An improved cell-free system for picornavirus synthesis

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.