1887

Journal of General Virology header logo

Volume 105, Issue 3

Cardiac glycosides inhibit early and late vaccinia virus protein expression Open Access

Abstract

Cardiac glycosides (CGs) are natural steroid glycosides, which act as inhibitors of the cellular sodium-potassium ATPase pump. Although traditionally considered toxic to human cells, CGs are widely used as drugs for the treatment of cardiovascular-related medical conditions. More recently, CGs have been explored as potential anti-viral drugs and inhibit replication of a range of RNA and DNA viruses. Previously, a compound screen identified CGs that inhibited vaccinia virus (VACV) infection. However, no further investigation of the inhibitory potential of these compounds was performed, nor was there investigation of the stage(s) of the poxvirus lifecycle they impacted. Here, we investigated the anti-poxvirus activity of a broad panel of CGs. We found that all CGs tested were potent inhibitors of VACV replication. Our virological experiments showed that CGs did not impact virus infectivity, binding, or entry. Rather, experiments using recombinant viruses expressing reporter proteins controlled by VACV promoters and arabinoside release assays demonstrated that CGs inhibited early and late VACV protein expression at different concentrations. Lack of virus assembly in the presence of CGs was confirmed using electron microscopy. Thus, we expand our understanding of compounds with anti-poxvirus activity and highlight a yet unrecognized mechanism by which poxvirus replication can be inhibited.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): compound , drug , infection , poxvirus , viral transcription and virion assembly
Funding
This study was supported by the:
  • Medical Research Council (Award MC_U12266B)
    • Principle Award Recipient: IanJ. White
  • Biotechnology and Biological Sciences Research Council (Award BB/X011607/1)
    • Principle Award Recipient: JasonMercer
  • Medical Research Council (Award MC_UU_00012/7)
    • Principle Award Recipient: JasonMercer
© 2024 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001971
2024-03-28
2024-04-27
Loading full text...

Full text loading...

/deliver/fulltext/jgv/105/3/jgv001971.html?itemId=/content/journal/jgv/10.1099/jgv.0.001971&mimeType=html&fmt=ahah

