小林 富成

Cell-free synthesis of an infectious virus is an ideal tool for elucidating the mechanism of viral replication and for development of antiviral drugs. In this chapter, the synthesis of Encephalomyocarditis virus (EMCV) from RNA and DNA in a HeLa cell extract-derived in vitro protein expression system is described. When a synthetic EMCV RNA with a hammerhead ribozyme sequence at its 5′-end is incubated with a HeLa cell extract using a dialysis system, EMCV particles are progressively synthesized. For EMCV synthesis from DNA, a plasmid harboring the full-length cDNA of EMCV with the T7 promoter/terminator unit is incubated in the HeLa cell extract supplemented with T7 RNA polymerase. © 2014 Springer Science+Business Media, LLC.

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.

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.

To study the relationship between translation and replication of encephalomyocarditisvirus (EMCV) RNA, we established a cell-free RNA replication system by employing a human cell extracts-based in vitro translation system. In this system, a cis-EMCV RNA replicon encoding the Renilla luciferase (R-luc) or GFP and the viral regulatory proteins efficiently replicated with simultaneous translation of the encoded protein. To examine how translation of the replicon RNA, but not the translated products, affected replication, a trans-EMCV RNA replicon encoding R-luc and the RNA replication elements was next constructed. The trans-replicon RNA replicated only in the presence of the regulatory proteins pre-expressed in trans. Incubation with cycloheximide, puromycin or a dominant-negative eukaryotic translation initiation factor 4A following expression of the regulatory proteins almost completely inhibited not only translation of the trans-replicon RNA but also replication of the RNA, suggesting that EMCV RNA translation promotes replication of the RNA. In conclusion, the cell-free RNA replication systems should become useful tools for the study of the viral RNA replication.

Cell-free synthesis of an infectious virus is an ideal tool for elucidating the mechanism of viral replication and for screening anti-viral drugs. In the present study, the synthesis of Encephalomyocarditis virus (EMCV) from its RNA in HeLa and 293-F cell extracts was enhanced by employing a dialysis system in combination with a ribozyme technology. Although translation and processing of the EMCV polyprotein were not accelerated greatly by the dialysis system, de novo synthesis of viral RNA was enhanced considerably by dialysis, leading to a greater than eight-fold increased titer of synthesized EMCV compared with a conventional batch system. Furthermore, a synthetic EMCV RNA with a hammerhead ribozyme sequence at its 5'-end served as an efficient template for viral synthesis in the dialysis system. Therefore, this system provides opportunities for mutational analyses of EMCV in vitro. (c) 2007 Elsevier B.V. All rights reserved.