Characterization of an RNA-Dependent RNA Polymerase Activity Associated with La France Isometric Virus†
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JOURNAL OF VIROLOGY, Mar. 1997, p. 2264–2269 Vol. 71, No. 3 0022-538X/97/$04.0010 Copyright q 1997, American Society for Microbiology Characterization of an RNA-Dependent RNA Polymerase Activity Associated with La France Isometric Virus† MICHAEL M. GOODIN,‡ BETH SCHLAGNHAUFER, TIFFANY WEIR, AND C. PETER ROMAINE* Department of Plant Pathology, Pennsylvania State University, University Park, Pennsylvania 16802 Received 12 August 1996/Accepted 19 November 1996 Purified preparations of La France isometric virus (LIV), an unclassified, double-stranded RNA (dsRNA) virus of Agaricus bisporus, were associated with an RNA-dependent RNA polymerase (RDRP) activity. RDRP activity cosedimented with the 36-nm isometric particles and genomic dsRNAs of LIV during rate-zonal centrifugation in sucrose density gradients, suggesting that the enzyme is a constituent of the virion. Enzyme activity was maximal in the presence of all four nucleotides, a reducing agent (dithiothreitol or b-mercapto- ethanol), and Mg21 and was resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D, a-amanitin, and rifampin). The radiolabeled enzyme reaction products were predominantly (95%) single- stranded RNA (ssRNA) as determined by cellulose column chromatography and ionic-strength-dependent sensitivity to hydrolysis by RNase A. Three major size classes of ssRNA transcripts of 0.95, 1.3, and 1.8 kb were detected by agarose gel electrophoresis, although the transcripts hybridized to all nine of the virion-associated dsRNAs. The RNA products synthesized in vitro appeared to be of a single polarity, as they hybridized to an ssDNA corresponding to one strand of a genomic dsRNA and not to the complementary strand. Similarly, reverse transcription-PCR with total cellular ssRNA as a template and strand-specific primers targeting a genomic dsRNA during synthesis of cDNA suggested that only the coding strand was transcribed in vivo. Our data indicate that the RDRP activity associated with virions of LIV is probably a transcriptase engaged in the synthesis of ssRNA transcripts corresponding to each of the virion-associated dsRNAs. Sinden and Hauser (31) were the first to describe a serious dsRNA strands remains intact following transcription. In con- infectious disorder of the cultivated mushroom Agaricus trast, a semiconservative mechanism is utilized by fungal vi- bisporus, known as La France disease. Today the disease is ruses in the Partiviridae, in which one of the parental strands of considered an important limiting factor worldwide in the com- dsRNA is displaced by the nascent strand (3, 4, 22). In this mercial cultivation of mushrooms (10). The most diagnostic study, we report on the properties of an RNA-dependent RNA symptoms of the disease include the degeneration and death of polymerase (RDRP) activity with respect to requirements for the mycelium, a delayed appearance of mushrooms, the devel- catalysis, nature of the RNA products synthesized in vitro, and opment of deformed mushrooms, premature opening of the association with virions of LIV. veil, and a reduced yield. The viral nature of La France disease was proposed in 1962 MATERIALS AND METHODS by Hollings (16) following the observation of virus-like parti- Source of tissue. Healthy and diseased mushrooms were collected at commer- cles (VLPs) in extracts from diseased basidiocarps. Since then, cial sites and stored at 2808C. a wealth of correlative evidence implicating La France isomet- Purification of virus. (i) Method I. A 100-g sample of mushroom tissue was ric virus (LIV) in the etiology of the disease has accumulated homogenized in a Waring blender with 300 ml of a solution containing 50 mM (6, 10, 12, 23, 25–27, 33, 34). LIV has 36-nm isometric particles Tris-HCl, 1 mM EDTA, 0.1% b-mercaptoethanol (BME), and 0.1% Nonidet P-40, pH 8.0. The homogenate was clarified by centrifugation at 6,000 3 g for 20 that encapsidate nine double-stranded RNAs (dsRNAs) of 0.8 min. The resultant supernatant was collected and incubated for 3 to 4 weeks at to 3.8 kb (12, 33, 34). Three of the dsRNAs have been com- 48C, after which time a precipitate enriched in virus was collected by centrifu- pletely sequenced, although functions have not been ascribed gation at 3,000 3 g for 10 min. The pellet was resuspended in a minimal volume to the putatively encoded polypeptides (15). Virions of LIV are of the supernatant and stored at 2808C. (ii) Method II. More highly purified preparations of LIV were obtained by a composed of three structural polypeptides which, depending modification of the subcellular fractionation procedures described by Rogers et on the method of purification, are estimated to be 63, 66, and al. (24) and Morten and Hicks (21). Mushrooms (100 g) were homogenized in a 