Elucidating the Antiviral Mechanism of Different MARCH Factors

  1. Supawadee Umthong
  2. Brian Lynch
  3. Uddhav Timilsina
  4. Brandon Waxman
  5. Emily B. Ivey
  6. Spyridon Stavrou

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Abstract

This study examines the mechanism utilized by different MARCH proteins to restrict retrovirus infection. MARCH proteins block the incorporation of envelope glycoproteins to the budding virions.

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  1. Version published to 10.1128/mbio.03264-20
  2. ScreenIT

    SciScore for 10.1101/2020.12.22.422953: (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
    Blots were probed using the following antibodies: goat anti-MLV gp70 (66), goat anti-MMTV polyclonal (67), rat anti-MLV transmembrane protein/p15E (clone 42/114, Kerafast)
    anti-MLV
    suggested: (Creative Diagnostics Cat# DCABH-2412, RRID:AB_2476319)
    anti-MMTV
    suggested: (Rockland Cat# 100-401-P12S, RRID:AB_2610798)
    anti-MLV transmembrane protein/p15E
    suggested: None
    Lysates were then resolved on 10% sodium dodecyl sulfate polyacrylamide gels and blots were probed using the following antibodies: rabbit anti-FLAG (Cell Signaling Technology), rabbit anti-V5 (Invitrogen), mouse anti-Flavivirus envelope 4G2 (BEI Resources, NIH, NIAID NR-50327), rabbit anti-AU1 (Novus Biologicals)
    anti-V5
    suggested: None
    anti-AU1
    suggested: None
    Cells were harvested and 1 × 105 cells were stained with 1:50 of rabbit anti-MARCH1 (Thermo Fisher Scientific Cat#PA5-69223) or 1:50 of rabbit IgG isotype antibody (Thermo Fisher Scientific Cat#02-6102) for 30 minutes at 4°C.
    rabbit IgG
    suggested: (Thermo Fisher Scientific Cat# 02-6102, RRID:AB_2532938)
    Blots were probed with the following antibodies: rabbit anti-MARCH1 (Invitrogen Cat#PA5-69223), rabbit anti-MARCH8 (Invitrogen Cat#30220)
    anti-MARCH8
    suggested: None
    Briefly, 50 μl proteinA Dynabeads (Thermo Fisher Scientific) were pre-incubated with 1:50 dilution of rabbit anti-MARCH1 (Invitrogen Cat#PA5-69223) or 1:25 dilution of rabbit anti-MARCH8 (Proteintech Cat#14119-1-AP) antibodies.
    anti-MARCH1
    suggested: (Thermo Fisher Scientific Cat# PA5-69223, RRID:AB_2689421)
    Cell lysates were incubated with antibodies-coated protein A dynabeads overnight at 4°C, washed, and eluted.
    antibodies-coated protein
    suggested: None
    Protein A Dynabeads were pre-incubated with anti-Myc (Cell Signaling Technology), or 1:20 of culture supernatant of 372 (ATCC CRL-1893) and then cell lysates were added to antibodies-bound protein A dynabeads and incubated at room temperature for 1 hour, washed, and eluted, followed by SDS-PAGE and western blot analysis detecting for MLV p15E or MARCH(myc).
    Protein A Dynabeads were pre-incubated with anti-Myc (Cell Signaling Technology)
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    EL4 cells (ATCC) were cultured in RPMI media with 10% FBS, P/S, and 0.05 mM β-mercaptoethanol (β-ME; Bio-Rad).
    EL4
    suggested: None
    In the case of human MARCH genes, hM1 cDNA was synthesized from HeLa cell RNA.
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    For the MMTV transfection experiments, we seeded 293T cells in a 6-well plate (0.5 × 106/well) and transfected them with 5 μg of MMTV hybrid provirus (HP) plasmid (34), 50ng of rat glycocorticoid receptor (RSVGR) construct and 1 μg mM1, 2, 3, 8 or E.
    293T
    suggested: None
    Interferon treatment of cells: 0.5 × 104 cells of MutuDC1940, EL-4, NIH3T3, BMDMs and BMDCs were seeded in a 96-well plate for 24 hours.
    EL-4
    suggested: None
    Virus preparation: MLV stocks were prepared by transfecting 293FT cells (Invitrogen) seeded in 10-cm-diameter cell culture dishes using Lipofectamine3000 (Thermo Fisher Scientific) with 25 μg of an MLV infectious clone (pLRB302) per manufacturer’s recommendation.
    293FT
    suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
    Infection assays: To examine the effect of MLV infection on MARCH gene expression, 0.5 × 104 cells of MutuDC1940, EL4, NIH3T3 were seeded in 96-well plate followed by infection with MLV (5 MOI) via spinoculation as previously described (68).
    NIH3T3
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    BMDCs and BMDMs were generated from 6-9 week C57BL6/N mice as previously described (55).
    C57BL6/N
    suggested: None
    We introduced in the NL4-3XhoI-EcoRI fragment two stop codons at residues 705 and 707 (R705Stop and R707Stop) of the envelope ORF located at the N’ terminus of the cytosolic tail, using the Phusion Site-Directed Mutagenesis Kit (Thermo Fisher Scientific).
    R705Stop
    suggested: None
    Software and Algorithms
    SentencesResources
    Stained populations from our FACS experiments were further analyzed using Flowjo software version 10.7.1.
    Flowjo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analysis: Statistical analyses were performed using GraphPad Prism software version 8.2.
    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 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.

    About SciScore

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  3. Version published to 10.1101/2020.12.22.422953v1 on bioRxiv