SARS ‐ CoV ‐2 nucleocapsid protein variants have differential RNA chaperone activity

Figures

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  Fig. 1. N protein stability and binding preferences. (A) Recombinant full‐length wild‐type N protein (N_WT), the receptor‐binding domain (N_RBD), and the C‐terminal domain (N_CTD) were expressed in E. coli and purified using a 6×His tag. Equal amounts (2 μg) of each protein were resolved on a 15% SDS gel. (B) The stability and quality of the purified proteins were assessed by differential scanning fluorometry (nanoDSF). The intrinsic tryptophan fluorescence of 10 μm of each protein was measured at 330 nm and 350 nm over a temperature gradient from 20 °C to 95 °C and vice versa (x‐axis). The y‐axis represents the rate of fluorescence ratio change per degree Celsius (°C). The RBD unfolding peak was observed at 45 °C, while the CTD unfolded at 68 °C. Moreover, all proteins showed proper refolding ability after heat denaturation. Number of replicates n = 4. (C) Comparative boxplots showing the binding affinities of three distinct SARS‐CoV‐2 RNAs (RNA3, RNA20, RNA22) in their single‐stranded (blue‐gray) and double‐stranded (red) forms with N_WT and N_CTD. Data are derived from MST analysis. Each boxplot summarizes four MST measurements, illustrating the interquartile range (IQR) with the…

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  Fig. 2. CoV‐2 N protein binds RNA and hybridizes ssRNA to dsRNA. (A) Sequences of ssRNA2 (nt 5′‐498‐520‐3′) and ssRNA7 (nt 3′‐498‐520‐5′ reverse). Complementary nucleotides for hybridization are marked bold. (B) Complementary ssRNA2‐Cy5 and ssRNA7 were mixed and incubated in the presence (indicated in Lanes 4, 6, 8, and 10), or absence (indicated in Lanes 2, 3, 5, 7, and 9) of N protein. Reactions were stopped with a sodium dodecyl sulfate (SDS) containing buffer to inactivate the protein. Samples were applied to a native 6% polyacrylamide (PAA) gel. RNA hybridization is enhanced in the presence of SARS‐CoV‐2 N protein. Lane 1 served as a control with ssRNA2‐Cy5 alone. Number of replicates n = 3. (C) Binding of the SARS‐CoV‐2 N protein toward hybridized RNA2 (Lane 2) was analyzed using a concentration range of 500–15 nM (Lanes 3–8) of N protein. Samples were run on a native 6% PAA TBE gel. A binding shift can be observed in the presence of N protein. Number of replicates n = 3.

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  Fig. 3. SARS‐CoV‐2 N exchanges mismatched RNA strand. (A) Strand‐exchange RNA chaperone assay: Single‐stranded (ss) RNA20‐Cy5 (Lane 1) or ssRNA20‐5′‐mismatch (Lane 5) was fully hybridized with ssRNA21 to form double‐stranded (ds) RNA complexes facilitated by wild‐type N protein (Lanes 2, 6). Competitor RNA20 was then either added directly to the dsRNA in the presence of the N protein (Lanes 4, 8), in the absence of N protein (Lane 3) or following the inactivation of N protein activity by sodium dodecyl sulfate (SDS), prior to incubation with an excess of competitor ssRNA20 (Lane 8). Whereas there is no change observable for the perfectly matching RNA20, the mismatched RNA was displaced from the double strand upon the addition of competitor RNA (Lane 7), with this removal being significantly enhanced by the presence of the N protein (lane 8). Number of replicates n = 3. (B) Each boxplot summarizes data from two biological replicates of the strand‐exchange RNA chaperone assays, as shown in Figure A. Additional replicates (n = 2) were performed at different RNA concentrations, yielding the same overall result, but could not be included in the box plot representation. The boxplots illustrate…

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  Fig. 4. SARS‐CoV‐2 N protein truncation proteins with their respective protein stability and RNA chaperoning ability. (A) Schematic representation of the generated SARS‐CoV‐2 N protein truncation variants and the corresponding nomenclature used. (B) SDS/PAGE gel analysis of purified recombinant N protein truncations. 2 μg of each protein were loaded onto a 10% SDS/PAGE gel. Protein bands observed align with the calculated molecular weights. (C) Protein stability assessment using differential scanning fluorometry (nanoDSF) measurements. Of each protein variant, 10 μm were loaded into standard capillaries and subjected to a temperature gradient ranging from 25 °C to 85 °C using the Prometheus NT.48 nanoDSF device. Unfolding was monitored in real‐time by measuring changes in intrinsic tryptophan fluorescence at 350 nm and 330 nm. The ratio of these wavelengths and the corresponding first derivative were used to determine melting points for the folded domains of the proteins. For N_WT (wild‐type), the structured RBD (receptor‐binding domain) and CTD (C‐terminal domain) domains unfold at 45 °C and 68 °C, respectively. Truncation of specific protein regions led to alterations in unfolding…

