Double-Stranded RNA Virus in the Human PathogenicFungus Blastomyces dermatitidis
SHIGERU KOHNO,1t TSUTOMU FUJIMURA,2 SHEN RULONG,3 AND K. J. KWON-CHUNGl*Clinical Mycology Section, Laboratory of Clinical Investigation, National Institute ofAllergy and Infectious Diseases,'
and Section of Genetics on Simple Eukaryotes, Laboratory of Biochemical Pharmacology, National Institute ofDiabetes and Digestive and Kidney Diseases,2 Bethesda, and Membrane Biology Section, Laboratory
of Mathematical Biology, National Cancer Institute-Frederick Cancer Research Center, Frederickl3 Maryland
Received 20 April 1994/Accepted 1 August 1994
Double-stranded RNA viruses were detected in a strain of Blastomyces demaitidis isolated from a patient inUganda. The viral particles are spherical (mostly 44 to 50 nm in diameter) and consist of about 25%double-stranded RNA (5 kb) and 75% protein (90 kDa). The virus contains transcriptional RNA polymeraseactivity; it synthesized single-stranded RNA in vitro in a conservative manner. The newly synthesizedsingle-stranded RNA was a full-length strand, and the rate of chain elongation was approximately 170nucleotides per min. The virus-containing strain shows no morphological difference from virus-free strains inthe mycelial phase. Although the association with the presence of the virus is unclear, the virus-infected strainconverts to the yeast form at 37°C, but the yeast cells fail to multiply at that temperature.
The occurrence of virus has been reported in over 60 speciesfrom more than 50 genera of true fungi. The majority of thesefungi are plant pathogens and saprophytes, and only a fewspecies are known to cause disease in humans (21, 34). Inhuman pathogens, however, proof of the occurrence of viralinfection has been tenuous at best. Reports have been basedon the observation of virus-like particles on electron micros-copy (1) or on the production of killer toxin (15, 16, 24).Virus-like particles were isolated from a strain of Aspergillusflavus, but biophysical characterization failed to detect anynucleic acid (35).
Blastomyces dermatitidis is a dimorphic fungus that causesblastomycosis, which is most prevalent in the southeasternUnited States and the Ohio-Mississippi river valleys (20). Thedisease is also known to be endemic to wide geographic areasof Africa. The African isolates of B. dermatitidis are antigeni-cally different from American isolates (17), and in tissue, theyeast cells of North American strains are mostly sphericalwhile those of African isolates tend to be more ovoid (20). Theconversion to the yeast form in vitro is much more readilyachieved with North American than with African isolates (20).African isolates and American isolates fail to produce teleo-morphs (the Ajellomyces state) upon sexual cross (19). Al-though this trend may be associated with the host rather thanthe fungus, Segretain noted that patients with African blasto-mycosis tend to develop more subcutaneous abscesses andfewer ulcerated cutaneous lesions than those with the NorthAmerican disease (26).
During our comparative study of rDNA restriction fragmentlength polymorphism between seven African isolates and fiveAmerican isolates of B. dermatitidis, we detected double-stranded (ds) RNA virus in one of the African isolates. To ourknowledge, this is the first report of the purification andcharacterization of a virus from fungi pathogenic to humansand animals.
Detection of a dsRNA in B. dermatitidis. A total of 13
t Present address: Second Department of Internal Medicine, Na-gasaki University Medical School, Nagasaki, Japan.
isolates, 7 from Africa, 5 from North America, and 1 fromIndia, were used in the study. The isolates either were obtainedfrom the American Type Culture Collection or were from theculture collection of our laboratory. The African isolates were6066 (South Africa), 6071 (Uganda), ATCC 48089 (Zaire),ATCC 56214 (Mozambique), ATCC 56217 (South Africa),ATCC 56218 (Algeria), and ATCC 56220 (Angola). The fiveAmerican isolates were ATCC 18187 (Wisconsin), ATCC14112 (?), 6059 (?), B-3227 (Tennessee), and B3228 (Arkan-sas). One isolate from India, ATCC 48938, was from the lungof a bat. Nucleic acids were extracted from mycelial cultures(28) grown in YEPD (1% yeast extract, 2% peptone, 2%glucose) broth at 30°C on a shaker for 10 days and electropho-resed in 1% agarose gel. Three African isolates, 6071, ATCC56217, and ATCC 56220, showed extrachromosomal bands of5 kb or less on an ethidium bromide-stained gel (not shown).No such bands were observed for other isolates. The largestand most prominent band (5 kb) was in strain 6071, which wasisolated from a patient in Uganda (not shown). To determinethe nature of the band, an aliquot of the nucleic acid samplefrom 6071 was electrophoresed with or without RNase andDNase treatment. The band disappeared with RNase treat-ment while it remained in the sample treated with DNase,suggesting that the band is RNA. To test whether the RNA issingle stranded (ss) or ds, the nucleic acid sample from strain6071 was treated with pancreatic RNase A under high and lowsalt concentrations. The RNA band persisted in the high-saltcondition even if the RNase concentration was 1 mg/ml. Underthe low-salt condition, however, the band disappeared within30 min (not shown) with addition of 0.1 mg of RNase per ml(not shown). Resistance of pancreatic RNase in a high-saltcondition is characteristic of dsRNA.
