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) 

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The Global Phosphorylation Landscape of SARS-CoV-2 583 Infection

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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

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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

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Structural basis of RNA 606 recognition by the SARS-CoV-2 nucleocapsid phosphoprotein. bioRxiv

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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

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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