title: Cell-free RNA replication systems based on a human cell extracts-derived in vitro translation system with the encephalomyocarditisvirus RNA.
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.
authors.affiliation: Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, Himeji 671-2280, Japan.
authors.forename: Tominari
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Kodai
authors.initials: K
authors.lastname: Machida
authors.forename: Satoshi
authors.initials: S
authors.lastname: Mikami
authors.forename: Mamiko
authors.initials: M
authors.lastname: Masutani
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Imataka
mesh_terms: D000818:Animals; D002457:Cell Extracts / Q000494:pharmacology*; D002474:Cell-Free System; D002478:Cells, Cultured; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D004686:Encephalomyocarditis virus / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D011691:Puromycin / Q000494:pharmacology; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects* / Q000235:genetics
chemical_list: D002457:Cell Extracts; D012367:RNA, Viral; D011691:Puromycin; D003513:Cycloheximide