JOURNAL OF VIROLOGY, Mar. 1997, p. 2264–2269 Vol. 71, No. 3 0022-538X/97/$04.00ϩ0 Copyright ᭧ 1997, American Society for Microbiology

Characterization of an RNA-Dependent RNA Polymerase Activity Associated with La France Isometric †

MICHAEL M. GOODIN,‡ BETH SCHLAGNHAUFER, TIFFANY WEIR, AND C. PETER ROMAINE* Department of 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 , a reducing agent (dithiothreitol or ␤-mercapto- ,ethanol), and Mg2؉ and was resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D ␣-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 -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 Ϫ80ЊC. 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% ␤-mercaptoethanol (BME), and 0.1% Nonidet P-40, pH 8.0. The homogenate was clarified by centrifugation at 6,000 ϫ g for 20 that encapsidate nine double-stranded (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- 4ЊC, after which time a precipitate enriched in virus was collected by centrifu- pletely sequenced, although functions have not been ascribed gation at 3,000 ϫ 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 Ϫ80ЊC. (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 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 belonging to the recognized taxonomic 1,300 ϫ g for 10 min at 4ЊC. The supernatant was centrifuged at 14,000 ϫ 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 ϫ g for 10 viruses classified in the Totiviridae use a conservative mode of min. The supernatant was centrifuged at 50,000 ϫ 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 ϫ g for 90 min at 4ЊC 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- ␮ of 50 l, 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 5 mM dithiothreitol (DTT), 2065. E-mail: [email protected]. 40 ␮g 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 ␮Ci of [3H]UTP (35 to 50 Ci/mmol; ICN ‡ Present address: Department of Plant Biology, University of Cal- Biomedical, Irvine, Calif.) or [␣-32P]UTP (Ͼ600 Ci/mmol; ICN), and, unless ifornia, Berkeley, Berkeley, CA 94720. stated otherwise, 2 ␮l 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 ϫ g) of healthy and diseased mushrooms were incubated for 4 weeks at 4ЊC and then centrifuged at 3,000 ϫ 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 ␮gof␣-amanitin per ml, 50 ␮g 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 37ЊC and was terminated by spotting (Stratagene) that had been transformed with the 1.7-kb cDNA subcloned in the ϩ Ϫ 10 ␮l 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 10ϫ SSC (10ϫ 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 50ЊC in capped scintillation vials (19) agarose gels. Prehybridization and hybridization were carried out at 42ЊCin containing 300 ␮l of a 90% aqueous solution of NCS tissue solubilizer (Amer- a solution containing 50% formamide, 6ϫ SSC, 5ϫ 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 ␮g 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 [␣-32P]UTP-labeled polymerase reaction [␣-32P]UTP was used in polymerase assays. products. Following hybridization, blots were washed twice at room temperature, For sizing the polymerase reaction products, virus purified by method II was twice at 42ЊC, and twice again at room temperature. Each wash was done for 15 used as a source of enzyme, the standard reaction cocktail was amended with 0.1 min in 0.1ϫ SSC containing 0.1% SDS, except for the final wash, in which SDS mM UTP and 50 ␮Ci of [␣-32P]UTP, and reactions were carried out for 3 to 12 h was excluded. at 30ЊC. Southern hybridization. Rescued ss-cDNAs corresponding to the complemen- Extraction of RDRP reaction products. Polymerase reaction products were tary strands of the 1.7-kb cDNA to the LIV 3.8-kb dsRNA were separated by extracted with