Multiple Roles of SARS-CoV-2 N Protein Facilitated by Proteoform-Specific Interactions with RNA, Host Proteins, and Convalescent Antibodies

Figures

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  Figure 1. SARS-CoV-2 N protein exists as an ensemble of proteoforms. (A) Scheme depicting the full-length construct for expression in E. coli and native mass spectrum of the full-length N protein. Four charge state distributions correspond to monomers and dimers of full-length N protein (red circle, MW 45 769 Da) and a proteoform of N protein (blue circle, MW 28 735 Da). (B) Mass spectrum of N protein after several days at room temperature reveals the coexistence of five distinct charge state distributions corresponding to N proteoforms. The chemical composition of each proteoform was determined using top-down MS (C,D). (C) Charge-reduced mass spectrum resulting from electron-transfer dissociation (ETD) of the selected 9+ charge state at m/z 2616.79. (D) Mass spectrum of sequence ions for the same parent ion generated by higher-energy collision induced dissociation (HCD). (E) Scheme representing the composition of protein domains for the observed proteoforms as determined by top-down MS. Five distinct proteoforms are observed: N1–209, N1–220, N1–273, N156–419, and N1–392. The exact site of cleavage, including the five residues flanking either side of each cleavage site, is indicated below…

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  Figure 2. RNA sequence influences the binding stoichiometry to N protein. (A) Mass spectrum of N1–209 after incubation with 4 × −GAUGG RNA oligonucleotides in a molar ratio of 1:4 RNA:protein. Two additional charge state distributions are observed that correspond to one and two RNA oligonucleotides bound to N1–209. The mass spectrum at m/z > 2700 was magnified 3× and offset for clarity (red trace). (B) Mass spectrum of N1–209 after incubation with 4 × −GAGAA RNA oligonucleotides in a molar ratio of 1:4. One additional charge state distribution is observed that corresponds to one RNA oligonucleotide bound to N1–209. The mass spectrum at m/z > 2700 was magnified 3× and offset for clarity (red trace). (C) Mass spectrum of NFL after incubation with 4 × −GAUGG RNA oligonucleotides in a molar ratio of 1:4. Monomers and dimers of NFL (red circles) and N156–419 (blue circles) are observed. An additional peak series between 4300 and 5600 m/z corresponds to two 4 × −GAUGG RNA oligonucleotides bound to NFL dimer. The scheme in the inset of (C) depicts NFL dimer bound to RNA as the functional unit of the ribonucleoprotein assembly.

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  Figure 3. N proteoforms directly interact with cyclophilin A. (A) Mass spectrum of N1–209 after incubation with cyclophilin A (CypA) in a 1:1 molar ratio. The mass spectrum at m/z > 3000 was magnified 5× and offset for clarity (red trace). Three charge state distributions that correspond to a low population of homodimers of N1–209, homodimers of CypA, and heterodimers of N1–209–CypA. (B) Mass spectrum of N1–209 after incubation with CypA and cyclosporin A (CsA) in a molar ratio of 1:1:2. CypA preferentially binds CsA as demonstrated by the charge state distribution centered at 8+, which corresponds to the CypA–CsA complex. The mass spectrum at m/z > 3500 was magnified 5× and offset (red trace) to highlight the exceedingly low abundance of N1–209–CypA heterodimers that persist after CsA treatment. The scheme (inset of B) depicts CsA competitively binding to CypA and abolishing the N1–209–CypA interaction.

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  Figure 4. N proteoforms show significantly different interactions with antibodies. (A) Mass spectrum of NFL after incubation with a monoclonal antibody raised against the full-length N protein in a molar ratio of 1:1. We observe five charge state distributions that correspond to NFL monomers (centered at 13+), NFL dimers (centered at 19+), a monomeric antibody (centered at 21+), an antibody bound to one NFL (centered at 24+), and a population of antibodies bound to NFL dimer (centered at 30+). (B) Mass spectrum of a mixture of N1–209, N1–220, and N1–273 incubated with a monoclonal antibody in a molar ratio of 1:1:1:1. Charge state distributions are observed for antibody monomers and dimers. No binding to the antibody is observed for N proteoforms. This result was confirmed by immunoblot (see inset). SDS-PAGE shows that NFL, N1–209, N1–220, N1–273, and N156–419 are detected in high abundance by Coomassie stain. The same proteins analyzed by immunoblot show that only NFL and N156–419 are detected by the antibody. The box-and-whisker plots depict the antibody response for (C) IgM antibodies, (D) IgG antibodies, and (E) IgA antibodies from plasma from eight patients collected >6 months…

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  Figure 5. Scheme depicting features of SARS-CoV-2 N protein during infection. N protein undergoes proteolysis to produce N1–209, N1–220, N1–273, N156–419, and N1–392. NFL and N proteoforms bind RNA with a preference for structured RNA, and NFL dimers are likely functional unit of assembly in ribonucleoprotein complexes. Immunophilin CypA binds directly to N1–209 but not NFL, and the interaction can be inhibited through addition of cyclosporin A (CsA). N156–419 and NFL interact with antibodies from convalescent plasma, while proteoforms N1–209, N1–220, and N1–273 fail to induce the same antibody response.