Characterization of Magnaporthe oryzae Chrysovirus 1 StructuralProteins and Their Expression in Saccharomyces cerevisiae

Syunichi Urayama,a Tomoko Ohta,a Nobuya Onozuka,a Hirofumi Sakoda,a Toshiyuki Fukuhara,a Tsutomu Arie,b Tohru Teraoka,b andHiromitsu Moriyamaa

Laboratories of Molecular and Cellular Biologya and Plant Pathology,b Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo,Japan

Magnaporthe oryzae chrysovirus 1 (MoCV1), which is associated with an impaired growth phenotype of its host fungus, harborsfour major proteins: P130 (130 kDa), P70 (70 kDa), P65 (65 kDa), and P58 (58 kDa). N-terminal sequence analysis of each pro-tein revealed that P130 was encoded by double-stranded RNA1 (dsRNA1) (open reading frame 1 [ORF1] 1,127 amino acids [aa]),P70 by dsRNA4 (ORF4; 812 aa), and P58 by dsRNA3 (ORF3; 799 aa), although the molecular masses of P58 and P70 were signifi-cantly smaller than those deduced for ORF3 and ORF4, respectively. P65 was a degraded form of P70. Full-size proteins of ORF3(84 kDa) and ORF4 (85 kDa) were produced in Escherichia coli. Antisera against these recombinant proteins detected full-sizeproteins encoded by ORF3 and ORF4 in mycelia cultured for 9, 15, and 28 days, and the antisera also detected smaller degradedproteins, namely, P58, P70, and P65, in mycelia cultured for 28 days. These full-size proteins and P58 and P70 were also compo-nents of viral particles, indicating that MoCV1 particles might have at least two forms during vegetative growth of the host fun-gus. Expression of the ORF4 protein in Saccharomyces cerevisiae resulted in cytological changes, with a large central vacuole as-sociated with these growth defects. MoCV1 has five dsRNA segments, as do two Fusarium graminearum viruses (FgV-ch9 andFgV2), and forms a separate clade with FgV-ch9, FgV2, Aspergillus mycovirus 1816 (AsV1816), and Agaricus bisporus virus 1(AbV1) in the Chrysoviridae family on the basis of their RdRp protein sequences.

Mycoviruses are widespread in filamentous fungi and yeast (7,9, 33). Whereas most mycoviruses are associated with latent

infection, some affect the phenotypes of their fungal hosts. Inplant-pathogenic fungi, several mycoviruses reduce the virulenceof their hosts, causing morphological and physiological changes.For example, hypovirus infection of the chestnut blight fungusCryphonectria parasitica results in persistent and stable pheno-typic changes: reduced pigmentation, suppressed asexual sporu-lation, and loss of female fertility and hypovirulence (23). In 2004,Hillman et al. also reported the characterization of a reovirus fromC. parasitica. The virus substantially reduces the virulence of thefungus and results in dramatically altered colony morphologywith respect to the isogenic virus-free strain (14). In white root rotfungus, three taxonomically different mycoviruses have been re-ported: Mycoreovirus 3 (MYRV3-RnW370) (17), Rosellinia neca-trix partitivirus 1 W8 (RnPV1-W8) (24) and Rosellinia necatrixmegabirnavirus 1 (RnMBV1) (2). Except for RnPV1, these virusesare a hypovirulence factor in Rosellinia necatrix.

Magnaporthe grisea (formerly known as Magnaporthe oryzae;anamorph, Pyricularia oryzae) is a filamentous heterothallic asco-mycete that causes rice blast disease. Like other plant-pathogenicfungi, the presence of some double-stranded RNAs (dsRNAs) orvirus-like particles in Magnaporthe grisea has been reported (4, 15,35). Two viruses in the family Totiviridae were found in M. grisea:M. oryzae virus 1 and 2 (MoV1 and MoV2); their complete se-quences have been determined (21, 36). Magnaporthe oryzaechrysovirus 1 (MoCV1) is also the mycovirus found in the Viet-namese isolate of M. grisea (19), which impairs growth of host cells(29). Besides, macroscopic and microscopic phenotypic altera-tions were induced by the virus infection and purified virus par-ticles. Phylogenetic analysis of the putative RNA-dependent RNApolymerase (RdRp) of MoCV1 showed that MoCV1 forms a sisterclade with chrysoviruses such as Penicillium chrysogenum virus

