Abstract
Virus particles are promising vehicles and templates for vaccination, drug delivery and material sciences. Although infectious picornaviruses can be synthesized from genomic or synthetic RNA by cell-free protein expression systems derived from mammalian cell extract, there has been no direct evidence that authentic viral particles are indeed synthesized in the absence of living cells. We purified encephalomyocarditis virus (EMCV) synthesized by a HeLa cell extract-derived, cell-free protein expression system, and visualized the viral particles by transmission electron-microscopy. The in vitro-synthesized EMCV particles were indistinguishable from the in vivo-synthesized particles. Our results validate the use of the cell-free technique for the synthesis of EMCV particles.
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References
Arnold E, Luo M, Vriend G, Rossmann MG, Palmenberg AC, Parks GD, Nicklin MJ, Wimmer E (1987) Implications of the picornavirus capsid structure for polyprotein processing. Proc Natl Acad Sci USA 84:21–25
Barton DJ, O’Donnell BJ, Flanegan JB (2001) 5′ cloverleaf in poliovirus RNA is a cis-acting replication element required for negative-strand synthesis. EMBO J 20:1439–1448
Herold J, Andino R (2000) Poliovirus requires a precise 5′ end for efficient positive-strand RNA synthesis. J Virol 74:6394–6400
Kitai Y, Fukuda H, Enomoto T, Asakawa Y, Suzuki T, Inouye S, Handa H (2011) Cell selective targeting of a simian virus 40 virus-like particle conjugated to epidermal growth factor. J Biotechnol 155:251–256
Kobayashi T, Mikami S, Yokoyama S, Imataka H (2007) An improved cell-free system for picornavirus synthesis. J Virol Methods 142:182–188
Kobayashi T, Machida K, Mikami S, Masutani M, Imataka H (2011) Cell-free RNA replication systems based on a human cell extracts-derived in vitro translation system with the encephalomyocarditisvirus RNA. J Biochem 150:423–430
Kobayashi T, Nakamura Y, Mikami S, Masutani M, Machida K, Imataka H (2012) Synthesis of encephalomyocarditis virus in a cell-free system: from DNA to RNA virus in one tube. Biotechnol Lett 34:67–73
Ludwig C, Wagner R (2007) Virus-like particles-universal molecular toolboxes. Curr Opin Biotechnol 18:537–545
Machida K, Masutani M, Kobayashi T, Mikami S, Nishino Y, Miyazawa A, Imataka H (2012) Reconstitution of the human chaperonin CCT by co-expression of the eight distinct subunits in mammalian cells. Protein Expr Purif 82:61–69
Mikami S, Kobayashi T, Yokoyama S, Imataka H (2006a) A hybridoma-based in vitro translation system that efficiently synthesizes glycoproteins. J Biotechnol 127:65–78
Mikami S, Masutani M, Sonenberg N, Yokoyama S, Imataka H (2006b) An efficient mammalian cell-free translation system supplemented with translation factors. Protein Expr Purif 46:348–357
Mikami S, Kobayashi T, Machida K, Masutani M, Yokoyama S, Imataka H (2010) N-terminally truncated GADD34 proteins are convenient translation enhancers in a human cell-derived in vitro protein synthesis system. Biotechnol Lett 32:897–902
Palmenberg AC (1990) Proteolytic processing of picornaviral polyprotein. Annu Rev Microbiol 44:603–623
Pestova TV, Hellen CU, Shatsky IN (1996) Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry. Mol Cell Biol 16:6859–6869
Strable E, Finn MG (2009) Chemical modification of viruses and virus-like particles. Curr Top Microbiol Immunol 327:1–21
Svitkin YV, Sonenberg N (2003) Cell-free synthesis of encephalomyocarditis virus. J Virol 77:6551–6555
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “RNA regulation” (20112006) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) to HI and a Grant-in-Aid for Scientific Research (C) (22560774) from MEXT to HI.
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Kobayashi, T., Yukigai, J., Ueda, K. et al. Purification and visualization of encephalomyocarditisvirus synthesized by an in vitro protein expression system derived from mammalian cell extract. Biotechnol Lett 35, 309–314 (2013). https://doi.org/10.1007/s10529-012-1086-1
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DOI: https://doi.org/10.1007/s10529-012-1086-1