Cell-free RNA replication systems based on a human cell extracts-derived in vitro translation system with the encephalomyocarditisvirus RNA |
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Authors: | Kobayashi Tominari Machida Kodai Mikami Satoshi Masutani Mamiko Imataka Hiroaki |
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Affiliation: | Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, Himeji 671-2280, Japan. |
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Abstract: | 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. |
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