(PcV) (16), Helminthosporium victoriae 145S virus (Hv145SV) (6),and Cryphonectria nitschkei BS122 strain (CnV1-BS122) (18) al-though the apparent molecular mass of the MoCV1 coat protein(P58) is smaller than that of the PcV coat protein (109 kDa) (16).The MoCV1 virions are isometric particles about 35 nm in diam-eter, with buoyant densities in CsCl ranging from 1.37 to 1.40 gcm�3, and each dsRNA segment is packaged separately. Thesetraits are similar to PcV, the type species of the genus Chrysovirus.However, the 5= untranslated region (UTR) of each MoCV1dsRNA genome lacks the CAA repeats that are conserved in the 5=UTR of dsRNA genomes of typical chrysoviruses and tobamovi-ruses (16). It is noteworthy that MoCV1 particles were detectablenot only in mycelial cells but also in cell-free cultured superna-tants (29).

Recently, two novel mycoviruses, Fusarium graminearum my-covirus-China 9 (FgV-ch9) and Fusarium graminearum virus 2(FgV2), which are closely related, were, respectively, found in Fus-arium graminearum strain China 9 (5) and strain 98-8-60 (37).These two mycoviruses have five dsRNA segments; the proteinsencoded by dsRNA1 possess motifs that are conserved in RdRp,and the viruses form a clade with Aspergillus mycovirus 1816(AsV1816) (13), Agaricus bisporus virus 1 (AbV1) (30), andMoCV1 (29).

In this paper, we report the characterization of MoCV1 viral

Received 8 April 2012 Accepted 15 May 2012

Published ahead of print 23 May 2012

Address correspondence to Hiromitsu Moriyama, hmori714@cc.tuat.ac.jp.

Supplemental material for this article may be found at http://jvi.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.00871-12

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proteins. Processing of MoCV1 structural proteins occurs afterthe expression of full-size viral proteins. The fifth dsRNA segmentof MoCV1, which we were not able to separate by agarose gelelectrophoresis in earlier experiments but could separate byPAGE, was sequenced in this study. We used a yeast (Saccharomy-ces cerevisiae) expression system to identify gene products ofMoCV1 involved in cytological damage to host cells; the openreading frame 4 (ORF4) protein of MoCV1 caused cytologicalchanges and growth inhibition in yeast. The data presented heresuggest that MoCV1 products may have roles in regulating fungalgrowth and development.

MATERIALS AND METHODSFungal strains and culture methods. The MoCV1-infected strainS-0412-II 1a (the original isolate) of Magnaporthe grisea and an MoCV1-free strain S-0412-II 1a (MoCV1-cured isolate) were grown on potatodextrose agar (PDA) for 2 weeks at 25°C. For liquid cultures, mycelialplugs were inoculated in 0.5% yeast extract and 2% glucose liquid broth(YG broth) with reciprocal shaking (60 strokes per min [spm]) at 25°C ina flask. Fermentation reactors (2.5 liters; Able Biott) were also used forfungal cultivation in YG broth (1.5 liters) at 25°C, with agitation at 100rpm and air introduced at 1.5 liters min�1.

Yeast strains and media. Saccharomyces cerevisiae strain W303-1A(MAT� ura3 his3 leu2 trp1 ade2 can1 [L-A]) was used in this study and wasa gift of A. Toh-e. We isolated an L-A virus-free strain of W303-1A byscreening about 200 colonies to identify spontaneous L-A-free colonies.We confirmed that the L-A-free W303-1A strain can maintain L-A virusby cytoduction experiments (32); no MAK (maintenance of killer) geneswere damaged, suggesting that the two strains are isogenic except for thepresence of L-A virus. Standard yeast media were used (25). Transforma-tion was by a variant of the lithium acetate method (11).

cDNA cloning. Detection and purification of dsRNAs from mycelia orpurified MoCV1 particles followed previously reported procedures (1,29). Purified dsRNA segments were separated by 5% PAGE and stainedwith ethidium bromide. The part of the gel containing the dsRNA5 bandwas excised. Purified dsRNA5 was used as the template for cDNA synthe-sis, and a series of overlapping cDNA clones was obtained. These cDNAclones were confirmed to be derived from the fifth dsRNA segment ofisolate S-0412-II 1a by Northern hybridization (data not shown). To ob-tain PCR clones that corresponded to the terminal regions of dsRNA5, 5=rapid amplification of cDNA ends (RACE) was used; the 5=-end regions ofthe dsRNA were amplified by inverse PCR using 5= phosphorylated prim-ers following the manufacturer’s protocol (5=-Full RACE core set; TaKaRaBio, Kyoto, Japan).

