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. 2023 Jul 21;15(7):1600.
doi: 10.3390/v15071600.

Human Post-Translational SUMOylation Modification of SARS-CoV-2 Nucleocapsid Protein Enhances Its Interaction Affinity with Itself and Plays a Critical Role in Its Nuclear Translocation

Vipul Madahar  1 , Runrui Dang  1 , Quanqing Zhang  2   3 , Chuchu Liu  1 , Victor G J Rodgers  1   4 , Jiayu Liao  1   2   4
Affiliations

Human Post-Translational SUMOylation Modification of SARS-CoV-2 Nucleocapsid Protein Enhances Its Interaction Affinity with Itself and Plays a Critical Role in Its Nuclear Translocation

Vipul Madahar et al. Viruses. .

Abstract

Viruses, such as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), infect hosts and take advantage of host cellular machinery for genome replication and new virion production. Identifying and elucidating host pathways for viral infection is critical for understanding the development of the viral life cycle and novel therapeutics. The SARS-CoV-2 N protein is critical for viral RNA (vRNA) genome packaging in new virion formation. Using our quantitative Förster energy transfer/Mass spectrometry (qFRET/MS) coupled method and immunofluorescence imaging, we identified three SUMOylation sites of the SARS-CoV-2 N protein. We found that (1) Small Ubiquitin-like modifier (SUMO) modification in Nucleocapsid (N) protein interaction affinity increased, leading to enhanced oligomerization of the N protein; (2) one of the identified SUMOylation sites, K65, is critical for its nuclear translocation. These results suggest that the host human SUMOylation pathway may be critical for N protein functions in viral replication and pathology in vivo. Thus, blocking essential host pathways could provide a novel strategy for future anti-viral therapeutics development, such as for SARS-CoV-2 and other viruses.

Keywords: SARS-CoV-2 N protein; SUMOylation; nuclear translocation; protein interaction dissociation constant KD; qFRET.

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Conflict of interest statement

The J.L. laboratory has received research support from Attaisina. J.L., R.D. and V.M. are inventors on patents and patent applications on using SUMOylation inhibitor for virus infections and cancer, owned by the University of California at Riverside, outside of the reported work. The funders had no role in the study’s design; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
SUMOylation of SARS-CoV-2 N protein. (A) Diagram of the fluorescence fusion protein CyPet-SUMO1 and YPet-N for the qFRET-based SUMOylation assay. (B) In vitro SUMOylation assay with the FRET as a reporter signal. The fusion protein CyPet-SUMO is first bound to E1 activating enzyme, for the intermediate E1-Cypet-SUMO1 thioester bond at Cys173 to Gly98 on SUMO1. The SUMO is then transferred to the catalytic Cys-93 of E2 conjugating enzyme. The E3 ligase and target protein are said to non-covalently interact with the E2-SUMO1 complex. The CyPet-SUMO1 is then shuttled to a lysine on the target protein, to be covalently bound by an isopeptide bond. (C) The Western-blot of SUMOylated N protein in the in vitro reaction containing E1, E2 and E3 using a monoclonal anti-SUMO1 antibody.
Figure 2
Figure 2
In vitro SUMOylation of SARS-CoV-2 N protein and SUMOylation site identification using Mass Spectrometry (MS). (A) The in vitro MS sample was measured for qFRET signal before processing for MS, with and without ATP or E3 PIAS1. (B) The illustration of the location of the three discovered lysines and a total of 31 lysines on the protein shown as yellow lines. (C) The spectrum was generated by Thermofisher Proteome DiscovererTM MS spectrum of peptide containing modified K61. (D) The spectrum of K65 peptide with trypsin cut on SUMO1, GG. (E) The spectrum of K347 was found in the same peptide, with SUMO1 peptide GG cut with trypsin. p values are p < 0.0001 *** and no significant difference (NS), n = 3.
Figure 3
Figure 3
qFRET assay and Western-blot of in vitro SUMOylation of N protein and its Lys mutants. (A). SUMOyaltion assay of wildtype and K mutants of N protein using qFRET assay. Comparison of no ATP (−ATP), with no E3 ligase PIAS1 (−E3), and a complete reaction with ATP and E3 ligase PIAS1 (+ATP+E3). The reactions were performed under the same conditions, and the measurements were taken on the same instrument, Molecular Devices SpectraMax3TM. One-way ANOVA was performed on the data sets of −ATP/−E3/+ATP+E3, the −ATP was the control group. Tukey test was used as the post hoc analysis. p values are p < 0.0001 ***, p < 0.05 *, and no significant difference (NS), n = 3. (B) In vitro SUMOyaltion assay of N protein was probed with anti-N protein antibody in the Western-blot analysis. (C) The Western blot of various K-to-R mutants of N protein was probed with the anti-SUMO1 antibody.
Figure 4
Figure 4
The KD determinations of SUMOylated or un-SUMOylated N protein—N protein interactions using qFRET assay and aggregation assay of N protein with or without SUMO modification. (A) The binding curve of wildtype of N protein with or without SUMOylation. The EmFRET signal fit of SUMO modified (Diamond/Green), and unmodified (Circle/Orange). (B) The summary of binding affinity of Un-SUMOylated and SUMOylated N protein to itself. (C) The aggregation assay of SARS-CoV-2 N protein with or without the SUMOs modification. The 10 μg of SARS-CoV-2 N protein was SUMOylated with SUMO1, Aos1/Uba2, Ubc9, and PIAS1 in the presence or absence of ATP, followed with disuccinimidyl suberate and Immunoblot with anti-N protein antibody. (D) The Binding curves of the SUMO-modified N protein mutants of (K61R). (E) The binding curves of the SUMO-modified N protein mutants of (K65R). (F) The binding curves of the SUMO-modified N protein mutants of (K347R). All plots and the fits were generated on GraphpadPrism5TM.
Figure 5
Figure 5
Subcellular localization determination of wildtype N protein and Lys mutants using a fluorescent microscope. The nuclear stain Hoechst was determined at 488 nm. The images of YPet-tagged-N proteins and their mutants were taken at 533 nm using the fluorescent microscope, Olympus BX43. Images were processed on ImageJTM.
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Grants and funding

This work was partially funded by the UCR Academic Senate Research Grant and Attaisina Gift grant to J.L.
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