RNA structure and multiple weak interactions balance the interplay between RNA binding and phase separation of SARS-CoV-2 nucleocapsid

Abstract The nucleocapsid (N) protein of SARS-CoV-2 binds viral RNA, condensing it inside the virion, and phase separating with RNA to form liquid–liquid condensates. There is little consensus on what differentiates sequence-independent N–RNA interactions in the virion or in liquid droplets from those with specific genomic RNA (gRNA) motifs necessary for viral function inside infected cells. To identify the RNA structures and the N domains responsible for specific interactions and phase separation, we use the first 1,000 nt of viral RNA and short RNA segments designed as models for single-stranded and paired RNA. Binding affinities estimated from fluorescence anisotropy of these RNAs to the two-folded domains of N (the NTD and CTD) and comparison to full-length N demonstrate that the NTD binds preferentially to single-stranded RNA, and while it is the primary RNA-binding site, it is not essential to phase separation. Nuclear magnetic resonance spectroscopy identifies two RNA-binding sites on the NTD: a previously characterized site and an additional although weaker RNA-binding face that becomes prominent when binding to the primary site is weak, such as with dsRNA or a binding-impaired mutant. Phase separation assays of nucleocapsid domains with double-stranded and single-stranded RNA structures support a model where multiple weak interactions, such as with the CTD or the NTD's secondary face promote phase separation, while strong, specific interactions do not. These studies indicate that both strong and multivalent weak N–RNA interactions underlie the multifunctional abilities of N.

Using these plots as a diagnostic, we elected to report a fit at n=4 in the main body of the manuscript, following the logic that it is the lowest integer value that fits the data well and reaches a low plateau in the χ 2 error surface.We expect N to have at least 3 RNA-binding sites based on our work here and in previous publications [13], therefore a stoichiometry of 4 is near our expectations, especially when considering additional possible binding sites in N's intrinsically disordered regions.

Figure S1 :
Figure S1: Temperature, pH and concentration ratio phase dependence of the nucleocapsid.

Figure S5 :
Figure S5: Analysis of the fluorescence anisotropy curve of FL-N binding to the ss-14mer.

Figure S7 :
Figure S7 : Chemical shift perturbations for all secondary-site residues from titration of ds-14mer into the NTD.

Figure S9 :
Figure S9 : Phase separation of the NTD at NMR conditions.

Figure S1 :
Figure S1: Temperature, pH and concentration ratio phase dependence of the nucleocapsid.Red fluorescence images of samples of FL-N, CTD and NTD with g1-1000 (buffer conditions of 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT).(a) 3.6 (left) or 9 (right) μM FL-N, CTD or NTD and 50 nM g1-1000, all images taken at 37°C.Scale bar in the top left panel is 200 μm and scale is the same for all images.(b) 3.6 μM FL-N, CTD or NTD and 50 nM g1-1000 at 25°C (top) and 37°C (botton).Samples in both panels were equilibrated at their experimental temperature for 90 minutes prior to imaging.Scale bar in the top left panel is 200 μm and scale is the same for all images.

Figure S4 :
Figure S4: Characterization of 14mer RNAs.(a,b) dot plots of the ss-14mer (a) and 14mer reverse complement (b).The region above the diagonal in the dot plot shows the relative probability of base pairs within the equilibrium ensemble.The size of the dots in this section are proportional to the probability of a base pair occurring.The region below the diagonal shows base pairs corresponding to the mean free energy structure.A lack of dots below the diagonal indicates that 14mer and reverse complement have no predicted secondary structure.(c,d) 1H NMR spectra of the ss-14mer (c) and ds-14mer (d) RNAs.The ds-14mer exhibits a pattern of peaks in the 5-6 PPM region that is distinct from the ss-14mer, indicating the presence of double-stranded RNA.In the 11-15 PPM region, peaks that are characteristic of paired bases appear in the ds-14mer spectrum, while none are present in the ss-14mer spectrum.