Phylogenetic analysis. Molecular phylogenetic analysis using the de-duced amino acid sequence of the putative RdRp gene of MoCV1 dsRNA1was carried out using ClustalX, GeneDoc, and MEGA4 software (27, 28).A bootstrap test was conducted with 100 resamplings for a neighbor-joining (NJ) tree.

Protein sequence analysis. For sequencing of internal peptides ofMoCV1 P130, gradient-purified MoCV1 proteins were separated by 8%SDS-PAGE and stained with Coomassie Brilliant Blue (Bio-Safe CBB;Bio-Rad). The part of the gel containing the P130 band was excised anddigested with lysyl endopeptidase at 37°C for 17 h; the digestion productswere separated by reverse-phase high-performance liquid chromatogra-phy (HPLC) on an analytical C18 column. One of the resolved peptideswas selected for amino acid sequencing by automated Edman degradation(LC-10Avp system; Shimadzu, Kyoto, Japan). For sequencing the N-ter-minal peptides of MoCV1 P70 and P58, purified MoCV1 proteins wereseparated by 8% SDS-PAGE and blotted to polyvinylidene difluoride(PVDF) membrane. After CBB staining, these proteins were cut out andsequenced by automated Edman degradation.

MALDI-TOF-MS analysis. MoCV1 protein (2 �g) was desalted usingZipTip C4 pipette tips (Millipore) according to the protocol provided by

the supplier. After a sample droplet of protein was added to the matrix-assisted laser desorption ionization (MALDI) plate and air dried, the ma-trix (10 mg ml�1 sinapinic acid in 0.1% trifluoroacetic acid [TFA] and50% MeCN solution) was also dropped on and air dried. The preparedplate was introduced into an Axima Performance mass spectrometer(MS) equipped with a 337.1-nm nitrogen laser (Shimadzu). MALDI-timeof flight (TOF)-MS was carried out in linear mode and operated in posi-tive-ion mode. The accelerating voltage applied was 20 kV. Spectra wereacquired by accumulation of 100 single laser shots.

Preparation of MoCV1 particles. For production of antiserum, ap-proximately 100 g of fresh mycelia was homogenized in a mixer with 10volumes of 0.1 M sodium phosphate buffer (pH 7.4) containing 0.2 M KCl(buffer A) at 4°C. The homogenate was centrifuged at 5,100 � g for 10min, and the supernatant was centrifuged at 34,700 � g for 1 h. Thesupernatant was ultracentrifuged at 148,400 � g for 1 h, and the resultantprecipitate was suspended in buffer A at 4°C. The suspension was appliedto sucrose density gradients (100 to 400 mg ml�1) in buffer A and centri-fuged at 112,700 � g for 2.5 h. The virus-containing fractions were com-bined and diluted with buffer A. The solution was ultracentrifuged(148,400 � g for 1 h), and the pellets were resuspended in the same buffer(29). Proteins of the purified viral preparation were analyzed by 8% SDS-PAGE with 25 mM Tris-glycine and 0.1% SDS at 15 mA for 2 h. Afterelectrophoresis, the gels were stained with Coomassie Brilliant Blue (Bio-Safe CBB; Bio-Rad). In order to obtain anti-MoCV1 antiserum, rabbitswere immunized with 0.75 mg of the gradient-purified MoCV1 proteins.

Purification of the virus particles associated with full-size ORF3 andORF4 was performed in the same manner, except that centrifugation at5,100 � g for 10 min and 34,700 � g for 1 h replaced centrifugation at22,000 � g for 15 min.

