Biochemical characterization of SARS-CoV-2 nucleocapsid protein

Abstract

The nucleocapsid (N) protein is an important antigen for coronavirus, which participate in RNA package and virus particle release. In this study, we expressed the N protein of SARS-CoV-2 and characterized its biochemical properties. Static light scattering, size exclusive chromatography, and small-angle X-ray scattering (SAXS) showed that the purified N protein is largely a dimer in solution. CD spectra showed that it has a high percentage of disordered region at room temperature while it was best structured at 55 °C, suggesting its structural dynamics. Fluorescence polarization assay showed it has non-specific nucleic acid binding capability, which raised a concern in using it as a diagnostic marker. Immunoblot assays confirmed the presence of IgA, IgM and IgG antibodies against N antigen in COVID-19 infection patients' sera, proving the importance of this antigen in host immunity and diagnostics.

Keywords: Antigenicity; COVID-19; Nucleocapsid protein; SARS-CoV-2; SAXS; Structure and function.

Fig. 1

Structural organization of SARS-CoV-2 N protein and sequence alignment. (A) Domain structure of SARS-CoV-2 N protein. The domain boundaries were shown on the top and the different domains were labeled in different colors. (B) The predicted structure of SARS-CoV-2 N protein was presented. The NTD and CTD were highlighted in red and blue, respectively.

Fig. 2

Oligomerization state and conformation analysis of the N protein. (A) Static light scattering analysis of the oligomerization of the N protein. The molecular weight was calculated by Astra software and is shown in red. (B) DSS cross-linking analysis of the oligomerization forms of the N-protein (1). The protein used for positive control was mCARD9-CARD with an MBP tag (52 kDa) which was reported to form dimers in solution (2) [33]. The MBP was used as a negative control (42 kDa) (3). (C) SAXS results for the protein. Scattering profile (points) and fitting with GNOM (solid lines). I, scattering intensity; q, scattering angle vector. Insert: the guinier region with fitting line of the scattering profile. (D) Dimensionless Kratky plot showed that the protein was partially extended in solution. (E) A representative CORAL model in which the NTDs are shown in yellow and purple, respectively, and the CTD dimer is shown in green. The coiled coil regions are represented as dots. (F) Results from GNOM showing the pairwise distance distribution [P(r)] and the maximum distance. The radius of gyration is fitted to 59 Å, and r represents the pairwise distances.

Fig. 3

Conformational and functional analysis of the N protein (A) CD spectrum analysis of the N protein (right) and thermal denaturation of the N protein monitored at Θ222 nm (left). (B) Fluorescence polarization analysis of the N protein. The concentration of 5′-FAM double stranded 14mer DNA was 20 nM, and the apparent Kd value was 191 ± 0.036 nM. (C) The electrostatic surface of the N protein generated by PyMOL, where the negatively charged region are represented in red, neutral regions in white, and positively charged regions in blue.

Fig. 4

Antigenicity of the N antigen Western Blot (A) and Dot Blot (B) analysis of specific IgA, IgM, IgG antibodies against the N -protein after incubated with different dilution of COVID-19 recovering patients’ serum pool using anti-human IgA-Fc/IgM-μ chain/IgG-Fc secondary antibodies, respectively.

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