Topoisomerase III-β is required for efficient replication of positive-sense RNA viruses

  1. K. Reddisiva Prasanth
  2. Minato Hirano
  3. W. Samuel Fagg
  4. Eileen T. McAnarney
  5. Chao Shan
  6. Xuping Xie
  7. Adam Hage
  8. Colette A. Pietzsch
  9. Alexander Bukreyev
  10. Ricardo Rajsbaum
  11. Pei-Yong Shi
  12. Mark T. Bedford
  13. Shelton S. Bradrick
  14. Vineet Menachery
  15. Mariano A. Garcia-Blanco

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Abstract

No abstract available

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  1. Version published to 10.1016/j.antiviral.2020.104874
  2. ScreenIT

    SciScore for 10.1101/2020.03.24.005900: (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: Rabbit monoclonal antibody against TDRD3 was obtained from CST (5942S).
    TDRD3
    suggested: (Cell Signaling Technology Cat# 5942, RRID:AB_2797626)
    Rabbit monoclonal antibody against TOP3B was obtained from ABcam (ab183520).
    Rabbit monoclonal antibody against TOP3B
    suggested: None
    antibody against TOP3B
    suggested: None
    Rabbit monoclonal antibodies against GFP were obtained from ThermoFisher Scientific (G10362).
    GFP
    suggested: (Thermo Fisher Scientific Cat# G10362, RRID:AB_2536526)
    G10362
    suggested: (Thermo Fisher Scientific Cat# G10362, RRID:AB_2536526)
    Mouse monoclonal antibodies against Actin were obtained from Santa Cruz Biotechnology (sc-47778).
    Actin
    suggested: (Santa Cruz Biotechnology Cat# sc-47778 HRP, RRID:AB_2714189)
    4G2 anti-Flavivirus envelope Mouse monoclonal antibody was isolated from the DI-4G2-4-15 hybridoma, ATCC.
    anti-Flavivirus envelope Mouse
    suggested: (Thermo Fisher Scientific Cat# MA1-71258, RRID:AB_962081)
    Monoclonal Mouse Anti-Enterovirus (clone 5-D8/1) antibody was purchased from Agilent Dako (Code M7064).
    Anti-Enterovirus
    suggested: (Agilent Cat# M7064, RRID:AB_2118128)
    The blots were then washed and incubated with anti-rabbit or anti-mouse fluorophore/DyLight FW 800-conjugated secondary antibody (Santa Cruz Biotechnology).
    anti-rabbit
    suggested: None
    anti-mouse
    suggested: None
    72 hpi cells were stained with 4G2 mAb (YFV) or VP1 (CVB3) primary antibody, Alexa647-conjugated anti-mouse IgG mAb as a secondary antibody and DAPI, and the infection rate was obtained from the average of the fthree areas and shown as % infected cells.
    VP1
    suggested: None
    Briefly, the cells were stained with 4G2 mAb as a primary antibody, Alexa647-conjugated anti-mouse IgG mAb as a secondary antibody and DAPI.
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells, and Viruses: Human hepatoma cells (HuH-7) and African green monkey kidney (Vero) cells were kindly provided by Charles Rice (Rockefeller University), and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C with 5% CO2.
    Vero
    suggested: None
    Human Embryonic kidney 293 with FLP-In site (HEK293 Flp-In T-REx) cells were purchased from Thermo Fisher Scientific, and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, zeocin (100 μg/ml) and blasticidin (15 μg/ml) at 37°C with 5% CO2.
    HEK293
    suggested: None
    Dengue virus-2 (DENV-2 New Guinea C), yellow fever virus (YFV 17D) or Coxsackie virus B3 (CVB3 Strain 20) were prepared as working stock after the virus propagation in C6/36 (DENV-2, YFV) or HeLa (CVB3), respectively 3.
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    Virus titration: To determine virus titer, plaque-forming assays of DENV were performed by using BHK21 cells.
    BHK21
    suggested: ATCC Cat# CRL-6282, RRID:CVCL_1914)
    Generation of TDRD3 and TOP3B knockout cells by the CRISPR/Cas9 system: To generate TDRD3 KO cells, HuH-7 cells were co-transfected with pX330-TDRD3 gRNA1, pX330-TDRD3 gRNA2, and puromycin plasmid.
    HuH-7
    suggested: None
    HuH-7 TOP3B KO cells were generated in the same manner.
    TOP3B
    suggested: None
    Immunoblotting: The HuH-7 wild-type (WT), HuH-7 TDRD3 KO, or HuH-7 TOP3B KO cells were lysed in RIPA buffer (Cell Signaling Technologies) with 1X protease inhibitor cocktail (Cell Signaling Technologies) and centrifuged at 10,000 rpm for 3 min to pellet membranous cell debris.
    HuH-7 TOP3B KO
    suggested: None
    In brief, for plaque assays, confluent monolayers of MDCK cells were washed once with DPBS.
    MDCK
    suggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)
    Experimental Models: Organisms/Strains
    SentencesResources
    Immunoblotting: The HuH-7 wild-type (WT), HuH-7 TDRD3 KO, or HuH-7 TOP3B KO cells were lysed in RIPA buffer (Cell Signaling Technologies) with 1X protease inhibitor cocktail (Cell Signaling Technologies) and centrifuged at 10,000 rpm for 3 min to pellet membranous cell debris.
    HuH-7
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistical analyses were conducted in Prism8 (GraphPad Software).
    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 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 10.1101/2020.03.24.005900v1 on bioRxiv