Cell cycle independent role of cyclin D3 in host restriction of SARS-CoV-2 infection

  1. Ravi K. Gupta
  2. Petra Mlcochova

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Abstract

The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) presents a great threat to human health. The interplay between the virus and host plays a crucial role in successful virus replication and transmission. Understanding host-virus interactions is essential for development of new COVID-19 treatment strategies. Here we show that SARS-CoV-2 infection triggers redistribution of cyclin D1 and cyclin D3 from the nucleus to the cytoplasm, followed by its proteasomal degradation. No changes to other cyclins or cyclin dependent kinases were observed. Further, cyclin D depletion was independent from SARS-CoV-2 mediated cell cycle arrest in early S phase or S/G2/M phase. Cyclin D3 knockdown by small interfering RNA specifically enhanced progeny virus titres in supernatants. Finally, cyclin D3 co-immunoprecipitated with SARS-CoV-2 Envelope and Membrane proteins. We propose that cyclin D3 inhibits virion assembly and is depleted during SARS-CoV-2 infection to restore efficient assembly and release of newly produced virions.

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  1. ScreenIT

    SciScore for 10.1101/2022.05.07.491022: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: Reagents: Cell lines: All cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 U ml−1 penicillin and 100 mg ml−1 streptomycin and regularly tested and found to be mycoplasma free.

    Table 2: Resources

    Antibodies
    SentencesResources
    Anti-rabbit IgG, HRP-linked Antibody (7074); Cyclin D3 Mouse mAb (DCS22, 2936); from Cell Signaling.
    Anti-rabbit IgG
    suggested: (Cell Signaling Technology Cat# 7074, RRID:AB_2099233)
    Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody: Alexa 488 (A-11001), Alexa 594 (A-11032), Alexa 647 (A-21236); Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody: Alexa 488 (A-11034), Alexa 405 (A-48254); Rabbit polyclonal SARS-CoV-2 Spike (PA1-41165); Rabbit monoclonal SARS-CoV-2 Nucleocapsid (MA5-29982) from Thermo Fisher Scientific.
    anti-Mouse IgG
    suggested: (Thermo Fisher Scientific Cat# A-11001, RRID:AB_2534069)
    Rabbit Polyclonal Cyclin A2 antibody (GTX103042); Rabbit Polyclonal Cyclin D1 antibody (N1C3, GTX108824); Rabbit Polyclonal Cyclin E1 antibody (GTX103045); Rabbit Polyclonal Cyclin B1 antibody (GTX100911); monoclonal SARS-CoV-2 Spike (GTX632604) from GeneTex.
    Cyclin A2
    suggested: (GeneTex Cat# GTX103042, RRID:AB_1949884)
    Cyclin D1
    suggested: (GeneTex Cat# GTX108824, RRID:AB_10618686)
    Cyclin E1
    suggested: (GeneTex Cat# GTX103045, RRID:AB_10731259)
    Cyclin B1
    suggested: (GeneTex Cat# GTX100911, RRID:AB_1949886)
    GTX632604
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    pre-cleared cell lysates were incubated with a-HA magnetic beads, MagStrep beads (IBA-Lifescience, Gottingen, Germany) or anti-cyclin D3 monoclonal antibody (sc-xx) bound Protein G Dynabeads for 1h at 4°C.
    anti-cyclin D3
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Following cells were a gift from: A549 ACE2/TMPRSS2 40 Massimo Palmerini, Vero E6 ACE2/TMPRSS2 from Emma Thomson, HeLa-ACE2 from James Voss, 293T (a human embryonic kidney cell line, ATCC CRL-3216).
    A549
    suggested: None
    Vero E6
    suggested: None
    293T GFP11 cells and Vero-GFP10 cells for Split GFP assay were a gift from Leo James41.
    293T GFP11
    suggested: None
    Vero-GFP10
    suggested: None
    293Tv cells were transfected with pEXN-MNCX-Fucci, CMVi and pMD2.G.
    293Tv
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmids: pBOB-EF1-FastFUCCI-Puro was a gift from Kevin Brindle & Duncan Jodrell (Addgene plasmid # 86849 ; http://n2t.net/addgene:86849 ; RRID:Addgene_86849) 29. pCMV5 cyclin D3 HA was obtained from MRC-PPU Reagents and Services. Rc/CMV cyclin D1 HA was a gift from Philip Hinds (Addgene plasmid # 8948 ; http://n2t.net/addgene:8948 ; RRID:Addgene_8948) 44. pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro (Addgene plasmid # 141385 ; http://n2t.net/addgene:141385 ; RRID:Addgene_141385); pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro (Addgene plasmid # 141386 ; http://n2t.net/addgene:141386 ; RRID:Addgene_141386).
    detected: RRID:Addgene_86849)
    pCMV5
    suggested: RRID:Addgene_15002)
    detected: RRID:Addgene_8948)
    detected: RRID:Addgene_141385)
    detected: RRID:Addgene_141386)
    pLVX-EF1alpha-SARS-CoV-2-nsp9-2xStrep-IRES-Puro (Addgene plasmid # 141375 ; http://n2t.net/addgene:141375 ; RRID:Addgene_141375); pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro (Addgene plasmid # 141391 ; http://n2t.net/addgene:141391 ; RRID:Addgene_141391) were a gift from Nevan Krogan 34. pEXN-MNCX, MLV vector encoding N-terminal double HA tag 45.
    detected: RRID:Addgene_141375)
    detected: RRID:Addgene_141391)
    pCAGGS_SARS-CoV-2_Spike was obtained from NIBS.
    pCAGGS_SARS-CoV-2_Spike
    suggested: None
    Cell cycle analysis using fluorescence ubiquitination cell cycle indicator (Fucci): Fucci cassete was cloned from pBOB-EF1-FastFucci-Puro vector to pEXN-MNCX using BamHI/NotI restriction sites.
    pEXN-MNCX
    suggested: None
    293Tv cells were transfected with pEXN-MNCX-Fucci, CMVi and pMD2.G.
    pEXN-MNCX-Fucci
    suggested: None
    pMD2.G
    suggested: RRID:Addgene_12259)
    Cell to cell fusion assay: 293T GFP11 cells were transfected with WT full length Spike, and/or with WT Envelope, Membrane, cyclin D3, and empty vector (pCDNA, to ensure equal amount of transfected DNA).
    pCDNA
    suggested: RRID:Addgene_66792)
    Software and Algorithms
    SentencesResources
    Harmony (PerkinElmer, Waltham, MA, USA) and ImageJ software were used to measure MFI for each protein in each region.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Cell populations positive or negative for SARS-CoV-2 nucleocapsid staining were gated and Cdt1-RFP positive (G1 phase), Geminin-GFP positive (S/G2/M phase), and Cdt1-RFP/ Geminin-GFP positive (early S phase) populations were identified using flow cytometry using LSRFortessa X-20 (BD Biosciences, UK) and FlowJo software (Tree Star, OR, USA).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2022.05.07.491022 on bioRxiv