Synthesis of Double-Stranded RNA in a Virus-Enriched Fraction from Agaricus bisporus

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Abstract
Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from healthy sporophores. Enzyme activity was dependent upon the presence of Mg2+ and the four nucleoside triphosphates and was insensitive to actinomycin D, .alpha.-amanitin, and rifampin. The 3H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity in hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 .times. 106 and 1.4 .times. 106; they corresponded in size and hybridized to the major dsRNAs detected in the virus preparation by ethidium bromide staining. Cs2SO4 equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 .times. 106 and 1.4 .times. 106. The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs.