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

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 Mg 2+ and the four nucleoside triphosphates and was insensitive to actinomycin D, α-amanitin, and rifampin. The 3 H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 × 10 6 and 1.4 × 10 6 ; they corresponded in size and hybridized to the major dsRNAs detected in the virus preparation by ethidium bromide staining. Cs 2 SO 4 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 × 10 6 and 1.4 × 10 6 . 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.