SUMOylation of SARS-CoV-2 Nucleocapsid protein enhances its interaction affinity and plays a critical role for its nuclear translocation

  1. Vipul Madahar
  2. Victor G. J. Rodgers
  3. Jiayu Liao

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Abstract

Viruses, such as SARS-CoV-2, infect hosts and take advantages of host cellular machinery for their genome replication and new virion production. Identification and elucidation of host pathways for viral infection are critical for understanding the viral life cycle and novel therapeutics development. SARS-CoV-2 N protein is critical for viral RNA(vRNA) genome packaging in new virion formation, Here, we report that identification of SUMOylation sites of SARS-CoV-2 N protein and role of SUMO modification in N protein interaction affinity with itself using our qFRET/MS coupled method. We found, for the first time, that the SUMO modification of N protein can significantly increase its interaction affinity with itself and may support its oligomer formation. One of the identified Lys residues, K65 was critical for N protein translocation to nucleus, where the vRNA replication and packaging take place. The in vitro assessment of the affinity of N protein to N protein with SUMO mutants provides insight of the oligomerized N protein formation after SUMO modification. These results suggest that the host human SUMOylation pathway may be very critical for N protein functions in viral replication. The host SUMOylation pathway may be a critical host factor for the SARS-CoV-2 virus life cycle. Identification and inhibition of critical host SUMOyaltion could provide a novel strategy for future anti-viral therapeutics development, such as SARS-CoV-2 and other viruses.

Importance

The SARS-CoV-2 virus N protein plays a critical role critical for viral RNA(vRNA) genome packaging in host cell nucleus for new virion formation. Therefore, deciphering the molecular mechanisms modulating N activity could be a strategy to identify potential targets amenable to therapeutics. Here, we identify a comprehensive SUMOylation sites of N proteins using an in vitro reconstitute SUMOyaltion assay containing SUMO E1 activating enzyme, E2 conjugating enzyme, and E3 ligase. We find that SUMOylation modification of N protein can significantly enhance it interaction affinity with itself, indicating an increased oligomerization capability, which is critical for N protein activity for vRNA genome packaging. In addition, we find one of SUMOylation sites of N protein is critical for its nucleus translocation, which is a critical for viral genome packaging. The SUMOylation modification may represent novel potential approach to design new antivirals with the ability to modulate SARS-CoV-2 virus replication.

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    SciScore for 10.1101/2022.05.31.494262: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Recombinant DNA
    SentencesResources
    , E2 conjugating enzyme UBC9, and E3 ligase PIAS1 were all cloned into pET28B vector for expression in BL21(DE3) cells.
    pET28B
    suggested: RRID:Addgene_73018)
    Final Gibson reaction of bacterial expression pET28B vector SALI and NOTI and for mammalian expression pCDNA3.1-FLAGtag-Nprotein- YPet were created.
    pCDNA3.1-FLAGtag-Nprotein-
    suggested: None
    Tabulated PCR primers are shown in Table 3 for pcDNA3.1. In-Vitro SUMOylation with qFRET Reporter for N Protein Mutants: The in-vitro SUMOylation assay of SARS-CoV-2 N protein mutants is an initial screening to determine impact of lysine sites on SUMOylation.
    pcDNA3.1
    suggested: RRID:Addgene_79663)
    Thus, five pairs of N protein genes, the wild type, Lys mutations on individual 61, 65, 347, and 355, were cloned into pET-28(b
    pET-28
    suggested: RRID:Addgene_141289)
    Software and Algorithms
    SentencesResources
    raw) raw data was analyzed on Thermofisher Proteome AnalyzerTM.
    Thermofisher Proteome
    suggested: None
    The cells were imaged on Olympus BX43, and images were stacked and analyzed using ImageJ software. qFRET KD of SUMOylated N Protein: The evaluation of N protein oligomerization by in vitro qFRET based KD affinity assay.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

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  2. Version published to 10.1101/2022.05.31.494262v1 on bioRxiv