In vitro synthesis of poliovirus

A HeLa cell-free extract has been prepared that efficiently uses full-length poliovirus-specific RNA, transcribed from plasmids with phage T7 RNA polymerase, or poliovirion RNA, for viral protein synthesis in vitro. Extensive proteolytic processing of the polyprotein in the extract produced viral enzymes that led to de novo viral RNA synthesis, and to the formation of infectious particles, as assayed on HeLa cell monolayers. The titre of plaque-forming units (p.f.u.) in the cell-free extract could be increased 70-fold when nucleoside triphosphates were added to the incubation mixture. Formation of infectious material was completely abolished if guanidine hydrochloride, an inhibitor of poliovirus RNA synthesis, but not of viral protein synthesis, was added; it was restored when the template used in the incubation was the RNA of a guanidine-resistant poliovirus mutant. Infectivity was completely inhibited by type-specific neutralizing antisera to poliovirus, and plaques were not formed if the HeLa cell monolayers were first treated with monoclonal antibodies to the poliovirus receptor. These results suggest de novo synthesis of poliovirus in a cell-free extract.