References

  1. Bertran M, Andrews N, Davison C, Dugbazah B, Boateng J et al. Effectiveness of one dose of MVA-BN smallpox vaccine against mpox in England using the case-coverage method: an observational study. Lancet Infect Dis 2023; 23:828–835 [View Article] [PubMed]
     [Google Scholar]
  2. Thornhill JP, Barkati S, Walmsley S, Rockstroh J, Antinori A et al. Monkeypox virus infection in uhmans across 16 countries - April-June 2022. N Engl J Med 2022; 387:679–691 [View Article] [PubMed]
     [Google Scholar]
  3. Blasco R, Moss B. Extracellular vaccinia virus formation and cell-to-cell virus transmission are prevented by deletion of the gene encoding the 37,000-Dalton outer envelope protein. J Virol 1991; 65:5910–5920 [View Article] [PubMed]
     [Google Scholar]
  4. Chinsangaram J, Honeychurch KM, Tyavanagimatt SR, Leeds JM, Bolken TC et al. Safety and pharmacokinetics of the anti-orthopoxvirus compound ST-246 following a single daily oral dose for 14 days in human volunteers. Antimicrob Agents Chemother 2012; 56:4900–4905 [View Article]
     [Google Scholar]
  5. Olson VA, Smith SK, Foster S, Li Y, Lanier ER et al. In vitro efficacy of brincidofovir against variola virus. Antimicrob Agents Chemother 2014; 58:5570–5571 [View Article] [PubMed]
     [Google Scholar]
  6. Yang G, Pevear DC, Davies MH, Collett MS, Bailey T et al. An orally bioavailable antipoxvirus compound (ST-246) inhibits extracellular virus formation and protects mice from lethal orthopoxvirus challenge. J Virol 2005; 79:13139–13149 [View Article] [PubMed]
     [Google Scholar]
  7. Jordan R, Goff A, Frimm A, Corrado ML, Hensley LE et al. ST-246 antiviral efficacy in a nonhuman primate monkeypox model: determination of the minimal effective dose and human dose justification. Antimicrob Agents Chemother 2009; 53:1817–1822 [View Article]
     [Google Scholar]
  8. Lanier R, Trost L, Tippin T, Lampert B, Robertson A et al. Development of CMX001 for the treatment of Poxvirus infections. Viruses 2010; 2:2740–2762 [View Article] [PubMed]
     [Google Scholar]
  9. Lederman ER, Davidson W, Groff HL, Smith SK, Warkentien T et al. Progressive vaccinia: case description and laboratory-guided therapy with vaccinia immune globulin, ST-246, and CMX001. J Infect Dis 2012; 206:1372–1385 [View Article] [PubMed]
     [Google Scholar]
  10. Smith TG, Gigante CM, Wynn NT, Matheny A, Davidson W et al. Resistance to anti-orthopoxviral drug tecovirimat (TPOXX ®) during the 2022 Mpox outbreak in the US. medRxiv 2023 [View Article]
     [Google Scholar]
  11. Levi AJ, Boyett MR, Lee CO. The cellular actions of digitalis glycosides on the heart. Prog Biophys Mol Biol 1994; 62:1–54 [View Article] [PubMed]
     [Google Scholar]
  12. Prassas I, Diamandis EP. Novel therapeutic applications of cardiac glycosides. Nat Rev Drug Discov 2008; 7:926–935 [View Article] [PubMed]
     [Google Scholar]
  13. Hansen O, Skou JC. A study on the influence of the concentration of Mg2+, Pi, K+, Na+, and tris on (Mg2+ + Pi)-supported g-strophanthin binding to (Na+ + K+)-activated ATPase from ox brain. Biochimica et Biophysica Acta 197351–66
     [Google Scholar]
  14. Lingrel JB. The physiological significance of the cardiotonic steroid/ouabain-binding site of the NA,K-ATPase. Annu Rev Physiol 2010; 72:395–412
     [Google Scholar]
  15. Yatime L, Mette Laursen J, Esmann M, Nissen P, Fedosova N. Structural insights into the high affinity binding of cardiotonic steroids to the Na+,K+-ATPase. J Struct Biol 2011; 174:296–306 [View Article]
     [Google Scholar]
  16. Kassardjian A, Kreydiyyeh S. JNK modulates the effect of caspases and NF-kappaB in the TNF-alpha-induced down-regulation of Na+/K+ATPase in HepG2 cells. J Cell Physiol 2008; 216:615–620
