SARS-CoV-2 viral budding and entry can be modeled using virus-like particles

  1. Caroline B. Plescia
  2. Emily A. David
  3. Dhabaleswar Patra
  4. Ranjan Sengupta
  5. Souad Amiar
  6. Yuan Su
  7. Robert V. Stahelin

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in December 2019 in Wuhan, China and expeditiously spread across the globe causing a global pandemic. While a select agent designation has not been made for SARS-CoV-2, closely related SARS-CoV-1 and MERS coronaviruses are classified as Risk Group 3 select agents, which restricts use of the live viruses to BSL-3 facilities. Such BSL-3 classification make SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the US; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. In this work, we assessed the four structural proteins from SARS-CoV-2 for their ability to form viruslike particles (VLPs) from human cells to form a competent system for BSL-2 studies of SARS-CoV-2. Herein, we provide methods and resources of producing, purifying, fluorescently and APEX2-labeling of SARS-CoV-2 VLPs for the evaluation of mechanisms of viral budding and entry as well as assessment of drug inhibitors under BSL-2 conditions.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.30.320903: (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
    Genetex); goat anti-rabbit IgG HRP (Abcam) and sheep anti-mouse IgG HRP (Abcam) secondary antibodies were used as appropriate.
    anti-rabbit IgG
    suggested: None
    anti-mouse IgG
    suggested: None
    At 24-hours post-transfection, the cells were fixed by 4% paraformaldehyde (PFA), permeabilized by 0.1-0.2% Triton X-100 in PBS, blocked by 2.5-3% FBS and 1% BSA in PBS and then incubated with the appropriate primary antibody overnight (Rabbit anti-SARS-CoV-2-N antibody, GeneTex #GTX135357, 1:500 or Rabbit anti-SARS-1-M, Rockland, 1:1000; mouse anti-GORAPS2 (Golgi marker), Sigma Aldrich, 1:500)
    anti-SARS-CoV-2-N
    suggested: None
    anti-SARS-1-M
    suggested: None
    anti-GORAPS2
    suggested: None
    Following overnight incubation, cells were washed and then incubated with the appropriate secondary antibody (anti-rabbit IgG-Atto 594, Millipore Sigma #77671-1ML-F
    anti-rabbit IgG-Atto 594
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Production of SARS-CoV-2 VLPs: HEK293 cells 70% confluent in 100mm dishes were transfected with 6 μg of each SARS-CoV-2 pcDNA3-M, pcDNA3-N, pCVM-3xFlag-E, and/or pCAGGS-S.
    HEK293
    suggested: None
    Scanning electron microscopy: Silica coverslips were placed on the bottom of 12-well plates before being seeded with HEK-293 cells to 30% confluency for transfection 24-hours after seeding.
    HEK-293
    suggested: None
    Software and Algorithms
    SentencesResources
    Images were analyzed using ImageJ.
    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:
    • 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.

  2. Version published to 10.1101/2020.09.30.320903 on bioRxiv