Abstract
Backgrounds
Coxsackievirus B3 (CVB3) is a member of the family Picornaviridae, and along with polio-viruses, belongs to the Enterovirus genus. The CVB3 genome is composed single-stranded RNA encoding polyproteins, which are cleaved to individual functional proteins by 2A and 3C proteases proteins which have been targeted for drug development. Here, we showed that protease activity required to activate a toxic protein may be used to prevent viral infection.
Methods
We modified the MazE-MazF antitoxin-toxin system of Escherichia coli to fuse a C-terminal fragment of MazE to the N-terminal end of toxin MazF with a linker having a specific protease cleavage site for CVB3. This fusion protein formed a stable dimer and was capable of inactivating the mRNA interferase activity of MazF which cleaves the ACA sequence in mRNA substrates.
Results
The incubation of 2A proteases with the fusion proteins induced cleavage between the MazE and MazF fragments from the fusion proteins; the subsequent release of MazF significantly inhibited virus replication. Additionally, we note that, CVB3 infected HeLa cells quickly died through a MazF toxin mediated effect before virus protein expression.
Conclusion
These findings suggest that the MazEF fusion protein has a strong potential to be developed as an anti-virus therapy following CVB3 infection.
Keywords: Coxsackievirus, MazEF, Antitoxin, Cardiotropic, Polyprotein, Proteases
Acknowledgements
Jung-Ho Park and Jin-Ho Park contributed equally to this study. This study was supported by grants from the National Research Foundation (NRF) of Korea provided by the Korean Government (No. NRF-2016R1D1A1A02937046, Lim BK), the Next-Generation BioGreen 21 Program (Project No. PJ01368601), Rural Development Administration, Republic of Korea, and the KRIBB Research Initiative Program.
Footnotes
Conflict of Interest
Jung-Ho Park, Jin-Ho Park, Wonho Choi, & Byung-Kwan Lim declares that they have no conflict of interest.
Human and animal rights
The article does not contain any studies with human and animal and this study was performed following institutional and national guidelines.
These authors contributed equally to this work.
References
- 1.Lim B K, et al. Coxsackievirus and adenovirus receptor (CAR) mediates atrioventricular-node function and connexin 45 localization in the murine heart. J Clin Invest. 2008;118:2758–2770. doi: 10.1172/JCI34777. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Baboonian C, Davies M J, Booth J C, McKenna W J. Coxsackie B viruses and human heart disease. Curr Top Microbiol Immunol. 1997;223:31–52. doi: 10.1007/978-3-642-60687-8_3. [DOI] [PubMed] [Google Scholar]
- 3.Huber S A, Lodge P A. Coxsackievirus B-3 myocarditis. Identification of different pathogenic mechanisms in DBA/2 and Balb/c mice. Am J Pathol. 1986;122:284–291. [PMC free article] [PubMed] [Google Scholar]
- 4.Feldman A M, McNamara D. Myocarditis. N Engl J Med. 2000;343:1388–1398. doi: 10.1056/NEJM200011093431908. [DOI] [PubMed] [Google Scholar]
- 5.Herskowitz A, Beisel K W, Wolfgram L J, Rose N R. Coxsackievirus B3 murine myocarditis: wide pathologic spectrum in genetically defined inbred strains. Hum Pathol. 1985;16:671–673. doi: 10.1016/S0046-8177(85)80149-0. [DOI] [PubMed] [Google Scholar]
