Polyamine Analog Diethylnorspermidine Restricts Coxsackievirus B3 and Is Overcome by 2A Protease Mutation In Vitro
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Generation of 2AS35 Mutants
2.3. Infection and Enumeration of Viral Titers
2.4. CVB3 Serial Passage
2.5. Drug Treatments
2.6. RNA Purification and cDNA Synthesis
2.7. DENSpm and DFMO Sensitivity Assays
2.8. Stability and Competition Assays
2.9. Protease Plasmid Cloning
2.10. Transfections
2.11. Luciferase Protease Assay
2.12. Western Blots
2.13. Plaque Size Measurement
2.14. Thin Layer Chromatography Determination of Polyamines
2.15. Statistical Analysis
3. Results
3.1. DENSpm Induces Polyamine Depletion and Limits Coxsackievirus B3 INFECTION
3.2. Coxsackievirus B3 Gains Resistance to DENSpm via 2A Mutation in 2A Protease
3.3. CVB3 2AS35G Confers Resistance to DENSpm
3.4. CVB3 2AS35G Exhibits Reduced Fitness Compared to Wildtype Virus In Vitro
3.5. DENSpm Reduces 2A Protease Activity
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Strikas, R.A.; Anderson, L.J.; Parker, R.A. Temporal and Geographic Patterns of Isolates of Nonpolio Enterovirus in the United States, 1970–1983. J. Infect. Dis. 1986, 153, 346–351. [Google Scholar] [CrossRef]
- Jubelt, B.; Lipton, H.L. Chapter 18—Enterovirus/Picornavirus infections. In Handbook of Clinical Neurology; Tselis, A.C., Booss, J., Eds.; Neurovirology; Elsevier: Amsterdam, The Netherlands, 2014; Volume 123, pp. 379–416. [Google Scholar]
- Pons-Salort, M.; Parker, E.P.K.; Grassly, N.C. The Epidemiology of Non-Polio Enteroviruses: Recent Advances and Outstanding Questions. Curr. Opin. Infect. Dis. 2015, 28, 479–487. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Chapman, N. Persistent Coxsackievirus Infection: Enterovirus Persistence in Chronic Myocarditis and Dilated Cardiomyopathy. Curr. Top. Microbiol. Immunol. 2008, 323, 275–292. [Google Scholar] [PubMed]
- Chuang, Y.-Y.; Huang, Y.-C. Enteroviral Infection in Neonates. J. Microbiol. Immunol. Infect. 2019, 52, 851–857. [Google Scholar] [CrossRef]
- Garmaroudi, F.S.; Marchant, D.; Hendry, R.; Luo, H.; Yang, D.; Ye, X.; Shi, J.; McManus, B.M. Coxsackievirus B3 Replication and Pathogenesis. Future Microbiol. 2015, 10, 629–653. [Google Scholar] [CrossRef] [PubMed]
- Mounce, B.C.; Olsen, M.E.; Vignuzzi, M.; Connor, J.H. Polyamines and Their Role in Virus Infection. Microbiol. Mol. Biol. Rev. 2017, 81, e00029-17. [Google Scholar] [CrossRef] [Green Version]
- Firpo, M.R.; Mounce, B.C. Diverse Functions of Polyamines in Virus Infection. Biomolecules 2020, 10, 628. [Google Scholar] [CrossRef] [Green Version]
- Gibson, W.; Roizman, B. Compartmentalization of Spermine and Spermidine in the Herpes Simplex Virion. Proc. Natl. Acad. Sci. USA 1971, 68, 2818–2821. [Google Scholar] [CrossRef] [Green Version]
- Mounce, B.C.; Cesaro, T.; Moratorio, G.; Hooikaas, P.J.; Yakovleva, A.; Werneke, S.W.; Smith, E.C.; Poirier, E.Z.; Simon-Loriere, E.; Prot, M.; et al. Inhibition of Polyamine Biosynthesis Is a Broad-Spectrum Strategy against RNA Viruses. J. Virol. 2016, 90, 9683–9692. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Mastrodomenico, V.; Esin, J.J.; Qazi, S.; Khomutov, M.A.; Ivanov, A.V.; Mukhopadhyay, S.; Mounce, B.C. Virion-Associated Polyamines Transmit with Bunyaviruses to Maintain Infectivity and Promote Entry. ACS Infect. Dis. 2020, 6, 2490–2501. [Google Scholar] [CrossRef]
- Mounce, B.C.; Cesaro, T.; Vlajnić, L.; Vidiņa, A.; Vallet, T.; Weger-Lucarelli, J.; Passoni, G.; Stapleford, K.A.; Levraud, J.-P.; Vignuzzi, M. Chikungunya Virus Overcomes Polyamine Depletion by Mutation of NsP1 and the Opal Stop Codon To Confer Enhanced Replication and Fitness. J. Virol. 2017, 91, e00344-17. [Google Scholar] [CrossRef] [Green Version]
