SARS CoV-2 Nucleoprotein Enhances the Infectivity of Lentiviral Spike Particles

  1. Tarun Mishra
  2. M. Sreepadmanabh
  3. Pavitra Ramdas
  4. Amit Kumar Sahu
  5. Atul Kumar
  6. Ajit Chande

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Abstract

The establishment of SARS CoV-2 spike-pseudotyped lentiviral (LV) systems has enabled the rapid identification of entry inhibitors and neutralizing agents, alongside allowing for the study of this emerging pathogen in BSL-2 level facilities. While such frameworks recapitulate the cellular entry process in ACE2+ cells, they are largely unable to factor in supplemental contributions by other SARS CoV-2 genes. To address this, we performed an unbiased ORF screen and identified the nucleoprotein (N) as a potent enhancer of spike-pseudotyped LV particle infectivity. We further demonstrate that the spike protein is better enriched in virions when the particles are produced in the presence of N protein. This enrichment of spike renders LV particles more infectious as well as less vulnerable to the neutralizing effects of a human IgG-Fc fused ACE2 microbody. Importantly, this improvement in infectivity is observed with both wild-type spike protein as well as the D614G mutant. Our results hold important implications for the design and interpretation of similar LV pseudotyping-based studies.

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  1. Version published to 10.3389/fcimb.2021.663688
  2. ScreenIT

    SciScore for 10.1101/2021.02.11.430757: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Detection of p24, beta actin, SARS CoV-2 spike glycoprotein, SARS CoV-2 genes, and ACE2-IgFc was carried out using mouse anti-p24 (NIH ARP), rabbit anti-beta actin (LI-COR Biosciences, Cat# 926-42210, RRID:AB_1850027)
    rabbit anti-beta actin
    detected: (LI-COR Biosciences Cat# 926-42210, RRID:AB_1850027)
    mouse anti-spike (Cat#ZMS1076, Sigma Aldrich), mouse anti-Strep (Qiagen, Cat# 34850), and mouse anti-Histidine (Invitrogen, Cat# MA1-21315), respectively, as primary antibodies.
    anti-spike
    suggested: None
    anti-Strep
    suggested: None
    anti-Histidine
    suggested: None
    Secondary antibodies used were either IR dye 680 goat anti-mouse, IR dye 800 goat mouse, or IR dye 800 goat anti-rabbit (LI-COR Biosciences Cat# 925-68070, RRID:AB_2651128, and LI-COR Biosciences Cat# 925-32211, RRID:AB_2651127).
    anti-mouse
    detected: (LI-COR Biosciences Cat# 925-68070, RRID:AB_2651128)
    detected: (LI-COR Biosciences Cat# 925-32211, RRID:AB_2651127)
    For Western Blotting, the proteins were transferred to a PVDF membrane, blocked in a 5% BSA solution in TBS, and primary mouse-derived anti-Histidine antibodies were incubated with the blot in a 1:4000 dilution, followed by goat-derived anti-mouse antibodies in a 1:5000 dilution.
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The SARS CoV-2 genes’ screen was performed by producing spike pseudotyped lentiviruses from HEK293T cells that were seeded in 12-well plate.
    HEK293T
    suggested: None
    Software and Algorithms
    SentencesResources
    Plasmids: The list of plasmids that were used in this study is provided below (refer to “Table S1: List of Plasmids”) and are available upon a reasonable request.
    Plasmids
    suggested: None
    Following this, gels were electro blotted on the PVDF membrane (Immobilon-FL, Merck-Millipore)
    Merck-Millipore
    suggested: None
    Software: All graphs were generated using GraphPad Prism (version 9.0).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • 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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.02.11.430757v1 on bioRxiv