- 6.Knowlton K U, Jeon E S, Berkley N, Wessely R, Huber S. A mutation in the puff region of VP2 attenuates the myocarditic phenotype of an infectious cDNA of the Woodruff variant of coxsackievirus B3. J Virol. 1996;70:7811–7818. doi: 10.1128/jvi.70.11.7811-7818.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Knowlton K U, Badorff C. The immune system in viral myocarditis: maintaining the balance. Circ Res. 1999;85:559–561. doi: 10.1161/01.RES.85.6.559. [DOI] [PubMed] [Google Scholar]
- 8.Martino T A, Liu P, Sole M J. Viral infection and the pathogenesis of dilated cardiomyopathy. Circ Res. 1994;74:182–188. doi: 10.1161/01.RES.74.2.182. [DOI] [PubMed] [Google Scholar]
- 9.Xiong D, et al. Inducible cardiac-restricted expression of enteroviral protease 2A is sufficient to induce dilated cardiomyopathy. Circulation. 2007;115:94–102. doi: 10.1161/CIRCULATIONAHA.106.631093. [DOI] [PubMed] [Google Scholar]
- 10.Woodruff J F. Viral myocarditis. A review. Am J Pathol. 1980;101:425–484. [PMC free article] [PubMed] [Google Scholar]
- 11.Yun S H, et al. Antiviral activity of coxsackievirus B3 3C protease inhibitor in experimental murine myocarditis. J Infect Dis. 2012;205:491–497. doi: 10.1093/infdis/jir745. [DOI] [PubMed] [Google Scholar]
- 12.Lim B K, et al. Foreign gene transfer to cardiomyocyte using a replication-defective recombinant coxsackievirus B3 without cytotoxicity. Intervirology. 2012;55:201–209. doi: 10.1159/000324541. [DOI] [PubMed] [Google Scholar]
- 13.Choi W, et al. Translation-dependent mRNA cleavage by YhaV in Escherichia coli. FEBS Letters. 2017;591:1853–1861. doi: 10.1002/1873-3468.12705. [DOI] [PubMed] [Google Scholar]
- 14.Choi W, Yoon M H, Park J H. Functional Characterization of the C-Terminus of YhaV in the Escherichia coli PrlF-YhaV Toxin-Antitoxin System. J Microbiol Biotechnol. 2018;28:987–996. doi: 10.4014/jmb.1803.03010. [DOI] [PubMed] [Google Scholar]
- 15.Zhang Y, et al. MazF cleaves cellular mRNAs specifically at ACA to block protein synthesis in Escherichia coli. Mol Cell. 2003;12:913–923. doi: 10.1016/S1097-2765(03)00402-7. [DOI] [PubMed] [Google Scholar]
- 16.Shimazu T, et al. NBK/BIK antagonizes MCL-1 and BCL-XL and activates BAK-mediated apoptosis in response to protein synthesis inhibition. Genes & Development. 2007;21:929–941. doi: 10.1101/gad.1522007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Kamada K, Hanaoka F, Burley S K. Crystal structure of the MazE/MazF complex: molecular bases of antidote-toxin recognition. Mol Cell. 2003;11:875–884. doi: 10.1016/S1097-2765(03)00097-2. [DOI] [PubMed] [Google Scholar]
- 18.Park J H, Yamaguchi Y, Inouye M. Intramolecular regulation of the sequence-specific mRNA interferase activity of MazF fused to a MazE fragment with a linker cleavable by specific proteases. Appl Environ Microbiol. 2012;78:3794–3799. doi: 10.1128/AEM.00364-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Lim B K, et al. Soluble coxsackievirus B3 3C protease inhibitor prevents cardiomyopathy in an experimental chronic myocarditis murine model. Virus Res. 2015;199:1–8. doi: 10.1016/j.virusres.2014.11.030. [DOI] [PubMed] [Google Scholar]
- 20.Xiong D, et al. Dystrophin deficiency markedly increases enterovirus-induced cardiomyopathy: a genetic predisposition to viral heart disease. Nat Med. 2002;8:872–877. doi: 10.1038/nm737. [DOI] [PubMed] [Google Scholar]
- 21.Lim B K, et al. Inhibition of Coxsackievirus-associated dystrophin cleavage prevents cardiomyopathy. J Clin Invest. 2013;123:5146–5151. doi: 10.1172/JCI66271. [DOI] [PMC free article] [PubMed] [Google Scholar]