- Roberts, S.; Ullman, B. Parasite Polyamines as Pharmaceutical Targets. Curr. Pharm. Des. 2017, 23, 3325–3341. [Google Scholar] [CrossRef] [PubMed]
- Casero, R.A.; Stewart, T.M.; Pegg, A.E. Polyamine Metabolism and Cancer: Treatments, Challenges and Opportunities. Nat. Rev. Cancer 2018, 18, 681–695. [Google Scholar] [CrossRef]
- Fabian, C.J.; Kimler, B.F.; Brady, D.A.; Mayo, M.S.; Chang, C.H.J.; Ferraro, J.A.; Zalles, C.M.; Stanton, A.L.; Masood, S.; Grizzle, W.E.; et al. A Phase II Breast Cancer Chemoprevention Trial of Oral α-Difluoromethylornithine: Breast Tissue, Imaging, and Serum and Urine Biomarkers. Clin. Cancer Res. 2002, 8, 3105–3117. [Google Scholar] [PubMed]
- Simoneau, A.R.; Gerner, E.W.; Nagle, R.; Ziogas, A.; Fujikawa-Brooks, S.; Yerushalmi, H.; Ahlering, T.E.; Lieberman, R.; McLaren, C.E.; Anton-Culver, H.; et al. The Effect of Difluoromethylornithine on Decreasing Prostate Size and Polyamines in Men: Results of a Year-Long Phase IIb Randomized Placebo-Controlled Chemoprevention Trial. Cancer Epidemiol. Biomark. Prev. 2008, 17, 292–299. [Google Scholar] [CrossRef] [Green Version]
- Firpo, M.R.; Mastrodomenico, V.; Hawkins, G.M.; Prot, M.; Levillayer, L.; Gallagher, T.; Simon-Loriere, E.; Mounce, B.C. Targeting Polyamines Inhibits Coronavirus Infection by Reducing Cellular Attachment and Entry. ACS Infect. Dis. 2020. [Google Scholar] [CrossRef] [PubMed]
- Dial, C.N.; Tate, P.M.; Kicmal, T.M.; Mounce, B.C. Coxsackievirus B3 Responds to Polyamine Depletion via Enhancement of 2A and 3C Protease Activity. Viruses 2019, 11, 403. [Google Scholar] [CrossRef] [Green Version]
- Bergeron, R.; Weimar, W.; Luchetta, G.; Streiff, R.; Weigand, J.; Perrin, J.; Schreier, K.; Porter, C.; Yao, G.; Dimova, H. Drug Metabolism and Disposition: The Biological Fate of Chemicals: Metabolism and Pharmacokinetics of N1,N11-Diethylnorspermine. Drug Metab. Dispos. 1995, 10, 1117–1125. [Google Scholar]
- Tate, P.M.; Mastrodomenico, V.; Mounce, B.C. Ribavirin Induces Polyamine Depletion via Nucleotide Depletion to Limit Virus Replication. Cell Rep. 2019, 28, 2620–2633. [Google Scholar] [CrossRef] [PubMed]
- Kenyon, T.K.; Lynch, J.; Hay, J.; Ruyechan, W.; Grose, C. Varicella-Zoster Virus ORF47 Protein Serine Kinase: Characterization of a Cloned, Biologically Active Phosphotransferase and Two Viral Substrates, ORF62 and ORF63. J. Virol. 2001, 75, 8854–8858. [Google Scholar] [CrossRef] [Green Version]
- Mousstache, N. Polyamines Stimulate DNA-Dependent RNA Synthesis Catalyzed by Vaccinia Virus. Biochim. Biophys. Acta 1985, 826, 113–120. [Google Scholar] [CrossRef]
- Saeed, M.; Kapell, S.; Hertz, N.T.; Wu, X.; Bell, K.; Ashbrook, A.W.; Mark, M.T.; Zebroski, H.A.; Neal, M.L.; Flodström-Tullberg, M.; et al. Defining the Proteolytic Landscape during Enterovirus Infection. PLoS Pathog. 2020, 16, e1008927. [Google Scholar] [CrossRef] [PubMed]
- Marcotte, L.L.; Wass, A.B.; Gohara, D.W.; Pathak, H.B.; Arnold, J.J.; Filman, D.J.; Cameron, C.E.; Hogle, J.M. Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-Dependent RNA Polymerase. J. Virol. 2007, 81, 3583–3596. [Google Scholar] [CrossRef] [Green Version]
- Palmenberg, A.C. Proteolytic Processing of Picornaviral Polyprotein. Annu. Rev. Microbiol. 1990, 44, 603–623. [Google Scholar] [CrossRef] [PubMed]
- Petersen, J.F.; Cherney, M.M.; Liebig, H.D.; Skern, T.; Kuechler, E.; James, M.N. The Structure of the 2A Proteinase from a Common Cold Virus: A Proteinase Responsible for the Shut-off of Host-Cell Protein Synthesis. EMBO J. 1999, 18, 5463–5475. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kim, B.-K.; Ko, H.; Jeon, E.-S.; Ju, E.-S.; Jeong, L.S.; Kim, Y.-C. 2,3,4-Trihydroxybenzyl-Hydrazide Analogues as Novel Potent Coxsackievirus B3 3C Protease Inhibitors. Eur. J. Med. Chem. 2016, 120, 202–216. [Google Scholar] [CrossRef] [PubMed]
