD-LKR in cis increases the binding affinity to the single digit nM Figure 3B ). This is potentially due, in 167 part, to the energetic penalty of unfolding the stem-loop structure. Furthermore, the 168 Narm and Carm may contribute more to slRNA binding than ssRNA because the impact 169 on N binding is more pronounced after removal of the Narm or Carm (Supp. Figure 3C) . While more phosphorylation events may occur, we focused on these three better-known 193 positions to evaluate if introduction of S176D/S188D/S206D mutations into N NTD-LKR (N NTD-LKR S176D/S188D/S206D ) will resolve N protein aggregation at the concentrations of 195 interest. With the introduction of the mutations on a shorter construct, N NTD-LKR S176D/S188D/S206D , 197 we were able to use sequential FXIII and pepsin digestion to recover 152 peptides, 198 resulting in 93.3% sequence coverage (Supp. Figure 4) , which enabled us to further However, oligomerization is an intrinsic property of N and is complicated by RNA 233 binding during copurification due to the high affinity of N proteins for RNA. We found 234 that bacterial RNA copurified even with increased ionic strength in purification buffer. residues for S176 by GSK-3) produced a reduced RNA-free peak (p3) and an increased 271 RNA-bound peak (p1) (Figure 4C ). Introduction of S176D to generate N S176D/S188D/S206D Similar observations were made for N S176D/S188D/S206D (Figure 4D ). To describe this 278 interaction further, we measured ssRNA binding to the N phosphomimics ( Figure 4E 279 and Supp. Figure 5A) . N S176D/S188D/S206D displays ~5-fold lower binding affinity to ssRNA Furthermore, binding to slRNA is also affected by these mutations (Supp. Figure 5B) . 284 Collectively, our data suggest that phosphorylation of the LKR region can impact N properties of N. Our data revealed that mutation of S176, S188, and S206 in the SR 365 motif to generate N phosphomimics resulted in decreased binding to RNA and a shift in We would like to thank Bruker for mass spectrometry technical and instrument support, The authors declare no competing interests. Inclusion and diversity 567 We worked to ensure sex balance in the selection of non-human subjects. One or more 568 of the authors of this paper self-identifies as living with a disability. One or more of the 569 authors of this paper received support from a program designed to increase minority 570 representation in science. N WT N S188D/S206D N S176D/S188D/S206D p1 p2 p3 N S188D/S206D p1 N S188D/S206D p2 2 m N S176D/188D/S206D p1 N S176D/188D/S206D p2 0.01 0.1 1 10 100 0 100 200 300 N WT N S176D/S188D/S206D N NTD-LKR N NTD-LKR S176D/S188D/S206D concentration ( M) High resolution 574 cryo-EM structure of the helical RNA-bound Hantaan virus nucleocapsid reveals its assembly 575 mechanisms SARS-CoV-2 (COVID-19) by the numbers Structural dissection of Ebola virus and its assembly determinants using cryo-electron 580 tomography The Global Phosphorylation Landscape of SARS-CoV-2 583 Infection Antigenic and cellular 585 localisation analysis of the severe acute respiratory syndrome coronavirus nucleocapsid protein using 586 monoclonal antibodies Phosphoregulation of phase separation by the SARS-CoV-2 N protein suggests a biophysical 589 basis for its dual functions The SARS coronavirus 591 nucleocapsid protein -Forms and functions Multiple Nucleic Acid Binding Sites and Intrinsic Disorder of Severe Acute Respiratory Syndrome 594 Coronavirus Nucleocapsid Protein: Implications for Ribonucleocapsid Protein Packaging Clinical evaluation of serological IgG antibody response on the Abbott Architect for 598 established SARS-CoV-2 infection The SARS-CoV-2 nucleocapsid protein is dynamic, 601 disordered, and phase separates with RNA Structural basis of RNA recognition by the SARS-CoV-2 nucleocapsid phosphoprotein Structural basis of RNA 606 recognition by the SARS-CoV-2 nucleocapsid phosphoprotein. bioRxiv Nucleocapsid proteins: roles beyond viral RNA packaging Human Coronavirus: Host-Pathogen Interaction Coronavirus N Protein N-Terminal Domain (NTD) Specifically Binds the Transcriptional Regulatory 613 Sequence (TRS) and Melts TRS-cTRS RNA Duplexes Electron microscopy studies 615 of the coronavirus ribonucleoprotein complex ORF8 and ORF3b antibodies are accurate serological 618 markers of early and late SARS-CoV-2 infection Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase 621 separate. bioRxiv SARS 623 CoV-2 nucleocapsid protein forms condensates with viral genomic RNA. bioRxiv Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential 627 unique drug targeting sites Functional Transcriptional Regulatory Sequence (TRS) RNA Binding and Helix Destabilizing Determinants of Murine Hepatitis Virus (MHV) The Architecture of SARS-CoV SSB-DNA Binding Monitored by Fluorescence 634 Intensity and Anisotropy SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and 638 uninfected controls When your cap matters: structural insights into self vs non-640 self recognition of 5' RNA by immunomodulatory host proteins Comprehensive Antibody Epitope Mapping of the Nucleocapsid Protein of Severe Acute Respiratory 643 Syndrome (SARS) Coronavirus: Insight into the Humoral Immunity of SARS Structural Basis for the 646 Identification of the N-Terminal Domain of Coronavirus Nucleocapsid Protein as an Antiviral Target Evaluation of Nucleocapsid and Spike Protein-Based Enzyme-Linked Immunosorbent Assays for 650 Detecting Antibodies against SARS-CoV-2 The immunodominant and neutralization linear epitopes for SARS-CoV-2 The SARS-CoV-2 Nucleocapsid 654 phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M 655 protein. bioRxiv SR-Rich Motif Plays a Pivotal Role in Recombinant 657 SARS Coronavirus Nucleocapsid Protein Multimerization Nucleocapsid Structure of Negative Strand RNA Virus. 