     [Google Scholar]
  17. Li Z, Cai T, Tian J, Xie JX, Zhao X et al. NaKtide, a Na/K-ATPase-derived peptide Src inhibitor, antagonizes ouabain-activated signal transduction in cultured cells. J Biol Chem 2009; 284:21066–21076 [View Article] [PubMed]
     [Google Scholar]
  18. Therien AG, Blostein R. Mechanisms of sodium pump regulation. Am J Physiol Cell Physiol 2000; 279:C541–66 [View Article] [PubMed]
     [Google Scholar]
  19. Xie Z. Molecular mechanisms of Na/K-ATPase-mediated signal transduction. Ann N Y Acad Sci 2003; 986:497–503 [View Article] [PubMed]
     [Google Scholar]
  20. Yudowski GA, Efendiev R, Pedemonte CH, Katz AI, Berggren PO et al. Phosphoinositide-3 kinase binds to a proline-rich motif in the Na+, K+-ATPase alpha subunit and regulates its trafficking. Proc Natl Acad Sci U S A 2000; 97:6556–6561 [View Article] [PubMed]
     [Google Scholar]
  21. Menger L, Vacchelli E, Adjemian S, Martins I, Ma Y et al. Cardiac glycosides exert anticancer effects by inducing immunogenic cell death. Sci Transl Med 2012; 4:143ra99 [View Article] [PubMed]
     [Google Scholar]
  22. Reddy D, Kumavath R, Barh D, Azevedo V, Ghosh P. Anticancer and antiviral properties of cardiac glycosides: a review to explore the mechanism of actions. Molecules 2020; 25:3596 [View Article] [PubMed]
     [Google Scholar]
  23. Kumavath R, Paul S, Pavithran H, Paul MK, Ghosh P et al. Emergence of cardiac glycosides as potential drugs: current and future scope for cancer therapeutics. Biomolecules 2021; 11:1275 [View Article] [PubMed]
     [Google Scholar]
  24. Deng L, Dai P, Ciro A, Smee DF, Djaballah H et al. Identification of novel antipoxviral agents: mitoxantrone inhibits vaccinia virus replication by blocking virion assembly. J Virol 2007; 81:13392–13402 [View Article] [PubMed]
     [Google Scholar]
  25. Schmidt FI, Bleck CKE, Reh L, Novy K, Wollscheid B et al. Vaccinia virus entry is followed by core activation and proteasome-mediated release of the immunomodulatory effector VH1 from lateral bodies. Cell Rep 2013; 4:464–476 [View Article] [PubMed]
     [Google Scholar]
  26. Schmidt FI, Bleck CKE, Helenius A, Mercer J. Vaccinia extracellular virions enter cells by macropinocytosis and acid-activated membrane rupture. EMBO J 2011; 30:3647–3661 [View Article] [PubMed]
     [Google Scholar]
  27. Samolej J, Mendonca DC, Upfold N, McElwee M, Landsberger M et al. Bisbenzimide compounds inhibit the replication of prototype and pandemic potential poxviruses. Microbiol Spectr 2024; e0407223: [View Article]
     [Google Scholar]
  28. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012; 9:676–682 [View Article] [PubMed]
     [Google Scholar]
  29. Doceul V, Hollinshead M, van der Linden L, Smith GL. Repulsion of superinfecting virions: A mechanism for rapid virus spread. Science 2010; 327:873–876 [View Article]
     [Google Scholar]
  30. Youngner JS, Furness G. One-step growth curves for vaccinia virus in cultures of monkey kidney cells. Virology 1959386–395 [View Article] [PubMed]
     [Google Scholar]
  31. McClatchey AI, Yap AS. Contact inhibition (of proliferation) redux. Curr Opin Cell Biol 2012; 24:685–694 [View Article] [PubMed]
     [Google Scholar]
  32. Klimanova EA, Tverskoi AM, Koltsova SV, Sidorenko SV, Lopina OD et al. Time- and dose dependent actions of cardiotonic steroids on transcriptome and intracellular content of Na+ and K+: a comparative analysis. Sci Rep 2017; 7:4503 [View Article]
     [Google Scholar]
  33. Mercer J, Helenius A. Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells. Science 2008; 320:531–535 [View Article] [PubMed]
     [Google Scholar]
