In vitro Synthesis of Viral RNA by the Poliovirus RNA Polymerase

Abstract

Infection of cultured mammalian cells by either Mengovirus or poliovirus causes the appearance of a new cytoplasmic RNA polymerase. The enzymes induced by the two viruses are quite similar. Both show a requirement for magnesium ions, are inhibited by manganese ions, require all four nucleoside triphosphates for maximal activity, and catalyze the incorporation of each of the nucleoside monophosphates into RNA. The two polymerases are found in particulate cytoplasmic structures and neither is sensitive to actinomycin, a compound which inhibits all normal RNA synthesis. A new cytoplasmic, virus-specific polymerase activity was originally sought because autoradiographic experiments had shown viral RNA synthesis to be an actinomycin-insensitive, cytoplasmic process. Since the polymerase activity which was found appeared in the cytoplasm of infected cells with approximately the kinetics of viral RNA synthesis and was insensitive to actinomycin, it was presumed to mediate viral RNA synthesis in the cytoplasm without the involvement of DNA. The present communication describes experiments which demonstrate that the bulk of the product of the in vitro reaction is poliovirus RNA and that a small proportion is double-stranded RNA. Experiments on the structural localization of the polymerase within the cytoplasm are also presented, and the mechanism of the reaction is discussed.