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Synthesis of encephalomyocarditis virus in a cell-free system: from DNA to RNA virus in one tube

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Abstract

Virus particles are used in vaccination, drug delivery, and material sciences. Here we devised a system where the RNA virus encephalomyocarditis virus (EMCV) is synthesized from DNA templates in vitro. When a plasmid or a PCR product harboring the full-length cDNA of EMCV in the T7 promoter/terminator unit was incubated in a HeLa cell extract supplemented with T7 RNA polymerase, EMCV was produced within 4 h at an efficiency of over 10-fold compared with the system programmed with the EMCV RNA. The EMCV RNA transcribed by the virally encoded RNA-dependent RNA polymerase was predominantly incorporated into the EMCV particle even in the presence of a larger amount of the EMCV RNA transcribed by T7 RNA polymerase from the plasmid.

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Acknowledgments

This study 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 H.I. and a Grant-in-Aid for Scientific Research (C) (22560774) from MEXT to H.I.

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Authors and Affiliations

  1. Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, Himeji, 671-2280, Japan

    Tominari Kobayashi, Yusuke Nakamura, Mamiko Masutani, Kodai Machida & Hiroaki Imataka

  2. RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan

    Satoshi Mikami, Mamiko Masutani & Hiroaki Imataka

  3. Molecular Nanotechnology Research Center, Graduate School of Engineering, University of Hyogo, Himeji, 671-2280, Japan

    Kodai Machida & Hiroaki Imataka

Authors
  1. Tominari Kobayashi
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  2. Yusuke Nakamura
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  3. Satoshi Mikami
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  4. Mamiko Masutani
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  5. Kodai Machida
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  6. Hiroaki Imataka
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Corresponding author

Correspondence to Hiroaki Imataka.

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Kobayashi, T., Nakamura, Y., Mikami, S. et al. Synthesis of encephalomyocarditis virus in a cell-free system: from DNA to RNA virus in one tube. Biotechnol Lett 34, 67–73 (2012). https://doi.org/10.1007/s10529-011-0744-z

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  • DOI: https://doi.org/10.1007/s10529-011-0744-z

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