Targeting of Protein Kinase CK2 Elicits Antiviral Activity on Bovine Coronavirus Infection

  1. Ailyn C. Ramón
  2. George V. Pérez
  3. Evelin Caballero
  4. Mauro Rosales
  5. Daylén Aguilar
  6. Dania Vázquez-Blomquist
  7. Yassel Ramos
  8. Arielis Rodríguez-Ulloa
  9. Viviana Falcón
  10. María Pilar Rodríguez-Moltó
  11. Ke Yang
  12. Yasser Perera
  13. Silvio E. Perea

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Abstract

Coronaviruses constitute a global threat to the human population; therefore, effective pan-coronavirus antiviral drugs are required to tackle future re-emerging virus outbreaks. Protein kinase CK2 has been suggested as a promising therapeutic target in COVID-19 owing to the in vitro antiviral activity observed after both pharmacologic and genetic inhibition of the enzyme. Here, we explored the putative antiviral effect of the anti-CK2 peptide CIGB-325 on bovine coronavirus (BCoV) infection using different in vitro viral infected cell-based assays. The impact of the peptide on viral mRNA and protein levels was determined by qRT-PCR and Western blot, respectively. Finally, pull-down experiments followed by Western blot and/or mass spectrometry analysis were performed to identify CIGB-325-interacting proteins. We found that CIGB-325 inhibited both the cytopathic effect and the number of plaque-forming units. Accordingly, intracellular viral protein levels were clearly reduced after treatment of BCoV-infected cells, with CIGB-325 determined by immunocytochemistry. Pull-down assay data revealed the physical interaction of CIGB-325 with viral nucleocapsid (N) protein and a group of bona fide CK2 cellular substrates. Our findings evidence in vitro antiviral activity of CIGB-325 against bovine coronavirus as well as some molecular clues that might support such effect. Altogether, data provided here strengthen the rationale of inhibiting CK2 to treat betacoronavirus infections.

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  1. Version published to 10.3390/v14030552
  2. ScreenIT

    SciScore for 10.1101/2021.06.08.447588: (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 Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Then, peroxidase-conjugated anti-rabbit secondary antibody 1:100 (Sigma, MO, USA) was incubated for 1 h at room temperature and washed 3 times with PBS.
    anti-rabbit
    suggested: None
    For western blot analysis, a human polyclonal IgG anti-SARS-CoV-2 (CIGB, Cuba) and a mouse monoclonal antibody against CK2α (Abcam, Cambridge, United Kingdom) were used as primary antibodies.
    a human polyclonal IgG anti-SARS-CoV-2 (CIGB, Cuba)
    suggested: None
    anti-SARS-CoV-2 (CIGB, Cuba)
    suggested: None
    CK2α
    suggested: None
    Primary antibodies against phospho-RPS6 (S235/236) and total RPS6 (Cell Signaling Technology, MA, USA) were used, and detection was performed with peroxidase-conjugated anti-rabbit IgG 1:5000 (Sigma, MO, USA). 2.13.
    phospho-RPS6
    suggested: None
    total RPS6
    suggested: None
    anti-rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Briefly, 60 000 MDBK cells were seeded in flat-bottom 96-wells plates per well in DMEM medium with 10% fetal bovine serum (FBS), and a curve of serial dilutions (1:2) of CIGB-325 (3.12-200 μM) were added by triplicate.
    MDBK
    suggested: None
    Software and Algorithms
    SentencesResources
    Protein identification based on MS/MS spectra was made against the Swiss-Prot database by using Mascot database search engine (version 2.5).
    Mascot
    suggested: (Mascot, RRID:SCR_014322)
    Protein-protein interaction (PPI) networks were generated using information retrieved form STRING database [30], and protein kinase CK2 substrates among CIGB-325 interacting proteins were identified based on Meggio and Pinna dataset [31], the list of high confidence CK2 substrates reported by Bian et al. [32] and the PhosphoSitePlus database (www.phosphosite.org, accessed June 6, 2021). 2.11. Confocal Microscopy: MDBK cells were plated on 8-well glass slide and incubated overnight at 37° C and 5% CO2.
    STRING
    suggested: (STRING, RRID:SCR_005223)
    Images were acquired with UPLSAPO 40× immersion objective and processed using Olympus FluoView software (v4.0) (Olympus, Tokyo, Japan).
    Olympus FluoView
    suggested: (Olympus Fluoview FV10-ASW, RRID:SCR_014215)
    Analyses were performed in GraphPad Prism (v6.01) software for Windows (GraphPad Software, Inc, San Diego, CA, USA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

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

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  3. Version published to 10.1101/2021.06.08.447588v1 on bioRxiv