659 Viruses 12 Morphology of 661 Marburg Virus NP-RNA The Coronavirus Nucleocapsid Is a Multifunctional Protein. 663 Viruses Filovirus pathogenesis and immune evasion: 665 insights from Ebola virus and Marburg virus A Liquid-to-Solid Phase Transition of the ALS Protein FUS 668 Accelerated by Disease Mutation Coronaviruses post-SARS: update on replication and pathogenesis. 670 Structure of the Rift Valley fever virus 672 nucleocapsid protein reveals another architecture for RNA encapsidation Nucleocapsid protein of 675 SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates Continuous and Discontinuous RNA Synthesis in 678 Coronaviruses Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for 681 Nucleocapsid-like Assembly Solution Structure of the C-terminal Dimerization Domain of SARS Coronavirus Nucleocapsid 684 Protein Solved by the SAIL-NMR Method Clinical Performance of the Roche SARS-CoV-2 Serologic Assay Clinical Performance of Two SARS-CoV-2 Serologic Assays Structure 692 and assembly of the Ebola virus nucleocapsid Měnglà Virus Proteins on Human and Bat Innate Immune Pathways Nucleocapsid Phosphorylation and RNA Helicase DDX1 697 Recruitment Enables Coronavirus Transition from Discontinuous to Continuous Transcription Glycogen Synthase Kinase-3 Regulates the Phosphorylation of Severe Acute 701 Respiratory Syndrome Coronavirus Nucleocapsid Protein and Viral Replication Architecture and self-assembly of the SARS-704 CoV-2 nucleocapsid protein 65 ± 44 µM (N CTD , blue 735 up triangle), 2.5 ± 0.5 µM (N NTD-LKR µM (N NTD-LKR-CTD-Carm , navy left triangle). F. Table 737 summarizes K D values (µM) for key constructs binding to ssRNA and slRNA. Numbers 738 are reported as average and standard deviation of two experiments Woods' 741 plot showing cumulative differential HDX and validating differences using global 742 significance limits. The horizontal bars depict the cumulative HDX differences between 743 the RNA-bound and unbound N NTD-LKR S176D/S188D/S206D . Standard deviations are shown 744 for each peptide. Peptides showing statistically significant differences are differentiated 745 by global significance limit using this standard error of the mean a t-values for a two-746 tailed The blue shade of 747 the peptide bar indicates differing statistical significance (light blue, medium blue, and 748 navy, respectively); gray peptide bars depict peptides where statistically significant 749 differences in HDX were not observed. Vertical bars show previously reported binding 750 sites (residues reported for RNA-binding CoV2 N-protein with the 759 exception of those reporting a statistically significant difference in teal. D. Electrostatic 760 potential calculated with APBS mapped on to the N NTD structure (PDB 6M3M) shows a 761 major positive charge groove. Red and blue represent negative and positive 762 electrostatic potential. The color scale is in kTe -1 units N-RNA forms liquid droplets and phosphorylation modulates N-RNA 766 interactions. A. Size exclusion chromatography of N constructs 25 mM HEPES, 500 mM NaCl, 2 mM TCEP, 5% 768 glycerol. Samples from peak 1 (p1) and p2 contain RNA whereas p3 are RNA-free 769 based upon absorbance from the 260/280 ratio. B. Negative stain electron microscopy 770 (EM) image of p1 and p2 for Samples were diluted into 150 mM NaCl before negative-staining fixation by uranyl 772 acetate. C. Size exclusion chromatography of N constructs N S176D/S188D/S206D , red) in 25 mM HEPES, 500 mM NaCl, 2 mM TCEP Negative stain electron microscope image of N S188D/S206D and N S176D/S188D/S206D in 150 775 mM NaCl. E. Fluorescence polarization binding curves of N mutants to a 20-nt ssRNA S176D/S188D/S206D , black circle), 0.505 ± 0.075 µM (N NTD-LKR N NTD-LKR S176D/S188D/S206D , orange down triangle). Numbers are reported as 779 average and standard deviation of two experiments. See also Figure S5 Figure 5. The CTD of N is a highly sensitive serological marker. A. ELISA data of Black solid line indicates 784 the mean OD 450 value for each population. **** p < 0.0001. B. ELISAs with the various 785 N constructs for patient IgG. ELISAs were performed on plasma samples from COVID-786 19 patients (n = 68) and negative controls (n = 28). The cut-off is represented by the 787 dotted line and calculated as the mean + 3 standard deviations of the negative 788 population. Mean values ± standard deviation of COVID-19 and negative groups are 789 shown. C. Sensitivity and specificity for each of the N domains calculated from the 790 ELISA results. D. Heat-map of ELISA results for N NTD-LKR-CTD-Carm , N CTD , and N NTD 791 constructs from COVID-19 samples (n = 67) Ratio of OD 450 for N CTD and N NTD-LKR-CTD-Carm for acute and convalescent time-points Mean values ± standard deviation of acute and convalescent COVID-19 samples are 795 shown. Experiments were repeated twice. Statistical significance was calculated by 796 unpaired Student's t-test, ****p < 0.0001. G. Inhibition of SeV-induced IFN promoter 797 activation by N constructs Three 799 transfection concentrations were used: 1.25, 12.5, and 125 ng/well. Statistical 800 significance was determined by performing a one-way ANOVA followed with Tukey 801 multiple comparison as compared to Sendai virus