Purification and characterization of the viral particles. Wesuspected that strain 6071 might be infected with a dsRNAvirus, as there is a wide range of reports on virus-infected fungi(21). The viral particles (VPs) were isolated as describedpreviously (11) with modifications. Mycelial mats of 6071harvested by filtering 10-day-old broth cultures through aWhatman filter (no. 1) were washed with buffer A (1 Msorbitol, 0.1 M Tris-Cl [pH 7.6], 1 mM EDTA), resuspended inthe same buffer with 48 mM 2-mercaptoethanol, and incubated
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for 15 min at 37°C. The mycelia were then treated with amixture of Zymolyase-20T (1.7 mg/ml; ICN Biochemicals Inc.,Costa Mesa, Calif.) and mureinase (3.3 mg/ml; United StatesBiochemical Corp., Cleveland, Ohio) for 2 h at 30°C (5). Theenzyme-treated sample was centrifuged for 5 min at 4,500 x g(4°C), and the pellet was suspended in an equal amount ofbuffer B (50 mM Tris-Cl [pH 7.6], 150 mM NaCl, 10 mMMgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl flu-oride) and then vortexed with an equal volume (usually5 ml)of sterile glass beads (0.45 mm) for 2 min at 4°C. The solutionwas then centrifuged for 1 min at 4,500 x g, 4°C, and thesupernatant collected was centrifuged for15 min at 12,000 x g,4°C. After the cell debris was removed, the VPs were pelletedby centrifugation for 2 h at 110,000 x g. The pellet wassuspended in buffer B and centrifuged at 13,000x g for 15 min.Cesium chloride was added to the supernatant (1.35 g/ml) andcentrifuged for 20 h at 160,000 x g. The samples werefractionated (0.8 ml) in 14 tubes, and all fractions weredialyzed for 3 h against buffer C (50 mM Tris-Cl [pH 7.6], 250mM NaCl, 5 mM EDTA [pH 8.0]) containing 20% (vol/vol)glycerol. The dsRNA content of each fraction was examined byelectrophoresis in an agarose gel stained with ethidium bro-mide. To detect viral protein, each fraction was dissolved inelectrophoresis loading buffer (1% sodium dodecyl sulfate[SDS], 10% glycerol, 10 mM Tris [pH 7.6], 0.005% bromophe-nol blue, 1% 2-mercaptoethanol) and boiled for 3 min. Elec-trophoresis was carried out in a 7.5% acrylamide gel with 0.1%SDS, and protein was stained with Coomassie brilliant blue. Inthis test for the presence of dsRNA as well as proteins, fraction5, with a density of 1,425 g/cm3, was the only fraction thatshowed the 5-kb RNA band on an ethidium bromide-stainedgel (Fig. 1A) and two major protein bands on an SDS-polyacrylamide gel electrophoresis (PAGE) gel (Fig. 1C). Theprotein bands were 90 and 81 kDa with similar densities. In arepeat experiment, however, the 90-kDa band was clearlydenser than the 81-kDa band. This suggests that the 81-kDaprotein is a degradation product of the 90-kDa protein.Fraction 5 was also the only fraction that produced a singlepeak of RNA polymerase activity, as shown in Fig. 1B (seebelow). A pellet obtained from fraction 5 by ultracentrifuga-tion was subjected to electron microscopic study. The pelletcontained numerous spherical particles, mostly 44 to 50 nm indiameter (Fig. 2). The particles consisted of a shell surround-ing an electron-dense core, which is typical of mycovirusesfound in various filamentous fungi and yeasts (4).
