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. 2023 Feb 18;6(1):193.
doi: 10.1038/s42003-023-04570-2.

Structural insights into ribonucleoprotein dissociation by nucleocapsid protein interacting with non-structural protein 3 in SARS-CoV-2

Xincheng Ni  1   2 , Yinze Han  1   2 , Renjie Zhou  1   2 , Yanmei Zhou  1   2 , Jian Lei  3   4
Affiliations

Structural insights into ribonucleoprotein dissociation by nucleocapsid protein interacting with non-structural protein 3 in SARS-CoV-2

Xincheng Ni et al. Commun Biol. .

Abstract

The coronavirus nucleocapsid (N) protein interacts with non-structural protein 3 (Nsp3) to facilitate viral RNA synthesis and stabilization. However, structural information on the N-Nsp3 complex is limited. Here, we report a 2.6 Å crystal structure of the N-terminal domain (NTD) of the N protein in complex with the ubiquitin-like domain 1 (Ubl1) of Nsp3 in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One NTD and two Ubl1s formed a stable heterotrimer. We performed mutational analysis to reveal the key residues for this interaction. We confirmed the colocalization of SARS-CoV-2 N and Nsp3 in Huh-7 cells. N-Ubl1 interaction also exists in SARS-CoV and Middle East respiratory syndrome coronavirus. We found that SARS-CoV-2 Ubl1 competes with RNA to bind N protein in a dose-dependent manner. Based on our results, we propose a model for viral ribonucleoprotein dissociation through N protein binding to Ubl1 of Nsp3.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. SARS-CoV-2 N protein and Nsp3 colocalize in Huh-7 cells.
a Schematic representation of the N protein as well as Nsp3, Nsp3Δ111, Nsp3Δ168, and Ubl1 expression vectors. Nsp3 non-structural protein 3, Ubl1 ubiquitin‐like domain 1, HVR hypervariable region, Mac1 macrodomain 1, TM transmembrane domain, Y1–CoV-Y domains of Y1 and CoV-Y. b Colocalization (see white-triangle arrows) between N protein and Nsp3 in Huh-7 cells. Localization of N protein and Nsp3 were analyzed with fluorescence labeling and viewed at 48 h post-transfection using an Olympus Spin co-focus fluorescence microscope. c Nsp3 colocalizes with N protein through its Ubl1 and HVR regions in Huh-7 cells. Nsp3Δ111: the deletion of Ubl1 (residues 1–111) in Nsp3; Nsp3Δ168: the deletion of Ubl1–HVR (residues 1–168) in Nsp3.
Fig. 2
Fig. 2. ITC measurement of the binding affinity between SARS-CoV-2 N and Ubl1.
a Schematic diagram of SARS-CoV-2 N and its truncations. N-arm N-terminal arm, NTD N-terminal domain, LKR the central linker region, SR Ser/Arg-rich motif, CTD C-terminal domain, C-tail C-terminal tail. b–f Affinity between N protein (b) N-arm–NTD–LKR (c) NTD–LKR (d) NTD (e) CTD–C-tail (f) and Ubl1, respectively. The raw calorimetric curve is shown in the top panel, while the fitted binding isotherm curve is displayed in the bottom panel. Kd: dissociation constant.
Fig. 3
Fig. 3. Crystal structure of SARS-CoV-2 NTD in complex with Ubl1.
a The NTD-Ubl1 complex protein was confirmed by SEC assay. NTD: ~14.3 kD. Ubl1: ~12.9 kD. NTD-Ubl1 complex: ~40.1 kD. The peak positions of three standard proteins are shown as gray dashed lines. b SEC-MALS analysis of Ubl1 in solution. The light scattering data is displayed in a black curve and the molar mass data is shown in a red curve. The calculated MW of Ubl1 is ~13.1 kD. c The crystal structure of the NTD in complex with Ubl1. NTD is shown in magenta, and two Ubl1s are displayed in cyan (chain B) and light blue (chain C). The N- and C- termini of NTD and Ubl1s are marked in the corresponding colors. Compared to chain-B Ubl1, the two extra 310 helices in chain-C Ubl1 are labeled as η′ and η″. d Cross-linking experiments of Ubl1 as well as NTD-Ubl1 complex. The compositions of the samples after cross-linking were identified by SDS-PAGE. Bands corresponding to the dimeric Ubl1 (theoretical MW: ~25.8 kD) and the heterotrimeric NTD-Ubl1 complex (~40.1 kD) are indicated by black and red arrows, respectively. e Dynamic light scattering analysis of the NTD-Ubl1 complex in three independent experiments (n = 3). Radius: ~2.7 nm; Calculated MW: 35.3 ± 1.0 kD. Figure c was prepared using Chimera (http://www.cgl.ucsf.edu/chimera/).
