CHARACTERIZATION OF AN RNA-DEPENDENT RNA-POLYMERASE ACTIVITY ASSOCIATED WITH LA FRANCE ISOMETRIC VIRUS

Purified preparations of La France isometric virus (LIV), an unclassif ied, double-stranded RNA (dsRNA) virus of Agaricus bisporus, were asso ciated with an RNA-dependent RNA polymerase (RDRP) activity. RDRP acti vity cosedimented with the 36-nm isometric particles and genomic dsRNA s of LIV during rate-zonal centrifugation in sucrose density gradients , suggesting that the enzyme is a constituent of the virion, Enzyme ac tivity was maximal in the presence of all four nucleotides, a reducing agent (dithiothreitol or beta-mercaptoethanol), and Mg2+ and was resi stant to inhibitors of DNA-dependent RNA polymerases (actinomycin D, a lpha-amanitin, and rifampin). The radiolabeled enzyme reaction product s were predominantly (95%) single-stranded RNA (ssRNA) as determined b y cellulose column chromatography and ionic-strength-dependent sensiti vity to hydrolysis by RNase A. Three major size classes of ssRNA trans cripts of 0.95, 1.3, and 1.8 kb were detected by agarose gel electroph oresis, although the transcripts hybridized to all nine of the virion- associated dsRNAs. The RNA products synthesized in vitro appeared to b e of a single polarity, as they hybridized to an ssDNA corresponding t o one strand of a genomic dsRNA and not to the complementary strand, S imilarly, reverse transcription-PCR with total cellular ssRNA as a tem plate and strand-specific primers targeting a genomic dsRNA during syn thesis of cDNA suggested that only the coding strand was transcribed i n vivo. Our data indicate that the RDRP activity associated with virio ns of LIV is probably a transcriptase engaged in the synthesis of ssRN A transcripts corresponding to each of the virion-associated dsRNAs.