Composition of VPs. The content of dsRNA was estimatedaccording to the following equation (22): F = Bn/Bv [(Bv -Bp)/(Bn - Bp)], where Bn = the density of RNA in CsCl (1.9g/ml) (27), Bp = the density in CsCl of VP protein (emptycapsid, 1.31 g/ml) (25), and Bv = the density of VPs in CsCl(1.425 g/ml). The VPs from strain 6071, therefore, wereestimated to have 25% dsRNA and 75% protein by weight. Ifwe assume that the virions contain one 5-kb dsRNA perparticle, the molecular masses of the dsRNA and protein perparticle can be calculated as 3.5 x 106 and 10.5 x 106 Da,respectively. From the latter value, it can be estimated that thevirions contain about 118 molecules of the 90-kDa coat proteinper particle (assuming that the 81-kDa protein is a degradationproduct of the 90-kDa protein). These features are quitesimilar to that of Saccharomyces cerevisiae L-A viruses. TheL-A virus contains one 4.6-kb dsRNA and about 120 copies ofthe major coat protein (76 kDa) per particle (7). The L-Avirion structure is icosahedral, with T = 1, and a dimer of themajor coat protein forms the asymmetric unit as deduced fromcryoelectron micrography and image reconstruction (27a).Sucrose gradient centrifugation revealed that the sedimenta-
A
Bottom
B
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2 3 4 5 6 7 8 9 10 11 12 13 14Fraction No
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Fraction No
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1 2 3 4 5 6 7 8 9 10 11 12 13 14Fraction No.
FIG. 1. CsCl gradient fractions showing VPs from isolate 6071. (A)RNA was extracted from each fraction of the CsCl gradient (total, 14fractions), separated in an agarose gel, and stained with ethidiumbromide. Fraction 1 corresponds to the bottom of the gradient. Onlyfraction 5 contained an RNA band of 5 kb. (B) RNA polymeraseactivity in the same fractions. (C) Coomassie brilliant blue-stainedSDS-PAGE gel of the protein showing two major bands of 90 and 81kDa, most markedly in fraction 5.
tion coefficient of the VPs isolated from fraction 5 was 180S asopposed to 160S for the L-A virus. From their sedimentationcoefficient (180S) and the diameter of the virus (44 to 50 nm),one can calculate (22) that the virions have a mass of about 15X 106 Da, in good agreement with the value obtained above.Therefore, the virus from B. dernatitidis consists of one 5-kbdsRNA and about 120 copies of the coat protein per particle.RNA polymerase activity of the VPs. After CsCl equilibrium
density gradient centrifugation, aliquots of each fraction wereincubated with an RNA polymerase reaction mixture and thetrichloroacetic acid-precipitated reaction products were mea-sured. Three microliters of VP samples was used for an RNA
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1 2 1 2 1 2
FIG. 2. Electron micrograph of the VPs isolated from CsCl gradi-ent fraction 5 showing spherical particles with a shell surrounding anelectron-dense core (bar = 100 nm). After being washed with phos-phate-buffered saline (pH 7.0) and fixed with 1.5% gluteraldehyde, thepellet on Formvar and carbon-coated copper grids was negativelystained with either 2% uranyl acetate or 2% phosphotungstate (pH7.0) (14). Micrographs were taken at a magnification of x70,000 on aPhilips 410 electron microscope.