  34. Qiu J, Gao H-Q, Li B-Y, Shen L. Proteomics investigation of protein expression changes in ouabain induced apoptosis in human umbilical vein endothelial cells. J Cell Biochem 2008; 104:1054–1064 [View Article] [PubMed]
     [Google Scholar]
  35. Cang C, Aranda K, Seo Y, Gasnier B, Ren D. TMEM175 is an organelle K+ channel regulating Lysosomal function. Cell 2015; 162:1101–1112 [View Article] [PubMed]
     [Google Scholar]
  36. Steinberg BE, Huynh KK, Brodovitch A, Jabs S, Stauber T et al. A cation counterflux supports lysosomal acidification. J Cell Biol 2010; 189:1171–1186 [View Article] [PubMed]
     [Google Scholar]
  37. Stiefel P, Schmidt FI, Dörig P, Behr P, Zambelli T et al. Cooperative vaccinia infection demonstrated at the single-cell level using FluidFM. Nano Lett 2012; 12:4219–4227 [View Article] [PubMed]
     [Google Scholar]
  38. Moss B. Poxvirus DNA replication. Cold Spring Harb Perspect Biol 2013; 5:a010199 [View Article] [PubMed]
     [Google Scholar]
  39. Mercer J, Snijder B, Sacher R, Burkard C, Bleck CKE et al. RNAi screening reveals proteasome- and Cullin3-dependent stages in vaccinia virus infection. Cell Rep 2012; 2:1036–1047 [View Article] [PubMed]
     [Google Scholar]
  40. Domi A, Beaud G. The punctate sites of accumulation of vaccinia virus early proteins are precursors of sites of viral DNA synthesis. J Gen Virol 2000; 81:1231–1235 [View Article] [PubMed]
     [Google Scholar]
  41. Novy K, Kilcher S, Omasits U, Bleck CKE, Beerli C et al. Proteotype profiling unmasks a viral signalling network essential for poxvirus assembly and transcriptional competence. Nat Microbiol 2018; 3:588–599 [View Article] [PubMed]
     [Google Scholar]
  42. Amarelle L, Katzen J, Shigemura M, Welch LC, Cajigas H et al. Cardiac glycosides decrease influenza virus replication by inhibiting cell protein translational machinery. Am J Physiol Lung Cell Mol Physiol 2019; 316:L1094–L1106 [View Article] [PubMed]
     [Google Scholar]
  43. Cohen T, Williams JD, Opperman TJ, Sanchez R, Lurain NS et al. Convallatoxin-induced reduction of methionine import effectively inhibits human cytomegalovirus infection and replication. J Virol 2016; 90:10715–10727 [View Article] [PubMed]
     [Google Scholar]
  44. Perne A, Muellner MK, Steinrueck M, Craig-Mueller N, Mayerhofer J et al. Cardiac glycosides induce cell death in human cells by inhibiting general protein synthesis. PLoS One 2009; 4:e8292 [View Article] [PubMed]
     [Google Scholar]
  45. Unger B, Traktman P. Vaccinia virus morphogenesis: A13 phosphoprotein is required for assembly of mature virions. J Virol 2004; 78:8885–8901 [View Article] [PubMed]
     [Google Scholar]
  46. Assarsson E, Greenbaum JA, Sundström M, Schaffer L, Hammond JA et al. Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes. Proc Natl Acad Sci USA 2008; 105:2140–2145 [View Article]
     [Google Scholar]
  47. Feng X, Wang C, Xu Y, Turley J, Xie Z et al. Topical digitoxigenin for wound healing: a feasibility study. Bioengineering (Basel) 2018; 5:21 [View Article] [PubMed]
     [Google Scholar]
  48. Cheung YY, Chen KC, Chen H, Seng EK, Chu JJH. Antiviral activity of lanatoside C against dengue virus infection. Antiviral Res 2014; 111:93–99 [View Article]
     [Google Scholar]
  49. Jin Y-H, Jeon S, Lee J, Kim S, Jang MS et al. Broad spectrum antiviral properties of cardiotonic steroids used as potential therapeutics for emerging coronavirus infections. Pharmaceutics 2021; 13:1839 [View Article]
     [Google Scholar]
  50. Laird GM, Eisele EE, Rabi SA, Nikolaeva D, Siliciano RF. A novel cell-based high-throughput screen for inhibitors of HIV-1 gene expression and budding identifies the cardiac glycosides. J Antimicrob Chemother 2014; 69:988–994 [View Article] [PubMed]
     [Google Scholar]