129 kDa (12) or 65, 115, and 120 kDa (33). Waring blender for 4 min with 200 ml of 100 mM Tris-HCl–0.2 mM EDTA, pH The complexity of the genome renders LIV distinct from 7.5 (Tris buffer), containing 15% sucrose. The homogenate was mixed with an equal volume of Tris buffer containing 1% (wt/vol) sucrose and centrifuged at other dsRNA viruses belonging to the recognized taxonomic 1,300 3 g for 10 min at 48C. The supernatant was centrifuged at 14,000 3 g for groups (22). Information on replication strategy can be used to 30 min, and the pellet was resuspended in 4 ml of Tris buffer with 20% sucrose, support the taxonomic position of viruses. For example, fungal extracted with 0.1 volume of chloroform, and centrifuged at 10,000 3 g for 10 viruses classified in the Totiviridae use a conservative mode of min. The supernatant was centrifuged at 50,000 3 g for 30 min, and the virus- enriched pellets were resuspended in 4 ml of Tris buffer with 20% sucrose. Two replication (11, 22, 30) such that the integrity of the parental milliliters of the virus preparation was layered onto a sucrose step gradient consisting of 18 ml each of 60 and 35% sucrose prepared in Tris buffer. Gradients were centrifuged at 64,000 3 g for 90 min at 48C and partitioned into 3-ml * Corresponding author. Mailing address: Department of Plant Pa- fractions (model 640 density gradient fractionator; ISCO, Lincoln, Nebr.). Each thology, 209 Buckhout Laboratory, Pennsylvania State University, fraction was analyzed for RDRP activity, dsRNA, protein, and VLPs. Polymerase assays. The standard reaction cocktail contained, in a final volume University Park, PA 16802. Phone: (814) 865-7132. Fax: (814) 863- m of 50 l, 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 5 mM dithiothreitol (DTT), 2065. E-mail: [email protected]. 40 mg of actinomycin D per ml, 60 U of RNasin (Pharmacia, Piscataway, N.J.), † Department of Plant Pathology contribution 2043. 1 mM (each) ATP, CTP, and GTP, 50 mCi of [3H]UTP (35 to 50 Ci/mmol; ICN ‡ Present address: Department of Plant Biology, University of Cal- Biomedical, Irvine, Calif.) or [a-32P]UTP (.600 Ci/mmol; ICN), and, unless ifornia, Berkeley, Berkeley, CA 94720. stated otherwise, 2 ml of virus purified by method I. In some experiments, the 2264 VOL. 71, 1997 LIV-ASSOCIATED RNA POLYMERASE 2265 FIG. 1. Analysis of extracts of healthy and diseased mushrooms for LIV by agarose gel electrophoresis of dsRNA (A) and SDS-PAGE of proteins (B). Clarified homogenates (supernatant obtained at 6,000 3 g) of healthy and diseased mushrooms were incubated for 4 weeks at 48C and then centrifuged at 3,000 3 g. The resultant supernatants and precipitates were analyzed for the LIV dsRNAs and virion-specific polypeptides. C, clarified homogenate; S, supernatant; P, precipitate. The sizes of the dsRNAs and polypeptides are indicated in the left and right margins, respectively. standard reaction cocktail was amended with 50 mgofa-amanitin per ml, 50 mg firmed by Northern hybridization to LIV-associated dsRNAs (7). Subsequently, of rifampin per ml, 2 to 10 mM BME, or 10 mM MnCl2. Typically, the enzyme strand-specific ss-cDNAs were rescued by phage R408 from E. coli XL1-Blue reaction was allowed to proceed for 1.5 h at 378C and was terminated by spotting (Stratagene) that had been transformed with the 1.7-kb cDNA subcloned in the 1 2 10 ml of the reaction cocktail onto 2.3-cm-diameter filter paper discs (3MM; same orientation at the SmaI site of pBluescript KS or KS (29). Whatman Ltd., Maidstone, England). Discs were washed batchwise five times for Northern hybridization. RNA was capillary transferred to Nytran membranes 10 min each time in cold 5% trichloroacetic acid (TCA) containing 0.02% uracil (Schleicher & Schuell, Keene, N.H.) in 103 SSC (103 SSC is 1.5 M NaCl plus and then washed twice for 5 min each in 95% ethanol. Following washing, discs 0.15 sodium citrate) from either formaldehyde-denaturing (18) or nondenaturing were air dried and heated for 20 min at 508C in capped scintillation vials (19) agarose gels. Prehybridization and hybridization were carried out at 428Cin containing 300 ml of a 90% aqueous solution of NCS tissue solubilizer (Amer- a solution containing 50% formamide, 63 SSC, 53 Denhardt’s solution (0.1% sham, Arlington Heights, Ill.). Ten milliliters of Ecoscint (National Diagnostics, Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 0.5% SDS, and Manville, N.J.) was added to each vial, and radioactivity was determined by liquid 100 mg of sheared salmon sperm DNA per ml. Blots were prehybridized for at scintillation counting on an LS 7000 instrument (Beckman, Fullerton, Calif.). least 2 h and hybridized for a minimum of 16 h with fresh hybridization solution TCA-precipitable radioactivity was determined by Cerenkov counting when containing the in vitro-synthesized [a-32P]UTP-labeled polymerase reaction [a-32P]UTP was used in polymerase assays.