Figure S5 :
Figure S5: Analysis of the fluorescence anisotropy curve of FL-N binding to the ss-14mer.Attempts to fit the curve for this interaction to a 1:1 binding model results in a poor fit (n=1 in (a) above), as N binds multivalently to the RNA [13].While the underlying mechanism of binding is likely complex, we fit the curve to a simple quadratic model [64] with an additional varied, fixed stoichiometry value (n), representing the number of RNA-binding sites on FL-N.(a) shows example fits at values of n from 1 to 10 (in shades of blue) overlayed with the experimental data (black circles).(b) is an 'error surface' plot of the reduced χ 2 value of the fit as a function of the fixed n value in increments of 0.1, and (c) is a plot of the fit K d as a function of the fixed n value in increments of 0.1.Using these plots as a diagnostic, we elected to report a fit at n=4 in the main body of the manuscript, following the logic that it is the lowest integer value that fits the data well and reaches a low plateau in the χ 2 error surface.We expect N to have at least 3 RNA-binding sites based on our work here and in previous publications[13], therefore a stoichiometry of 4 is near our expectations, especially when considering additional possible binding sites in N's intrinsically disordered regions.

Figure S6 :
Figure S6: NMR assignments and dynamics of Y109A NTD.(a) 1 H-15 N HSQC spectrum collected at 25°C and a field strength of 800 MHz of the Y109A NTD labeled with assignments.A109 and the resides adjacent to it in sequence are indicated in red.(b) chemical shift perturbations induced by the Y109A mutation relative to the WT NTD.(c) 15 N R 1 (top), 15 N R 2 (middle), and { 1 H}-15 N Heteronuclear NOE (bottom) measurements for the NTD collected at a field strength of 800 MHz.Values for the WT NTD are shown as blue bars, and the Y109A NTD as green circles.Comparing the WT to the Y109A NTD reveals their dynamic properties are largely the same, suggesting that the mutation does not have any global impact on the domain's dynamics.The largest difference is in R 1 values, which are larger in the Y109A mutant in the 90-110 region that constitutes the NTD's beta hairpin.This is consistent with a slight increase in flexibility of this region, indicating the Y->A mutation may decrease the rigidity of the hairpin.

Figure S7 :
Figure S7: Additional NMR experiments on the ds-14mer binding to NTD.(a) 1 H NMR spectra of the ds-14mer (100μM) in the 5.2-6 ppm region, titrated with the NTD (up to 150μM).Titrations are from the same experiment as Fig. 3h.(b) Chemical shift perturbations for all secondary-site residues from titration of ds-14mer into the NTD.Plots are the profiles of CSPs as a function of RNA concentrations for titration of the ds-14mer into 100 μM WT NTD.Residues were selected from the protein based on the linear relationship between CSP and concentration of RNA.These residues cluster together on the NTD structure (See Fig. 4i), indicating that they represent a secondary weak RNA-binding site on the NTD.

Figure S8 :
Figure S8: NMR analyses of Y109A NTD interaction with RNA.(a) Titrations of Y109A NTD into the ss-14mer (left) and ds-14mer (right).Experimental conditions are the same as in Fig. 3g-i: 100 μM ss-or ds-14mer in a buffer of 20 mM NaPO 4 pH 6.5, 20 mM NaCl was titrated with the Y109A NTD, up to a 1.5:1 NTD:RNA ratio.(b) Average peak intensities for the 5 selected peaks (marked with black and red arrows in a), for the titration of NTD into the ss-14mer (black) and ds-14mer (red).(c)CSPs as shown in Fig. 5a,b of the Y109A NTD by residue following titration with the ss-14mer (top) and ds-14mer (bottom).Two standard deviations of the CSP population is drawn as a black dashed line, and residues that disappear due to intermediate exchange are labeled with a purple star.(d)Chemical shift perturbations for residues with linear chemical shift perturbation (as in Fig. 5d) profiles for titration of the ss-14mer into 100 μM Y109A NTD.(e) Chemical shift perturbations for residues with linear chemical shift perturbation (as in Fig. 5f) profiles for titration of the ds-14mer into 100 μM Y109A NTD.

Figure S9 :
Figure S9: Phase separation of the NTD at NMR conditions.Brightfield images of WT and Y109A NTD at NMR conditions (100 μM NTD, 20 mM NaPO 4 pH 6.5, 20 mM NaCl), with 100, 150, and 200 μM of RNA (ss-and ds-14mer) added.Images in the top panel are collected at 25°C (used for our NMR experiments) and those in the bottom panel are at 37°C.Scale bar shown in the top left of both panels is 100 μm, and scale is the same for all images.