phenol (18) and resuspended in water or 10 mM Tris-HCl–1 mM electrophoresis on 1% agarose gels, denatured in 1.5 M NaCl–0.5 M NaOH, EDTA, pH 7.5 (TE). neutralized in 1 M Tris-HCl–1.5 M NaCl, pH 7.4, and capillary transferred in Electrophoresis of . RDRP reaction products were analyzed on 10ϫ SSC to Nytran membranes. The conditions for hybridization and washing formaldehyde-denaturing 1.2% agarose gels (18). LIV dsRNAs were resolved on were as described for Northern blots. either nondenaturing 1% agarose gels prepared in Tris-acetate-EDTA buffer RT-PCR. Reverse transcription (RT)-PCR with primers targeting the putative (18) or formaldehyde-denaturing agarose gels. coding strand (reverse primer) or the noncoding strand (forward primer) of the Electrophoresis of protein. Polypeptides were separated by sodium dodecyl LIV 1.3-kb dsRNA was carried out essentially as described by Romaine and sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a 7.5% stacking gel Schlagnhaufer (27). The only modification of the procedure was that the forward as described previously (12). and reverse primers were used separately to initiate synthesis of cDNA to a total Cellulose column chromatography. RDRP reactions were terminated by the cellular ssRNA fraction prepared from diseased mushrooms by the acid guani- addition of 1 volume of 2ϫ STE (1ϫ STE is 50 mM Tris, 0.1 M NaCl, and 1 mM EDTA, pH 7.0) containing 1% SDS. The reaction mixture was incubated for 10 min at 65ЊC, and nucleic acids were extracted with phenol (18) and resuspended in water or TE. The nucleic acid preparation was modified to include STE containing 35% ethanol (STE-35) by the addition of a 5ϫ STE stock and absolute ethanol and loaded onto a column containing1gofCF-11 cellulose (Whatman) equilibrated with STE-35. The column was washed with 100 ml of STE-35 to remove and unincorporated radionucleotides, then with 36 ml of STE containing 15% ethanol to release single-stranded RNA (ssRNA), and finally with 36 ml of STE to elute dsRNA. Nuclease sensitivity assays. The sensitivity of the polymerase reaction prod- ucts to hydrolysis by DNase was determined in a solution containing 10 mM Tris-HCl, 10 mM MgCl2, 2 mM CaCl2 [pH 7.5], 0.6 U of RNasin per ml, and 1 U of RNase-free DNase I (Promega, Madison, Wis.). RNase sensitivity trials were carried out in the presence of 1 ␮g of RNase A (Sigma Chemical Co., St. Louis, Mo.) per ml under high (0.3 M NaCl)-salt and low (0.03 M NaCl)-salt conditions (34). Nuclease reactions were conducted in a final volume of 100 ␮l for 30 min at 37ЊC. Synthesis of cDNA. The LIV-specific 3.8-kb dsRNA was isolated from dis- eased basidiocarps by phenol extraction and cellulose column chromatography (20, 34) and purified by agarose gel electrophoresis (18). The gel-purified dsRNA was denatured with methylmercury hydroxide (1) and converted into blunt- ended cDNA according to the method of Gubler and Hoffman (14) as described by Klickstein et al. (17), with the only modification being the omission of RNase A in the blunt-ending reaction. Blunt-ended cDNAs were ligated to SmaI- FIG. 2. Kinetics of RDRP activity in the precipitates from diseased (LIV- ϩ digested pBlueScript KS phagemid and transformed into Escherichia coli enriched) and healthy tissues. Activity is defined as the incorporation of SURE according to the manufacturer’s instructions (Stratagene, La Jolla, Calif.). [3H]UTP into TCA-precipitable product. Time course experiments were carried Putative transformants were screened for DNA inserts by the alkaline out with the standard reaction cocktail described in Materials and Methods. Data procedure (18). A 1.7-kb cDNA corresponding to the 3.8-kb dsRNA was con- points represent the means of two identical experiments. 2266 GOODIN ET AL. J. VIROL.