- Lim, B.-K.; Yun, S.-H.; Ju, E.-S.; Kim, B.-K.; Lee, Y.-J.; Yoo, D.-K.; Kim, Y.-C.; Jeon, E.-S. Soluble Coxsackievirus B3 3C Protease Inhibitor Prevents Cardiomyopathy in an Experimental Chronic Myocarditis Murine Model. Virus Res. 2015, 199, 1–8. [Google Scholar] [CrossRef] [Green Version]
- Cai, Q.; Yameen, M.; Liu, W.; Gao, Z.; Li, Y.; Peng, X.; Cai, Y.; Wu, C.; Zheng, Q.; Li, J.; et al. Conformational Plasticity of the 2A Proteinase from Enterovirus 71. J. Virol. 2013, 87, 7348–7356. [Google Scholar] [CrossRef] [Green Version]
- Kandolf, R.; Hofschneider, P.H. Molecular Cloning of the Genome of a Cardiotropic Coxsackie B3 Virus: Full-Length Reverse-Transcribed Recombinant CDNA Generates Infectious Virus in Mammalian Cells. Proc. Natl. Acad. Sci. USA 1985, 82, 4818–4822. [Google Scholar] [CrossRef] [Green Version]
- Schneider, C.A.; Rasband, W.S.; Eliceiri, K.W. NIH Image to ImageJ: 25 Years of Image Analysis. Nat. Methods 2012, 9, 671–675. [Google Scholar] [CrossRef]
- Madhubala, R. Thin-Layer Chromatographic Method for Assaying Polyamines. In Polyamine Protocols; Morgan, D.M.L., Ed.; Methods in Molecular BiologyTM; Humana Press: Totowa, NJ, USA, 1998; pp. 131–136. ISBN 978-1-59259-565-5. [Google Scholar]
- Desmyter, J.; Melnick, J.L.; Rawls, W.E. Defectiveness of Interferon Production and of Rubella Virus Interference in a Line of African Green Monkey Kidney Cells (Vero). J. Virol. 1968, 2, 955–961. [Google Scholar] [CrossRef] [Green Version]
- Urin, V.; Shemesh, M.; Schreiber, G. CRISPR/Cas9-Based Knockout Strategy Elucidates Components Essential for Type 1 Interferon Signaling in Human HeLa Cells. J. Mol. Biol. 2019, 431, 3324–3338. [Google Scholar] [CrossRef]
- Li, K.; Chen, Z.; Kato, N.; Gale, M.; Lemon, S.M. Distinct Poly(I-C) and Virus-Activated Signaling Pathways Leading to Interferon-β Production in Hepatocytes*. J. Biol. Chem. 2005, 280, 16739–16747. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Farhadian, S.; Shareghi, B.; Saboury, A.A. Exploring the Thermal Stability and Activity of α-Chymotrypsin in the Presence of Spermine. J. Biomol. Struct. Dyn. 2017, 35, 435–448. [Google Scholar] [CrossRef]
- Agbowuro, A.A.; Huston, W.M.; Gamble, A.B.; Tyndall, J.D.A. Proteases and Protease Inhibitors in Infectious Diseases. Med. Res. Rev. 2018, 38, 1295–1331. [Google Scholar] [CrossRef] [PubMed]
- Chang, K.-O.; Kim, Y.; Lovell, S.; Rathnayake, A.D.; Groutas, W.C. Antiviral Drug Discovery: Norovirus Proteases and Development of Inhibitors. Viruses 2019, 11, 197. [Google Scholar] [CrossRef] [Green Version]
- Field, H.J.; Goldthorpe, S.E. Antiviral Drug Resistance. Trends Pharmacol. Sci. 1989, 10, 333–337. [Google Scholar] [CrossRef]
- Goyal, L.; Supko, J.G.; Berlin, J.; Blaszkowsky, L.S.; Carpenter, A.; Heuman, D.M.; Hilderbrand, S.L.; Stuart, K.E.; Cotler, S.; Senzer, N.N.; et al. Phase 1 Study of N(1),N(11)-diethylnorspermine (DENSPM) in Patients with Advanced Hepatocellular Carcinoma. Cancer Chemother. Pharm. 2013, 72, 1305–1314. [Google Scholar] [CrossRef] [PubMed]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Hulsebosch, B.M.; Mounce, B.C. Polyamine Analog Diethylnorspermidine Restricts Coxsackievirus B3 and Is Overcome by 2A Protease Mutation In Vitro. Viruses 2021, 13, 310. https://doi.org/10.3390/v13020310
Hulsebosch BM, Mounce BC. Polyamine Analog Diethylnorspermidine Restricts Coxsackievirus B3 and Is Overcome by 2A Protease Mutation In Vitro. Viruses. 2021; 13(2):310. https://doi.org/10.3390/v13020310
Chicago/Turabian StyleHulsebosch, Bridget M., and Bryan C. Mounce. 2021. "Polyamine Analog Diethylnorspermidine Restricts Coxsackievirus B3 and Is Overcome by 2A Protease Mutation In Vitro" Viruses 13, no. 2: 310. https://doi.org/10.3390/v13020310