Plasmids. For expression vectors in Escherichia coli, pET-22b(�)-ORF3 was prepared by cloning ORF3 without a stop codon from a full-length cDNA clone of the MoCV1 genome into the NdeI/XhoI sites ofpET-22b(�) (Novagen, Madison, WI). pET-22b(�)-ORF4 was con-structed in the same manner by cloning ORF4 into the NdeI/NotI sites.The NdeI/XhoI sites were added to the ORF3 PCR product by amplifyingit using primers MO3N5 and MO3Xh3, and NdeI/NotI sites were addedto the ORF4 product by amplifying it using primers MO4N5 and MO4N3.pColdI-ORF3 or pColdI-ORF4 were prepared by cloning ORF3 or ORF4into the NdeI/XbaI or NdeI/EcoRI sites of pColdI (TaKaRa Bio), respec-tively. NdeI/XbaI sites were added to the ORF3 PCR product by amplify-ing it using oligonucleotides MO3N5 and MO3Xb3, and NdeI/EcoRI siteswere added to the ORF4 PCR product by amplifying it using oligonucle-otides MO4N5 and MO4E3 (see Tables S1 and S2 in the supplementalmaterial).

Plasmids pRSA315 and pRSA316 were constructed by insertion of theADH1 promoter and terminator cassette (31) into pRS315 and pRS316,respectively (26). Plasmids pRSA315-ORF3 and pRSA316-ORF4 wereconstructed by insertion of, respectively, ORF3 with added SalI and XbaIsites (using primers MO3S5 and MO3Xb3) and ORF4 with added KpnI/HpaI sites (using primers MO4K5 and MO4H3). A strong TDH3 pro-moter and terminator cassette was constructed in pUC19 (22). PCR prod-ucts obtained using TDH3p5 and TDH3p3 or TDH3t5 and TDH3t3 werecloned into pUC19. The cloned PCR products were digested by BlnI andBamHI and ligated. Plasmids pRST315, pRST316, and pRST425 wereconstructed by insertion of a NotI/ApaI fragment of a TDH3 cassette intopRS315, pRS316, and pRS425, respectively (3). pRST426 was constructedby the same method, except that XhoI was used in place of ApaI.pRST315-ORF3 or pRST425-ORF3 was prepared by cloning ORF3 intothe SalI/XbaI sites of pRST315 or the SalI/BlnI sites of pRST425, respec-tively. pRST316-ORF4 or pRST426-ORF4 was prepared by cloning ORF4into the EcoRI/HpaI sites of pRST316 or pRST426, respectively (see Ta-bles S1 and S3 in the supplemental material).

Purification of recombinant ORF3 and ORF4 in E. coli. E. coli hostBL21(DE3) harboring the ORF3 expression vector pET-22b(�)-ORF3 orthe ORF4 expression vector pET-22b(�)-ORF4 was cultured in Luria-

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Bertani medium containing 50 �g ml�1 ampicillin until mid-log phaseand then induced by 1 mM isopropyl-�-D-thiogalactopyranoside (IPTG)for 3 h. After the cells were collected by centrifugation, they were dis-rupted by BugBuster Protein Extraction Reagent (Novagen). Inclusionbodies were collected by centrifugation for 20 min at 20,000 � g andsolubilized in binding buffer (10 mM Tris, 1 mM EDTA, pH 8.0) with 6 Murea. After centrifugation for 20 min at 20,000 � g, the solutions wereapplied to a Ni-nitrilotriacetic acid (NTA) agarose column (Qiagen,Hilden, Germany). After the column was washed with 50 mM sodiumphosphate buffer (pH 8.0) and 300 mM NaCl, the proteins were eluted byimidazole (250 mM).

The expression of recombinant ORF3 or ORF4 was induced in thepColdI-ORF3 or pColdI-ORF4 transformants, respectively, according tothe pColdI vector manufacturer’s instructions (TaKaRa Bio). After thecells were collected by centrifugation, purification of the recombinantproteins was performed in the same manner.

Detection of MoCV1 viral proteins. A fungal mat of rice blast funguscultured for 9, 15, or 28 days was collected and pulverized in a mortar withpestle in liquid nitrogen. The resultant powder was added to 0.1 M sodiumphosphate buffer (pH 7.4) containing protease inhibitor cocktail (com-plete mini EDTA-free; Roche Diagnostics, Mannheim, Germany) andcentrifuged for 10 min at 20,000 � g at 4°C; the supernatant fraction wasretained. Each extract contained about 2 �g �l�1 protein, and 10 �l ofeach one was subjected to SDS-PAGE.