TABLE 1. Characteristics of the RNA polymerase activitya Relative Modification of incorporation complete medium (%) None...... 100b ϪActinomycin D ...... 109 ϩ␣-Amanitin (50 ␮g/ml)...... 88 ϩRifampin (50 ␮g/ml)...... 87 ϪMg, ϩMn (10 mM)...... Ͻ1 ϪMg...... Ͻ1 ϪATP, ϪCTP, ϪGTP ...... Ͻ1 ϪDTT ...... 14 ϪDTT, ϩBME (2 mM) ...... 42 ϪDTT, ϩBME (5 mM) ...... 62 ϪDTT, ϩBME (10 mM) ...... 84

a All reactions were carried out at 37ЊC for 90 min in 50 ␮l of a standard reaction cocktail with the addition (ϩ) or omission (Ϫ) of components as indi- cated. Values are the means of two experiments. FIG. 3. Characterization of the LIV polymerase products synthesized in vitro b Incorporation in complete medium was 43,000 cpm. by cellulose column chromatography. Phenol-extracted 3H-labeled polymerase product (53,000 cpm) was loaded onto a column containing1gofcellulose resin. The column was washed exhaustively with STE-35 to remove unincorporated nucleotides and oligonucleotides. ssRNA was eluted from the column by washing dinium-phenol method (5) and cellulose column chromatography. For subse- with STE-15, and dsRNA was eluted by washing with STE. Six 3-ml fractions quent PCR amplification, both primers were included in the reaction mixture. were collected for each eluant, and radioactivity was determined for 1 ml of each fraction by liquid scintillation counting. RESULTS

Analysis of LIV-enriched precipitates. Precipitates (3,000 ϫ precipitable product occurred, presumably due to the activity g) derived from clarified homogenates of diseased mushrooms of endogenous ribonucleases. that had been incubated for 4 weeks at 4ЊC (method I) were Requirements for catalysis. The RDRP activity was largely highly enriched in LIV. The LIV-associated dsRNAs in the insensitive to polymerase inhibitors actinomycin D, ␣-aman- range of 0.8 to 3.8 kb were detected by agarose gel electro- itin, and rifampin, suggesting that the observed activity was not catalyzed by cellular transcriptases (Table 1). Maximal poly- phoretic analysis of clarified homogenates as well as the de- ϩ merase activity required the presence of Mg2 , which could rived precipitates (Fig. 1). The lack of detectable dsRNA in the ϩ postprecipitation supernatant suggested quantitative recovery not be replaced by Mn2 , the four triphosphates, of virus from solution. No dsRNA was recovered from com- and a reducing agent, either DTT or BME. parable preparations of healthy mushrooms. Similarly, SDS- Characterization of polymerase products. The polymerase PAGE analysis revealed that precipitates from La France products synthesized in vitro by the RDRP activity were insen- disease-affected mushrooms contained primarily the three LIV-specific polypeptides of 63, 66, and 129 kDa (Fig. 1). Precipitates prepared from clarified homogenates of healthy mushrooms lacked the three polypeptides and, in fact, showed a paucity of protein. Electron microscopic examination re- vealed that the precipitates from diseased mushrooms con- tained aggregates of 36-nm isometric particles characteristic of LIV, whereas those from healthy mushrooms were devoid of VLPs but contained amorphous material (13). In vitro polymerase activity. RDRP activity was detected in LIV-enriched precipitates prepared from diseased mush- rooms, whereas no appreciable enzyme activity was detected in comparable preparations from healthy mushrooms (Fig. 2). Incorporation of radiolabeled UMP increased steadily for 3 h, after which time a decrease in the accumulation of TCA-

TABLE 2. Nuclease sensitivity of the in vitro-synthesized polymerase producta Relative radio- Treatment activity (%) FIG. 4. Northern hybridization of [␣-32P]UTP-labeled polymerase reaction Water...... 100 products to the disease-specific (D) LIV dsRNAs. dsRNA isolated from healthy DNase I...... 72 (H) mushrooms was included as a control. The dsRNAs were separated under RNase A (0.03 M NaCl) ...... 5 nondenaturing conditions and transferred to nylon membranes by capillary blot- RNase A (0.3 M NaCl) ...... 16 ting in 10ϫ SSC following incubation in a solution containing 50% formamide, 9% formaldehyde, and 40 mM Tris acetate (pH 8.6) containing 2 mM EDTA for a Aliquots of phenol-extracted radiolabeled polymerase product (5,600 cpm) 30 min at 55ЊC. Hybridization conditions were as described in Materials and were treated for 30 min at 37ЊC with water, DNase I, RNase A under low-salt Methods. Shown are the original ethidium bromide (EtBr)-stained gel (A) and conditions (0.03 M NaCl), and RNase A under high-salt conditions (0.3 M autoradiograms of the radioactive membranes exposed for 2 (B) and 24 (C) h. NaCl). Values are the means of two experiments. The sizes of the dsRNAs are indicated in the left margin. VOL. 71, 1997 LIV-ASSOCIATED RNA POLYMERASE 2267

