Variant mutation G215C in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation

Copyright and License

© 2025 Kubinski et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding

This study was supported by an American Heart Association predoctoral fellowship (HWD; 10.58275/AHA.23PRE1020524.pc.gr.161030), George Mason University Fast Grant (PJB), NIH (NIAID) Award AI165075 (PJB), NIH (NIAID) Award 1R01AI153602-01 (VDM), Investigators in the Pathogenesis of Infectious Disease, Burroughs Wellcome Fund (VDM), and NIH (NIGMS) award P20GM125498 (EAB) and UVM start-up funds (EAB). The protein mass spectrometry was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103449. The funders played no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of Interest

I have read the journal’s policy and the authors of this manuscript have the following competing interests: HCK, HWD, BAJ, VDM, and EAB have filed a patent on the use of mutations in the nucleocapsid linker as a means of increasing nucleocapsid protein levels. VDM has filed a patent on the reverse genetics system and reporter SARS-CoV-2. Other authors declare no competing interests.

Data Availability

All raw sequencing data are available in the NCBI Bioproject ID PRJNA1083584. Individual peptides identified in proteomics experiments are included in S2 Data. Raw proteomics data are available in jPOSTrepo (JPST003506). Plasmids will be provided upon request to the corresponding author, after completion of a material transfer agreement. Infectious virus will be provided after completion of a material transfer agreement and confirmation of suitable BSL-3 facilities and IBC approval to safely handle infectious SARS-CoV-2.

Acknowledgement

We would like to thank Drs Matthew Poynter, Brian Cunniff, Nate Shannon, John Salogiannis, Dimitry Krementsov, and Joyce Oetjen for technical assistance.

The authors thank the Caltech Beckman Institute CryoEM Facility for use and maintenance of the Tecnai T12 TEM.