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  Fig. 5. Effect of N‐RNA LLPS inhibitors on phase separation, protein stability and N‐mediated RNA chaperone activity. (A) Liquid–liquid phase separation (LLPS) experiments were conducted using 100 nM of single‐stranded RNA3 (ssRNA3) labeled with Cy5, incubated with 2 μm of N protein, in the presence or absence of 1.25 μm of inhibitors A7, G22 and N13. After a 2‐h incubation at room temperature, droplet formation was assessed by fluorescent microscopy at 40× magnification. No droplet formation is detectable in presence of A7, G22 and N13. Scale bar = 25 μm. Number of replicates n = 3. (B) The stability of N protein was measured in absence (dark red) and presence of the inhibitors using Differential Scanning Fluorometry (nanoDSF). A mixture of 10 μm N protein and 1.25 μm of the respective compound (A7 (light blue), G22 (dark blue), N13 (purple)) was loaded into capillaries and subjected to a temperature gradient ranging from 25 °C to 85 °C using the Prometheus NT.48 nanoDSF. Protein unfolding was monitored to assess the impact of the inhibitors on N protein stability. RBD (receptor‐binding domain) and CTD (C‐terminal domain) unfolding peaks are displayed over the temperature range. CTD…

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  Fig. 6. N protein‐mediated RNA chaperoning is reduced at high salt concentration. (A) Protein stability of wild‐type N protein was assessed using Differential Scanning Fluorometry (nanoDSF) with a temperature gradient ranging from 25 °C to 80 °C at different salt concentrations. 10 μm N were loaded into standard capillaries and subjected to a temperature gradient using the Prometheus NT.48 nanoDSF device. Unfolding was monitored in real‐time by measuring changes in intrinsic tryptophan fluorescence at 350 nm and 330 nm. The ratio of these wavelengths and the corresponding first derivative were used to determine melting points for the folded domains of the proteins. The stability of the C‐terminal domain (CTD) was found to be affected by high salt concentration. Number of replicates n = 3. (B) RNA chaperone activity of WT N protein was tested under varying salt concentrations in the reaction buffer. 100 nM RNA mix (ssRNA6‐Cy5, ssRNA3) was incubated with in absence (Lanes 2, 4, 6, 8) or presence (Lanes 3, 5, 7, 9) of 500 nM N at 50 mm (Lanes 8, 9), 125 mm (Lanes 6, 7), 250 mm (Lanes 4, 5) and 500 mm NaCl (Lanes 2, 3). ssRNA signal disappears in presence of N protein, except at 500 mm salt.…

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  Fig. 7. Pseudo‐phosphorylation of SARS‐CoV‐2 N leads to a decrease in stability and changed chaperone activity of BA.5 N. (A) Schematic representation of sequence differences between wild‐type N and Omicron BA.5 N (P13L, Δ31‐33, E136D, R203K, G204R, S413R) and amino acid exchanges, indicating sites of pseudo‐phosphorylation (WT N: S23, T141, S176, S180, T198, S201, S202, S206; BA.5 N: S23, T138, S173, S177, S180, T195, S198, S202, S203) by substituting serine/threonine residues with aspartic acid (blue dots). (B) Recombinant unmodified and pseudo‐phosphorylated WT and BA.5 N proteins were expressed in E. coli and purified using Ni‐NTA. Equal amounts (2 μg) of each protein were resolved on a 10% SDS/PAGE gel, demonstrating their molecular weights. Protein stability was evaluated using unfolding and refolding experiments conducted on a Prometheus NT.48 Differential Scanning Fluorometry (nanoDSF) device applying 10 μm of each protein, with temperature ranging from 20 °C to 90 °C. Pseudo‐phosphorylated proteins display a reduced stability of the RBD in both WT (light red) and BA.5 (light blue) N proteins. Unmodified WT (dark red) and BA.5 (dark blue) N show the expected unfolding properties.…