FIG. 5. Southern hybridization of [␣-32P]UTP-labeled polymerase product to complementary strands of a 1.7-kb cDNA clone corresponding to the 3.8-kb ϩ Ϫ dsRNA. The cDNA was subcloned into the pBluescript vectors KS and KS in the same orientation with respect to the multiple- site, allowing recovery of the complementary strands by phage R408-mediated rescue. Equivalent amounts of each strand of cDNA were electrophoresed in nondenaturing aga- rose gels and visualized by ethidium bromide staining (A) and autoradiography after hybridization with the ssRNA probes (B). The size of the helper phage DNA is indicated in the left margin. The relative positions of the phage and LIV-specific are indicated in the right margin. sitive to hydrolysis by DNase I but were hydrolyzed by RNase A under high- and low-salt conditions, indicating that the prod- FIG. 7. Separation of the in vitro-synthesized transcription products by elec- ucts were ssRNA (Table 2). Moreover, approximately 95% of trophoresis on denaturing formaldehyde-agarose gels. Sucrose gradient-purified the radiolabeled products were eluted from a cellulose column virus (method II) was incubated in a standard polymerase reaction cocktail ␮ ␣ 32 Њ in the ssRNA fraction, with the remainder being eluted in the containing 0.1 mM UTP and 50 Ci of [ - P]UTP at 21 C for 12 h. Following phenol extraction and ethanol precipitation, transcripts were separated by elec- dsRNA fraction (Fig. 3). trophoresis and then transferred to Nytran membranes by capillary blotting. Northern hybridization analysis disclosed that transcripts Membranes were dried and exposed to X-ray film at Ϫ80ЊC. The sizes (in corresponding to all nine of the LIV-associated dsRNAs were kilobases) of the transcripts are indicated in the left margin. transcribed in vitro, with the hybridization signal being most intense for the 1.3-kb and 2.8- to 3.1-kb dsRNAs (Fig. 4). In contrast, the labeled transcription products failed to hybridize hybridization of the RDRP products to one strand of an to a 2.4-kb dsRNA found in healthy mushrooms. Southern ssDNA corresponding to the 3.8-kb LIV dsRNA, but not to the complementary strand, demonstrated that only transcripts of a single polarity were transcribed (Fig. 5). An experiment was carried out to determine if the single polarity transcripts synthesized in vitro by the RDRP activity reflected the nature of the products synthesized in vivo. Total cellular ssRNA from diseased mushrooms served as a template for RT-PCR in which primers directed against the putative coding or noncoding strand of the 1.3-kb dsRNA were used individually to prime RT prior to PCR amplification with both primers. The predicted PCR product of 128 bp was generated only when the reverse primer, which annealed to the putative coding strand, was used to initiate synthesis of cDNA (Fig. 6). Sizing of RNA products. Several high-molecular-weight RNA products were synthesized in vitro with sucrose gradient-puri- fied viral preparations (method II) as a source of enzyme in the RDRP reaction mixture. Three major size classes of ssRNA with estimated molecular sizes of 0.95, 1.3, and 1.8 kb, as well as higher-molecular-weight ssRNA, were observed on autora- diograms of electrophoretically separated transcripts (Fig. 7). Association of RDRP activity with LIV. Following rate-zonal centrifugation in sucrose density gradients, RDRP activity was associated with the gradient fraction containing the LIV-asso- ciated dsRNAs (fraction 4 [Fig. 8]) and the virion-specific polypeptides and 36-nm isometric VLPs (13). The correspond- ing fractions of gradients containing extracts from healthy mushrooms were devoid of RDRP activity, dsRNA, and VLPs.

