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Abstract
The SARS-CoV-2 nucleocapsid (N) protein is a highly immunogenic viral protein that plays essential roles in replication and virion assembly. Here, using native mass spectrometry, we show that dimers are the functional unit of ribonucleoprotein assembly and that N protein binds RNA with a preference for GGG motifs, a common motif in coronavirus packaging signals. Unexpectedly, proteolytic processing of N protein resulted in the formation of additional proteoforms. The N-terminal proteoforms bind RNA, with the same preference for GGG motifs, and bind to cyclophilin A, an interaction which can be abolished by approved immunosuppressant cyclosporin A. Furthermore, N proteoforms showed significantly different interactions with IgM, IgG, and IgA antibodies from convalescent plasma. Notably, the C-terminal proteoform exhibited a heightened interaction with convalescent antibodies, suggesting the antigenic epitope is localized to the C-terminus. Overall, the different interactions of N proteoforms highlight potential avenues for therapeutic intervention and identify a stable and immunogenic proteoform as a possible candidate for immune-directed therapies.
© 2021 The Authors. Published by American Chemical Society.
Conflict of interest statement
The authors declare no competing financial interest.
Figures
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 each construct.
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.
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.
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
following initial COVID-19 diagnosis. The antibody response was determined
using the absorbance following colorimetric detection of a sandwich
ELISA where the immobilized antigen was NFL, N1–209, N1–220, N1–273, or N156–419. The squares represent the mean, the center line represents the
median, and the box represents the first quartile (25–75%)
of the distributed data. Asterisks represent statistically significant
differences when compared to NFL; p-values
denoted by asterisks are defined as * p < 0.05,
** p < 0.01, and *** p < 0.001.
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.
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