A Systemic and Molecular Study of Subcellular Localization of SARS-CoV-2 Proteins

  1. Jing Zhang
  2. Ruth Cruz-cosme
  3. Meng-Wei Zhuang
  4. Dongxiao Liu
  5. Yuan Liu
  6. Shaolei Teng
  7. Pei-Hui Wang
  8. Qiyi Tang

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Coronavirus possesses the largest RNA genome among all the RNA viruses. Its genome encodes about 29 proteins. Most of the viral proteins are non-structural proteins (NSP) except envelop (E), membrane (M), nucleocapsid (N) and Spike (S) proteins that constitute the viral nucleocapsid, envelop and surface. We have recently cloned all the 29 SARS-CoV-2 genes into vectors for their expressions in mammalian cells except NSP11 that has only 14 amino acids (aa). We are able to express all the 28 cloned SARS-CoV-2 genes in human cells to characterize their subcellular distributions. The proteins of SARS-CoV-2 are mostly cytoplasmic but some are both cytoplasmic and nuclear. Those punctate staining proteins were further investigated by immunofluorescent assay (IFA) using specific antibodies or by co-transfection with an organelle marker-expressing plasmid. As a result, we found that NSP15, ORF6, M and ORF7a are related to Golgi apparatus, and that ORF7b, ORF8 and ORF10 colocalize with endoplasmic reticulum (ER). Interestingly, ORF3a distributes in cell membrane, early endosome, endosome, late endosome and lysosome, which suggests that ORF3a might help the infected virus to usurp endosome and lysosome for viral use. Furthermore, we revealed that NSP13 colocalized with SC35, a protein standing for splicing compartments in the nucleus. Our studies for the first time visualized the subcellular locations of SARS-CoV-2 proteins and might provide novel insights into the viral proteins’ biological functions.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2020.08.02.233023: (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
    Antibodies: Anti-Giantin (ab80864) for visualizing Golgi body, and anti-CoxIV (ab16056) for showing mitochondria were purchased from Abcam (Cambridge, MA).
    Anti-Giantin
    suggested: (Abcam Cat# ab80864, RRID:AB_10670397)
    anti-CoxIV
    suggested: (Abcam Cat# ab16056, RRID:AB_443304)
    Anti-Tubulin (
    Anti-Tubulin
    suggested: None
    Anti-FLAG antibody (monoclonal), M2, and the anti-SC35 antibody (S4045) were purchased from Sigma.
    Anti-FLAG
    suggested: None
    anti-SC35
    suggested: None
    S4045
    suggested: None
    Immunofluorescent assay (IFA): Immunostaining was performed on cells grown on coverslips after fixation with 1% paraformaldehyde (10 min at room temperature) and permeabilization in 0.2% Triton (20 min on ice) by sequential incubation with primary and Texas red (TR)-labeled secondary antibodies (Vector Laboratories, Burlingame, Calif.) for 30 min each (all solutions in PBS).
    Texas red (TR)-labeled secondary antibodies
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Plasmids and transfection: To show a protein’s location in the Hep-2 cells, the plasmid constructed from “molecular cloning” is co-transfected with one of the following plasmids that expresses a cell marker.
    Hep-2
    suggested: CLS Cat# 300397/p694_Hep-2, RRID:CVCL_1906)
    Software and Algorithms
    SentencesResources
    http://www.addgene.org).
    http://www.addgene.org
    suggested: (Addgene, RRID:SCR_002037)

    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 10.1101/2020.08.02.233023 on bioRxiv