FIG. 6. Verification by RT-PCR of the single-stranded nature of the LIV DISCUSSION transcripts synthesized in vivo. Total cellular RNA isolated from diseased mush- rooms was fractionated into ssRNA and dsRNA by cellulose column chroma- We have characterized an RDRP activity associated with tography. PCR primers were specific for the 1.3-kb LIV dsRNA. Either the virions of LIV, the suspected etiologic agent of La France forward or the reverse primer, which hybridized to the noncoding or the coding disease of A. bisporus. The requirements for catalysis of the strand, respectively, was used to prime cDNA synthesis. The second primer was then added to the reaction mixture for PCR amplification. The expected 128-bp LIV-associated enzyme were generally similar to those de- amplicon was generated in reactions where the reverse primer was used to prime scribed for other viral RDRPs (2, 11, 35). It should be noted cDNA synthesis. A 123-bp marker is shown. that Sriskantha et al. (32) associated an RDRP activity with 2268 GOODIN ET AL. J. VIROL.

FIG. 8. Association of the polymerase with virions of LIV following rate-zonal centrifugation in sucrose density gradients. The UV absorbance profiles of discontinuous sucrose gradients containing preparations from healthy (dashed line) or diseased (solid line) mushrooms are shown. Gradient fractions were assayed for polymerase activity (histogram in panel A) and LIV-specific dsRNAs (B). Polymerase activity cosedimented with the LIV dsRNAs (fraction 4). The sizes (in kilobases) of the dsRNAs are shown in the left margin of panel B.

35-nm isometric VLPs from La France disease-affected mush- a second family of dsRNA viruses known as the Partiviridae rooms. However, this polymerase synthesized in vitro two replicate by way of a semiconservative mechanism whereby dsRNAs that were homologous to and the same size as the nascent transcripts remain hybridized to the parental template genomic dsRNAs of 2.1 and 6.5 kb. Therefore, the viral poly- while the complementary parental strand is displaced (3, 4, 22). merases, and hence the viruses themselves (described in the Telltale of this strategy for replication is the generation of present study and by Sriskantha and coworkers), are distinct. labeled dsRNA molecules during transcription. We found that All lines of evidence support the role of LIV in the etiology of the preponderance of the labeled products synthesized in vitro La France disease (6, 10, 12, 23, 25–27, 33, 34), whereas the were ssRNA transcripts and that, in the case of at least one of biological relevance of the 35-nm virus characterized by Sris- the genomic dsRNAs, they were of a single polarity, presum- kantha et al. (32) has not been determined. ably positive strand. Moreover, RT-PCR analysis revealed that We found that RDRP activity cosedimented with 36-nm for one of the genomic dsRNAs, transcripts corresponding isometric particles of LIV during rate-zonal centrifugation in only to the positive strand were synthesized in vivo. Taken sucrose density gradients, demonstrating that the enzyme is a together, these findings seem to indicate that free negative constituent of the virion. We also established that under con- strands are not generated during replication but rather that ditions of centrifugation that were identical to those used by positive strands are synthesized and, ultimately, encapsidated Morten and Hicks (21), mitochondria sedimented to the same and converted to dsRNAs within the virion. However, our data position as LIV particles that were obtained by chloroform do not directly address the question of whether transcription extraction of preparations from diseased mushrooms (13). occurs by a conservative or semiconservative mechanism, be- Therefore, the association of LIV dsRNA with mitochondria, cause although traces of the in vitro-synthesized product were as proposed by Morten and Hicks (21), remains equivocal. eluted in the dsRNA fraction, we could not detect label in We determined that three major size classes of ssRNA tran- dsRNA