polymerase assay. The RNA polymerase reaction mixture (33)contained 50 mM Tris-Cl (pH 7.6); 5 MM MgCl2; 0.1 mMMEDTA; 20 mM NaCl; 5 mM KCI; 10 mM 2-mercaptoethanol;40 mg of bentonite per ml (9); 0.5 mM (each) ATP, CTP, andGTP; and 20~z.M [ct_32p]UTP (New England Nuclear, Boston,Mass.). The reaction mixture (25 LIi) was incubated for 1.5 h at30'C. A peak of RNA polymerase activity was found in fraction5, which contained VPs (Fig. iB). The phenol-extracted RNApolymerase product of the VPs was ss as judged by itssensitivity to pancreatic RNase in a high-salt condition (Fig.3). Furthermore, time course and pulse-chase experimentsshowed that, with increasing incubation time, the ssRNAproducts grew in length, reaching full length by 30 min, but thatthe dsRNA template was not labeled during the experiments(not shown). These results indicate that the ssRNA is tran-scribed in a conservative manner. We measured the length ofssRNA transcripts in glyoxal denaturing gels (23). The in vitrotranscripts comigrated with genomic RNA labeled with[32p]pCp and T4 RNA ligase, indicating full length (notshown). Thus, the average rate of chain elongation in ssRNAsynthesis in vitro calculated from these data is about 170nucleotides per min. To test whether the ssRNA synthesized invitro is homologous to the dsRNA of VPs, two Northern(RNA) experiments were performed according to the previous
1 2 3 4 5
FIG. 3. Effect of RNase treatment on the RNA synthesized in vitroby the VPs from 6071 . 32p-labeled RNA products were made in vitroby the VPs and treated with RNase A under high- or low-saltconditions (11). After treatment, the products were phenol extractedand analyzed in an agarose gel. An autoradiogram of the gel is shown.Lanes 1, no RNase; 2, 20 p.g of RNase per ml under a high-saltcondition; 3, 4 pRg of RNase per ml under a high-salt condition; 4, 20p.g of RNase per ml under a low-salt condition; 5, 4 p.g of RNase perml under a low-salt condition.
4wl: .
A B CFIG. 4. dsRNA of the VPs from isolate 6071 (lanes 1) and a
mixture of the L-A dsRNA and the positive-strand ssRNA (lanes 2)were separated in an agarose gel. Ethidium bromide staining of the gelis shown in panel A. After electrophoresis, the RNAs were denaturedin the gel, blotted onto a nylon membrane, and hybridized with either32P-labeled polymerase products of the VPs from 6071 (B) or 32p_labeled L-A positive-strand-specific probe (C). Hybridization wasdetected by autoradiography. Panel A contains HindIll-digestedlambda DNA marker in the first lane.
method (10) with minor modifications; the dsRNA frompurified VPs was separated in an agarose gel (1.8%) anddenatured by incubating the gel in 5% formamide-9% form-aldehyde-10 mM MOPS (morpholinepropanesulfonic acid)(pH 7.0)- 1 mM EDTA for 30 min at 55°C. The gel was thenwashed for 20 min in 18x SSC (lx SSC is 0.15 M NaCl plus0.015 M sodium citrate) (pH 7.0), and the RNAs weretransferred onto a Nytran membrane (Schleicher & Schuell,Keene, N.H.). The 32P-labeled ssRNA of the RNA polymeraseproduct as described above was used as a probe. We alsoincluded the L-A dsRNA and the plus-strand ssRNA as acontrol. The L-A plus-strand-specific probe was made by T3RNA polymerase with PvuII-cut pLM1 (12). The blottedRNAs were hybridized with either 32P-labeled in vitro tran-scripts from the VPs or the L-A plus-strand-specific probe.Following hybridization, the membrane was washed twice with6x SSPE (lx SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1mM EDTA [pH 7.7])-0.2% SDS for 15 min and once with 1xSSPE-0.2% SDS at room temperature. The final wash waswith the same buffer for 1 h at 55°C. As shown in Fig. 4, the5-kb dsRNA from purified VPs hybridized only with the invitro transcripts and not with the L-A probe. On the otherhand, the L-A specific probe detected denatured dsRNA of theL-A as well as the L-A plus-strand ssRNA but not the 5-kbdsRNA. These results confirmed that the ssRNA was synthe-sized by the VPs. When the same radiolabeled in vitrotranscripts were used as a probe for hybridization with theRNAs extracted from the original 13 isolates, the probehybridized only with the 5-kb dsRNA from strain 6071 and notwith the smaller bands (<2 kb) found in isolates 56217 and56220 (not shown). This result indicates that the 5-kb dsRNAin the cell extract is identical to the one found in the purifiedVPs (Fig. 1A) and that the 5-kb dsRNA from strain 6071 hasno homology with the smaller bands found in the other twostrains.Dimorphism in strain 6071. Mycelia of isolate 6071 grown