Supplemental Material

S1 Fig. Absence of cysteines in the linker region of Coronaviridae nucleocapsid proteins.
Sequences for the indicated coronavirus nucleocapsid protein sequences were obtained through the NIH Protein Databank and aligned using Clustal Omega from EMBL-EBI (Clustal 0 (1.2.4)). The navy box highlights the linker region (residues 175−247 in SARS-CoV-2). Shown in red is the only cysteine in the displayed sequences, which occurs immediately before the linker region in Erinaceus betacoronavirus.
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S2 Fig. SARS-CoV-2 N dimer formation.
VeroE6-TMPRSS2 cells were infected, or mock infected, with the indicated SARS-CoV-2 variants (or WA1, termed Wild type) at an MOI of 0.01 for 24 h. (A) Cell lysates were harvested under reducing conditions (10-mM DTT) before visualization by SDS-PAGE and western blot using antibodies recognizing SARS-CoV-2 N (red) and β-actin (green). Alternatively, unreduced lysates from cells infected as above were visualized by SDS-PAGE and western blot using two different antibodies recognizing SARS-CoV-2 N: (B) Invitrogen anti-N mouse antibody (MA5-35943, in green) and (C) Sino Biological anti-N rabbit antibody (40143-R001 in red). (D) Lysates were stained and imaged simultaneously with the two anti-N antibodies and the overlay is shown in yellow. Note minor bands that are seen with the Invitrogen but not the Sino-Biological antibody. The MW of the ~100-kDa band observed in Beta, Delta, and Iota samples under non-reducing conditions is indicated by a *. A representative gel from three (A) or two (C) independent biological replicates is shown. (E) Vero-TMPRSS2 cells were infected with WA1 or Delta SARS-CoV-2 at an MOI of 0.01. Twenty-four hpi cells were harvested, lysed, and N was affinity purified using immunoprecipitation. N and associated proteins were run on an SDS-PAGE gel, and bands corresponding to the monomer and dimer were cut and processed for mass spectrometry. (F) The number of peptides for each SARS-CoV-2 viral protein found in the monomer or dimer portion of the gel, for either WT (WA1) or Delta, is shown. The data underlying this Figure can be found in S1 Data and S2 Data.
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S3 Fig. Quantification of NEM treatment on N dimer formation.
A) VeroE6-TMPRSS2 cells were infected with WT or the G215C virus at an MOI of 0.01 for 24 h. Cell lysates were harvested in standard triton lysis buffer (lanes 1, 5) or in the presence of increasing concentrations of N-ethylmaleimide (1, 10, 100 mM). (B) A parallel experiment was performed in which cells were pre-treated by incubating for 30 min at 37°C in increasing concentrations (1, 10, 100 mM) of NEM in PBS. Cells were then lysed as in (A), with NEM present in the lysis buffer as well as the pre-incubation. Lysates were visualized by SDS-PAGE and western blot using antibodies to SARS-CoV-2 N and actin. (C and D) Quantification of the N dimer to monomer ratios in (A and B) is shown for two independent experiments. The data underlying this Figure can be found in S1 Data.
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S4 Fig. The G215C mutation increases levels of RNA bound to nucleocapsid.VeroE6-TMPRSS2 cells were infected with WT or the G215C viruses at an MOI of 0.01 for 48 h. Clarified viral supernatants were concentrated by binding to PEG and centrifuging at 10,000G for 30 min at 4°C. Concentrated virus was lysed and nucleocapsid proteins from each flask were affinity purified. Western blots were used to verify equal inputs of N from all conditions (inputs), and saturation of the beads (flow-through). The data underlying this Figure can be found in S1 Data.
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S5 Fig.  Antigen staining of lung tissue from infected hamsters.Section of lung tissue from infected hamsters was stained for viral antigen (nucleocapsid). Sections were then blinded and scored on a 4 point scale for parenchyma, airway, and overall staining (A). Each data point represents the average score form two lung section from each hamster in the group (n > 4). Horizontal lines represent the group mean and error bars are ±SD. Significance determined by student T test. (B) Representative images are shown for WT and N:215C infected animals for 2, 4, and 7 dpi. (. [p = 0.05−0.1], * [p < 0.05]). The data underlying this Figure can be found in S1 Data.
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S6 Fig. The G215S mutation decreases packaging of N and growth in primary differentiated human bronchial cells.(A) VeroE6-TMPRSS2 cells were infected with the WT or N:G21SC viruses at an MOI of 0.0005 for 1 h, viral supernatants were collected at 8, 24, 32, 48, and 72 h post infection and titered by focus forming assay (FFU; focus forming unit). (B) Human bronchial epithelial cells were infected with WT or N:G215S viruses at an MOI of 0.5 for 1 h, apical washes were collected sequentially from the same well at 8, 24, 32, 48, and 72 h post infection and titered by focus forming assay (FFU). (C) High titer viral stocks of the WT or N:G215S viruses were concentrated by binding to 10% polyethylene glycol then centrifugated at 10,000G for 30 min at 4ºC. Unreduced lysates were collected 24 h post transfection directly from the concentrated viral pellet and N and M were visualized by SDS-PAGE. (D) The ratio of N to M is shown, as well as (E) the ratio of N to FFU. Mean ± SEM is plotted, N = 6 from two biological experiments (A), N = 6 (B), and N = 3 (C–E) is shown. Statistical comparisons were conducted using a two-tailed T test, (. [p = 0.05−0.1], * [p < 0.05]). Limit of detection (LoD) is 20 PFU/mL and the y-axis minimum is set as the LoD. The data underlying this Figure can be found in S1 Data.
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S7 Fig. N:G215C virions are enlarged.Vero-TMPRSS2 cells were infected with WT or N:G215C viruses at an MOI of 0.1. The following day cells were prepared for electron microscopy by high-pressure freezing and freeze-substitution, then sectioned and imaged by dual-axis electron tomography. Virus-containing exit compartments were located in both samples, and 20 virions that had completely separated from cellular membranes were randomly selected and imaged for each virus.
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S1 Data. The individual datapoints underlying each graph can be found in this dataset.
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S2 Data. The proteolytic peptides identified in the mass spectrometry experiment in S2 Fig can be found in this dataset.
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S1 Raw Images. The uncropped western blots shown in figures and quantifications can be found in this dataset.
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