molecules by nondenaturing gel electrophoresis in en- scripts with estimated molecular sizes of 0.95, 1.3, and 1.8 kb, zyme time course studies (13). We do not know whether the as well as higher-molecular-weight ssRNA, were synthesized in labeled ssRNAs obscured small amounts of dsRNA that may vitro by the virion-associated RDRP activity. Assuming that have been synthesized or whether labeled dsRNA was absent the observed hybridization signal is proportional to the level of altogether. Further, polymerase assays carried out in the pres- transcription, the predominant transcripts corresponded to the ence of RNase A did not yield sufficient precipitable labeled 1.3-kb and 2.8- to 3.1-kb dsRNAs, although transcripts of all of product to be detected by gel electrophoresis. Continued in- the dsRNAs were detected. The RDRP specifically catalyzed vestigation into the replication and genome organization of LIV RNA, because the enzyme products failed to hybridize to LIV should aid in the understanding of its taxonomy, strategy an endogenous 2.4-kb dsRNA associated with fungal vesicles for replication, and pathogenicity. from healthy mushrooms (28). We are uncertain at this time if our inability to synthesize discrete full-length transcripts of ACKNOWLEDGMENTS each of the LIV dsRNAs can be attributed to contaminating ribonucleases or to suboptimal reaction conditions. The inabil- We thank Douglas Furtek, Ross Hardison, Richard Arteca, and ity to synthesize full-length transcripts with isolated viral poly- David Schultz for technical counsel. merases has been noted elsewhere. White and Wang (35) ob- This work was supported in part by a grant from the Pennsylvania Department of Agriculture. served heterogeneity in the sizes of the transcripts synthesized by the RDRP associated with the Giardia lamblia virus. In the REFERENCES case of the RDRP activities associated with yeast L-A virus, 1. Bailey, J. M., and N. Davidson. 1976. Methylmercury as a reversible dena- full-length transcription requires a host factor or specific chem- turing agent for agarose gel electrophoresis. Anal. Biochem. 70:75–85. ical agents (8, 9). 2. Ben-Tzvi, B.-S., Y. Koltin, M. Mevarech, and A. Tamarkin. 1984. RNA Conservative transcription occurs in members of the Toti- polymerase activity in virions from Ustilago maydis. Mol. . Biol. 4:188– viridae 194. (8, 11, 22, 30) and is characterized by the accumulation 3. Buck, K. W. 1978. Semiconservative replication of double-stranded RNA by of radiolabel in ssRNA molecules while the integrity of the a virion-associated RNA polymerase. Biochem. Biophys. Res. Commun. 84: parental dsRNA template is preserved. Members belonging to 639–645. VOL. 71, 1997 LIV-ASSOCIATED RNA POLYMERASE 2269

4. Buck, K. W., M. A. Romanos, J. S. P. MacFadden, and C. J. Rawlinson. 1981. stranded RNA from virus-infected plant and fungal tissue. Phytopathology In vitro transcription of double-stranded RNA by virion-associated RNA 69:854–858. polymerases of viruses from Gaeumannomyces graminis. J. Gen. Virol. 37: 21. Morten, K. J., and R. G. T. Hicks. 1992. Changes in double-stranded RNA 215–219. profiles in Agaricus bisporus. FEMS Microbiol. Lett. 91:159–164. 5. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation 22. Murphy, F. A., C. M. Fauquet, D. H. L. Bishop, S. A. Ghabrial, A. W. Jarvis, by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Bio- G. P. Martelli, M. A. Mayo, and M. D. Summers (ed.). 1995. Virus taxonomy. chem. 162:156–159. Classification and nomenclature of viruses. Sixth report of the International 6. Dieleman-van Zaayen, A., and J. H. M. Temmink. 1968. A virus disease of Committee on Taxonomy of Viruses. Springer-Verlag, New York, N.Y. cultivated mushrooms in the Netherlands. Neth. J. Plant Pathol. 74:48–51. 23. Passmore, E. L., and R. R. Frost. 1979. The detection of virus-like particles 7. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNA in mushrooms and mushroom spawns. Phytopathol. Z. 80:85–87. restriction endonuclease fragments. Anal. Biochem. 132:6–13. 