on a YEPD agar slant for 10 days were transferred to brainheart infusion (BHI) agar, blood glucose-cysteine agar, andcotton seed agar (20) slants and incubated at 37°C with orwithout 10% CO2 for up to 4 weeks. The isolates from NorthAmerica and India converted from mycelial to yeast form onBHI agar and blood glucose-cysteine agar at 37°C within 3weeks. The isolates from Africa, however, generally convertedpoorly to the yeast form even at 4 weeks of incubation. Strain
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6071 showed partial conversion by the fourth week on BHIagar but not on blood glucose-cysteine agar or on cotton seedagar. Incubation in 10% CO2 did not enhance the conversion.The yeast-like cells obtained from the BHI agar culture of 6071showed many single oblong to dumbbell-shaped cells with noevidence of bud separation (not shown). The partially con-verted yeast-like cells from a BHI agar slant were transferredinto BHI broth and incubated on a gyratory shaker (200 rpm)for 4 weeks at 37°C with no evidence of growth.A previous report by Adler suggested that some isolates of
Histoplasma capsulatum also contain virus-like particles (1).The spherical particles assumed to be VPs extracted from theisolates of H. capsulatum ranged from 40 to 130 nm. Theparticles were in a higher concentration in the yeast formcompared with the mycelial form of the same isolate. Althoughthe particles were referred to as a dsRNA virus, molecular andchemical characterization of the particles was not attempted.Our demonstration of the dsRNA virus in B. dermatitidis is thefirst such case among the fungi pathogenic to humans.The VPs of B. dermatitidis 6071 consist of 5-kb dsRNA and
90-kDa protein with a ratio of 1:3. The VPs have an RNA-dependent RNA polymerase activity and, as expected, lackreverse transcriptase activity (data not shown). The density ofthe VPs on CsCl gradient is 1.425 g/ml, and the sucrosegradient sedimentation coefficiency is 180S. The B. dermatitidisVPs are mostly between 44 and 50 nm, but some rare particlesreached 90 nm in diameter with a morphology typical of that ofthe spherical VPs isolated from various fungi belonging to thedivisions Deuteromycota and Ascomycota (32). The sphericalVPs isolated from fungi are mostly isometric, and their sizesrange from 25 to 40 nm (4). The physicochemical properties ofthe VPs from 6071 are similar to those of L-A viral particlesisolated from killer strains of S. cerevisiae. The L-A VPs are 40nm in size, having 4.6-kb dsRNA (23.3%) and a 76-kDa capsidprotein with CsCl gradient density of 1.41 g/ml and a 160Ssedimentation coefficiency (7, 11). L-A VPs synthesize plus-strand ssRNA, and the strands are extruded from the particles.Some of the plus-strand (message-strand) ssRNA moleculesare encapsulated by the coat protein, so that a particle withplus-strand ssRNA is formed. The minus strand is then syn-thesized in the particle by using the plus strand as a template(11). In the case of B. dermatitidis, the product of in vitrosynthesis was a full-length ssRNA, although we do not knowwhether it is a plus or a minus strand. The manner of RNAtranscription in the VPs of B. dermatitidis is conservative as isthe case with L-A VPs.
In general, fungal viruses are known to be avirulent or latentand cause no obvious phenotypic changes in the host fungi (4,21). However, some fungal viruses cause phenotypic changesdetrimental or beneficial to the survival of the host. The virusinfecting Agaricus bisporus impairs mycelial growth and se-verely affects the production of mushrooms (30). The diseasedmushrooms often manifest abnormal cap morphology orstripes (30). Some viruses cause lytic plaque formation in fungisuch as Penicillium chrysogenum or Schizophyllum commune (3,18). Virus-infected strains of Cryphonectria parasitica, a funguscausing chestnut blight, or the plant pathogenic fungus Hel-minthosporium victoriae become hypovirulent (13, 29) while avirus was found to be associated with hypervirulence in strainsof Rhizoctonia solani (8). The killer phenomenon due to thepresence of dsRNA is well known in S. cerevisiae (2, 6, 31, 34)and in Ustilago maydis (27).The virus-infected isolate of B. dermatitidis did not show any
detectable phenotypic differences compared with uninfectedisolates of African origin. The conversion to the yeast form invitro was more difficult in African strains than in North
American strains. The mycelia of the virus-infected strain,6071, partially converted to the yeast form, but the yeast cellsfailed to multiply by continuous budding. It was, however, notpossible to determine whether the characteristic is due to viralinfection since a virus-cured subpopulation from 6071 was notavailable.
We thank R. B. Wickner and J. E. Bennett for encouragement andsupport.
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