  51. Okuyama-Dobashi K, Kasai H, Tanaka T, Yamashita A, Yasumoto J et al. Hepatitis B virus efficiently infects non-adherent hepatoma cells via human sodium taurocholate cotransporting polypeptide. Sci Rep 2015; 5:17047 [View Article] [PubMed]
     [Google Scholar]
  52. Yang C-W, Lee Y-Z, Hsu H-Y, Jan J-T, Lin Y-L et al. Inhibition of SARS-CoV-2 by highly potent broad-spectrum anti-coronaviral tylophorine-based derivatives. Front Pharmacol 2020; 11:606097 [View Article] [PubMed]
     [Google Scholar]
  53. Gardner TJ, Cohen T, Redmann V, Lau Z, Felsenfeld D et al. Development of a high-content screen for the identification of inhibitors directed against the early steps of the cytomegalovirus infectious cycle. Antiviral Res 2015; 113:49–61 [View Article]
     [Google Scholar]
  54. Cho J, Lee YJ, Kim JH, Kim SI, Kim SS et al. Antiviral activity of digoxin and ouabain against SARS-CoV-2 infection and its implication for COVID-19. Sci Rep 2020; 10:16200 [View Article] [PubMed]
     [Google Scholar]
  55. Dodson AW, Taylor TJ, Knipe DM, Coen DM. Inhibitors of the sodium potassium ATPase that impair herpes simplex virus replication identified via a chemical screening approach. Virology 2007; 366:340–348 [View Article]
     [Google Scholar]
  56. Kapoor A, Cai H, Forman M, He R, Shamay M et al. Human cytomegalovirus inhibition by cardiac glycosides: evidence for involvement of the HERG gene. Antimicrob Agents Chemother 2012; 56:4891–4899 [View Article] [PubMed]
     [Google Scholar]
  57. Ashbrook AW, Lentscher AJ, Zamora PF, Silva LA, May NA et al. Antagonism of the sodium-potassium ATPase impairs chikungunya virus infection. mBio 2016; 7:e00693-16 [View Article] [PubMed]
     [Google Scholar]
  58. Barrows NJ, Campos RK, Powell ST, Prasanth KR, Schott-Lerner G et al. A screen of FDA-approved drugs for inhibitors of zika virus infection. Cell Host Microbe 2016; 20:259–270 [View Article]
     [Google Scholar]
  59. Hartley C, Hartley M, Pardoe I, Knight A. Ionic Contra-Viral Therapy (ICVT); a new approach to the treatment of DNA virus infections. Arch Virol 2006; 151:2495–2501 [View Article]
     [Google Scholar]
  60. Hoffmann H-H, Palese P, Shaw ML. Modulation of influenza virus replication by alteration of sodium ion transport and protein kinase C activity. Antiviral Res 2008; 80:124–134 [View Article]
     [Google Scholar]
  61. Kouznetsova J, Sun W, Martínez-Romero C, Tawa G, Shinn P et al. Identification of 53 compounds that block Ebola virus-like particle entry via a repurposing screen of approved drugs. Emerg Microbes Infect 2014; 3:e84 [View Article] [PubMed]
     [Google Scholar]
  62. Wong RW, Balachandran A, Ostrowski MA, Cochrane A. Digoxin suppresses HIV-1 replication by altering viral RNA processing. PLoS Pathog 2013; 9:e1003241 [View Article] [PubMed]
     [Google Scholar]
  63. Zhyvoloup A, Melamed A, Anderson I, Planas D, Lee C-H et al. Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation. PLoS Pathog 2017; 13:e1006460 [View Article] [PubMed]
     [Google Scholar]
  64. Dowall SD, Bewley K, Watson RJ, Vasan SS, Ghosh C et al. Antiviral screening of multiple compounds against ebola virus. Viruses 2016; 8:277 [View Article]
     [Google Scholar]
  65. Nagai Y, Maeno K, Iinuma M, Yoshida T, Matsumoto T. Inhibition of virus growth by ouabain: effect of ouabain on the growth of HVJ in chick embryo cells. J Virol 1972; 9:234–243 [View Article] [PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.001971
Loading
Cardiac glycosides inhibit early and late vaccinia virus protein expression
J. Gen. Virol. 105, 001971 (2024); https://doi.org/10.1099/jgv.0.001971
/content/journal/jgv/10.1099/jgv.0.001971
/content/journal/jgv/10.1099/jgv.0.001971
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error