24. Rogers, H. J., K. W. Buck, and C. M. Brasier. 1987. A mitochondrial target 8. Fujimura, T., and R. B. Wickner. 1989. Reconstitution of template depen- for double-stranded RNA in diseased isolates of the that causes dent in vitro transcriptase activity of a yeast double-stranded RNA virus. Dutch elm disease. Nature (London) 329:558–560. J. Biol. Chem. 264:10872–10877. 25. Romaine, C. P., and B. Schlagnhaufer. 1989. Prevalence of double-stranded 9. Fujimura, T., and R. B. Wickner. 1988. Replicase of L-A virus-like particles RNAs in healthy and La France disease-affected basidiocarps of Agaricus of Saccharomyces cerevisiae. J. Biol. Chem. 263:454–460. bisporus. Mycologia 81:822–825. 10. Ghabrial, S. A. 1994. New developments in fungal virology. Adv. Virus Res. 26. Romaine, C. P., and B. Schlagnhaufer. 1991. Hybridization analysis of the 43:303–387. single-stranded RNA bacilliform virus associated with La France disease of 11. Ghabrial, S. A., and W. M. Havens. 1989. Conservative transcription of Agaricus bisporus. Phytopathology 81:1336–1340. Helminthosporium victoriae 190S double-stranded RNA in vitro. J. Gen. 27. Romaine, C. P., and B. Schlagnhaufer. 1995. PCR analysis of the viral Virol. 70:1025–1035. complex associated with La France disease of Agaricus bisporus. Appl. En- 12. Goodin, M. M., B. Schlagnhaufer, and C. P. Romaine. 1992. Encapsidation viron. Microbiol. 61:2322–2325. of the La France disease-specific double-stranded RNAs in 36-nm isometric 28. Romaine, C. P., B. Schlagnhaufer, and M. M. Goodin. 1994. Vesicle-asso- virus-like particles. Phytopathology 82:285–290. ciated double-stranded ribonucleic acid genetic elements in Agaricus 13. Goodin, M. M., and C. P. Romaine. Unpublished data. 14. Gubler, U., and B. J. Hoffman. 1983. A simple and very effective method for bisporus. Curr. Genet. 25:128–134. generating cDNA libraries. 25:263–269. 29. Russell, M., S. Kidd, and M. R. Kelly. 1986. An improved filamentous helper 15. Harmsen, M. C., B. Tolner, A. Kram, S. J. Go, A. deHaan, and J. G. H. phage for generating single-stranded DNA. Gene 45:333–338. Wessels. 1991. Sequences of three dsRNAs associated with La France dis- 30. Sclafani, R. A., and W. L. Fangman. 1984. Conservative replication of dou- ease of the cultivated mushroom (Agaricus bisporus). Curr. Genet. 20:137– ble-stranded RNA in Saccharomyces cerevisiae by displacement of progeny 144. single strands. Mol. Cell. Biol. 4:1618–1626. 16. Hollings, M. 1962. Viruses associated with dieback disease of cultivated 31. Sinden, J. W., and E. Hauser. 1950. Report of two new mushroom diseases. mushrooms. Nature (London) 196:962–965. Mushroom Sci. 1:96–100. 17. Klickstein, L. B., R. L. Neve, E. A. Golemis, and G. Gyuris. 1987. Conversion 32. Sriskantha, A., M. P. Wach, B. Schlagnhaufer, and C. P. Romaine. 1986. of mRNA into double-stranded cDNA, p. 5.5.2–5.5.14. In F. M. Ausubel, R. Synthesis of double-stranded RNA in a virus-enriched fraction from Agaricus Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. bisporus. J. Virol. 57:1004–1009. Struhl (ed.), Current protocols in molecular biology. Greene Publishing 33. Van der Lende, T. R. 1994. Double-stranded RNAs and proteins associated Associates-Wiley-Interscience, New York, N.Y. with the 34 nm virus particles of the cultivated mushroom Agaricus bisporus. 18. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a J. Gen. Virol. 75:2533–2536. laboratory manual, p. 363–402. Cold Spring Harbor Laboratory, Cold Spring 34. Wach, M. P., A. Srikantha, and C. P. Romaine. 1987. Double-stranded Harbor, N.Y. RNAs associated with La France disease of the cultivated mushroom. Phy- 19. Matsumoto, Y., R. Fishel, and R. B. Wickner. 1990. Circular single-stranded topathology 77:1321–1325. RNA in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 87: 35. White, T. C., and C. C. Wang. 1990. RNA-dependent RNA polymerase 7628–7632. activity associated with the double-stranded RNA virus of Giardia lamblia. 20. Morris, T. J., and J. A. Dodds. 1979. Isolation and analysis of double- Nucleic Acids Res. 18:553–559.