Identification of the SHREK Family of Proteins as Broad-Spectrum Host Antiviral Factors

  1. Deemah Dabbagh
  2. Sijia He
  3. Brian Hetrick
  4. Linda Chilin
  5. Ali Andalibi
  6. Yuntao Wu

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Abstract

Mucins and mucin-like molecules are highly glycosylated, high-molecular-weight cell surface proteins that possess a semi-rigid and highly extended extracellular domain. P-selectin glycoprotein ligand-1 (PSGL-1), a mucin-like glycoprotein, has recently been found to restrict HIV-1 infectivity through virion incorporation that sterically hinders virus particle attachment to target cells. Here, we report the identification of a family of antiviral cellular proteins, named the Surface-Hinged, Rigidly-Extended Killer (SHREK) family of virion inactivators (PSGL-1, CD43, TIM-1, CD34, PODXL1, PODXL2, CD164, MUC1, MUC4, and TMEM123) that share similar structural characteristics with PSGL-1. We demonstrate that SHREK proteins block HIV-1 infectivity by inhibiting virus particle attachment to target cells. In addition, we demonstrate that SHREK proteins are broad-spectrum host antiviral factors that block the infection of diverse viruses such as influenza A. Furthermore, we demonstrate that a subset of SHREKs also blocks the infectivity of a hybrid alphavirus-SARS-CoV-2 (Ha-CoV-2) pseudovirus. These results suggest that SHREK proteins may be a part of host innate immunity against enveloped viruses.

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  1. Version published to 10.3390/v13050832
  2. ScreenIT

    SciScore for 10.1101/2021.02.02.429469: (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
    1×108 beads / 0.25 ml) were conjugated with one of the following antibodies, mouse anti-PSGL-1 Antibody (KPL-1) (BD Pharmingen), mouse anti-CD43 Antibody (1G10) (BD Biosciences), mouse anti-CD164 Antibody (67D2) (Biolegend), mouse anti-TMEM123 Antibody (297617) (Invitrogen), mouse anti-PODXL1 Antibody (222328) (R & D Systems), mouse anti-PODXL2 Antibody (211816) (R & D Systems) or mouse anti-CD34 Antibody (563) (BD Biosciences) for 30 minutes at room temperature.
    anti-PSGL-1
    suggested: None
    anti-CD43
    suggested: None
    anti-PODXL1
    suggested: None
    anti-PODXL2
    suggested: (Thermo Fisher Scientific Cat# MA5-23932, RRID:AB_2576899)
    anti-CD34
    suggested: None
    The attachment of HIV-1 virus produced in the presence of TMEM123 was also separately tested by pre-incubating the virus with mouse monoclonal anti-TMEM123 antibody (297617) (ThermoFisher) (25ug/ml) at 37°C for 1 h, followed by incubation of the virus with HeLaJC.
    anti-TMEM123
    suggested: (Thermo Fisher Scientific Cat# MA5-24007, RRID:AB_2606735)
    Proteins were denatured by boiling in sample buffer and subjected to SDS-PAGE, transferred to nitrocellulose membrane, and incubated overnight at 4°C with one of the following primary antibodies: mouse anti-HIV-1 p24 monoclonal antibody (183-H12-5C) (NIH AIDS Reagent Program) (1:1000
    anti-HIV-1
    suggested: (NIH AIDS Reagent Program Cat# 3537, RRID:AB_2832923)
    , human anti-HIV-1 gp41 (2F5) (1:1000 dilution) (NIH AIDS Reagent Program), mouse anti-MUC1antibody (HMPV) (BD Biosciences) (1:1000
    anti-HIV-1 gp41
    suggested: (bNAber Cat# bNAberID_11, RRID:AB_2491015)
    anti-MUC1antibody
    suggested: None
    dilution), mouse monoclonal anti-MUC4 antibody (1G8) (ThermoFisher) (1:1000
    anti-MUC4
    suggested: (Thermo Fisher Scientific Cat# 35-4900, RRID:AB_2533206)
    dilution), mouse monoclonal anti-TMEM123 antibody (297617) (ThermoFisher) (1:1000 dilution), or anti-GAPDH goat polyclonal antibody (Abcam) (1:1000 dilution).
    anti-GAPDH
    suggested: None
    Membranes were incubated with HRP-labeled goat anti-mouse IgG (KPL) (1:2500 dilution) for 60 min at room temperature, or goat anti-human 800cw-labeled antibodies (Li-Cor Biosciences) (1:2500 dilution) for 30 min at room temperature.
    anti-mouse IgG
    suggested: (Rockland Cat# 610-445-020, RRID:AB_1961635)
    anti-human 800cw-labeled
    suggested: None
    At 48 hours post transfection, 0.5–1 million cells were stained with one of the following primary antibodies: mouse anti-PSGL1 antibody (KPL-1) (BD Pharmingen),
    anti-PSGL1
    suggested: (Fitzgerald Industries International Cat# 10R-7618, RRID:AB_11194030)
    mouse anti-CD43 antibody (1G10) (BD Biosciences), mouse anti-CD164 antibody (67D2) (Biolegend)
    anti-CD164
    suggested: None
    , mouse anti-CD34 antibody (563) (BD Biosciences), mouse anti-TIM1 antibody (219211) (R & D Systems)
    anti-TIM1
    suggested: None
    ), mouse monoclonal anti-PODXL1 antibody (222328) (R & D systems), or mouse monoclonal anti-PODXL2 antibody (211816) (R & D Systems), followed by staining with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Invitrogen).
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and cell culture: HEK293T (ATCC), MDCK (ATCC), Calu-3 (ATCC), and HeLaJC.53 (kindly provided by Dr. David Kabat) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) containing 10% heat-inactivated FBS and 1x penicillin-streptomycin (Invitrogen).
    Calu-3
    suggested: None
    HEK293T(ACE2/TMPRSS2) cells (kindly provided by Virongy LLC) were maintained in DMEM (Invitrogen) supplemented with penicillin-streptomycin, puromycin and hygromycin B as instructed by the manufacturer.
    HEK293T(ACE2/TMPRSS2
    suggested: None
    Hybrid alphavirus-SARS-CoV-2 (Ha-CoV-2) particles were produced by cotransfection of HEK293T cells in 6-well plates with SARS-CoV-2 S, M, N, and E expression vectors (0.3 µg each), Ha-CoV-2(Luc) (1.2 µg), and each individual SHREK-expressing vector or an control empty vector (1.6 µg).
    HEK293T
    suggested: None
    Virions were harvested at 48 hours and used to infect MDCK cells (3 × 104 cells per infection).
    MDCK
    suggested: None
    Software and Algorithms
    SentencesResources
    The IC50 inhibition curves were generated using GraphPad Software, La Jolla, California, USA.
    GraphPad
    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

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  3. Version published to 10.1101/2021.02.02.429469v1 on bioRxiv