Fig. 4
Fig. 4. Interaction mode of SARS-CoV-2 NTD binding to Ubl1.
a The electrostatic surface (−5 kBT/e (red) – +5 kBT/e (blue)) of the NTD-Ubl1 complex. Residues R92, R95, and R107 of NTD comprise the positively charged center. Residues E26, E95, and D110 of Ubl1 form the negatively charged center. b Three salt bridges R92-D110, R95-E26, R107-E95, and one hydrogen bond (R95---Y103) are shown. Fo – Fc density maps (σ = 2.5; gray mesh) for these residues are displayed. Figures (a, b) were prepared using PyMOL (https://pymol.org).
Fig. 5
Fig. 5. The N-Nsp3 interaction exists in SARS-CoV and MERS-CoV.
a The Ubl1s of SARS-CoV, MERS-CoV, and SARS-CoV-2 present the lower elution volumes in SEC assays. The peak positions of two standard proteins (13.7 and 29.0 kD) are shown as gray dashed lines. b Affinity measurements of N and Ubl1 in SARS-CoV and MERS-CoV.
Fig. 6
Fig. 6. SARS-CoV-2 Ubl1 inhibits N protein binding to RNA.
a Ubl1 disrupts N-RNA binding in a dose-dependent manner according to EMSA assay. Lane C: ssRNA/ssDNA; Lane U: Ubl1 + ssRNA/ssDNA, Lane 0: N + ssRNA/ssDNA. Lanes 1–5, ssRNA/ssDNA plus different molar ratios of Ubl1 to N protein (from 1:5 to 5:5). The amount of free ssRNA/ssDNA from the N-RNA complex increases with increasing concentrations of Ubl1 in Lanes 1–5. b Curve fit of MST traces for determination of the N-ssRNA binding affinity. c–e The binding affinity of N protein and ssRNA decreases with the addition of 1 μM (c), 5 μM (d) and 10 μM (e) of Ubl1. No interaction between Ubl1 and ssRNA was detected (c–e, right). The error bar represents the standard error of the mean of three independent experiments (n = 3).
Fig. 7
Fig. 7. A proposed model for NTD–LKR in complex with Ubl1.
Superimposing the previously published NMR structure (PDB code: 7PKU) to our crystal structure (PDB code: 7WZO) enables the illustration of a hypothetical model of NTD–LKR plus Ubl1 complex. The NTD, LKR, chain-B Ubl1, and chain-C Ubl1 are colored in magenta, green, cyan, and light blue, respectively. The N- and C- termini of chain-B Ubl1 are indicated. Chain-C Ubl1 is displayed as the surface view. The SR region (orange dashed lines) is still missing in this model. The NTD interacts with the side near the β-sheets in two Ubl1s, while the LKR binds to the side of the α-helices of Ubl1 (chain C). The CTD following the LKR is indicated by black dashed circles. Figures were prepared using PyMOL (https://pymol.org).
Fig. 8
Fig. 8. A hypothetical model of viral RNP dissociation through N interacting with Ubl1.
a Original viral RNP is recruited to the molecular pore located on the DMV surface by N binding to Ubl1 of Nsp3. b Two Ubl1 molecules of adjacent Nsp3s interact with N protein to disrupt the N-RNA binding. N protein successively dissociates from the RNP through the dynamic N-Nsp3 (Ubl1) interactions, leading to the delivery of gRNA into DMV. c Intact gRNA enters the DMV and serves as a template for viral RNA replication. Free N proteins are released into the cytosol. RNP ribonucleoprotein, DMV double-membrane vesicle, gRNA genomic RNA.
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