S. cerevisiae strain W303-1A (MATa leu2 his3 ura3 trp1 ade2 can1[L-A-o]) was used for the following experiments. Transformants ofW303-1A (L-A-o) harboring the appropriate expression vector werestreaked to form single colonies and grown at 30°C on plates containingsynthetic complete solid medium lacking either uracil or leucine (SC-Uraor SC-Leu, respectively) for 4 to 5 days. Some of the single colonies weregrown in the selective liquid medium at 28°C for 14 h. The transformantswere suspended in 0.1 M sodium phosphate buffer (pH 7.4) containingprotease inhibitor cocktail and lysed by sonication; then extracts werecentrifuged in the same manner as the fungal powder; these extracts alsocontained about 2 �g �l�1 protein, and 10 �l of each one was subjected toSDS-PAGE.

Samples were separated by 8% SDS-PAGE using 25 mM Tris-glycineand 0.1% SDS at 15 mA for 2 h, transferred to PVDF membranes, andsubjected to Western blotting using an ECL Plus detection system (GEHealthcare). To enhance signals in immunoassays, we used Can Get Sig-nal (Toyobo, Osaka, Japan) during immunoreactions. Rabbit antiserumraised against a synthetic peptide corresponding to the C-terminal 14amino acids (aa) of the protein encoded by ORF3 and guinea pig antise-rum raised against recombinant ORF3 or ORF4 were used to detectMoCV1 viral proteins.

Light microscopy and DAPI nuclear staining of yeast cells. S. cerevi-siae strain W303-1A (L-A-o) was transformed with pRSA315-ORF3,pRST315-ORF3, pRST425-ORF3, pRSA316-ORF4, pRST316-ORF4, orpRST426-ORF4. The transformants were streaked to form single coloniesand grown at 30 or 37°C on plates containing SC-Ura/Leu solid mediumfor 3 days. Some of the single colonies were suspended in water. Then, cellswere examined at �1,000 magnification with a light microscope (Olym-pus IX71; Tokyo, Japan) and differential interference contrast (DIC) op-tics. The cells were rinsed with 1 ml of 50 mM PBS (2.0% [wt/vol] poly-vinylpyrrolidone and 1.0% [wt/vol] bovine serum albumin, pH 7.4), andthen fixed in methanol for 10 min at room temperature. After three rinseswith PBS, the mycelia were placed on a glass slide, stained with 1 ml of4=,6-diamidino-2-phenylindole (1.0 mg/ml DAPI in water; Wako, Osaka,Japan) for 10 min, rinsed three times with PBS, covered with a coverslip,and examined with a light microscope equipped with a fluorescence exci-tation filter (360 to 370 nm) and emission filter (470 to 495 nm).

Nucleotide sequence accession number. The sequence of theMoCV1 dsRNA5 was deposited in the GenBank under accession num-ber AB700631.

RESULTSNucleotide sequence of dsRNA5 associated with MoCV1 parti-cles. In our previous experiments, four separated dsRNA bandswere visible following agarose gel electrophoresis (29); however, afifth dsRNA (dsRNA5) band was found by PAGE in this study(Fig. 1A). dsRNAs purified from MoCV1 particles were electro-phoresed in 5% (wt/vol) polyacrylamide gels, and the dsRNA5molecule was collected from the gels and used as a template forcDNA synthesis. The complete nucleotide sequence of thedsRNA5 was determined (AB700631); it has a single ORF (Fig.1B). The untranslated sequences conserved among the other four

FIG 1 Migration patterns of five dsRNA genomes derived from purifiedMoCV1. (A) dsRNA was extracted and electrophoresed in 5% (wt/vol) poly-acrylamide gels. (B) Genome organization of MoCV1. (C) Phylogenetic anal-ysis of the RdRp sequences of MoCV1 and 35 selected viruses of the Chryso-viridae, Totiviridae, and Partitiviridae families. An unrooted phylogenetic treebased on the neighbor-joining method was created using the MEGA4 program(27). Virus abbreviations: MoCV1, Magnaporthe oryzae chrysovirus 1;AsV1816, Aspergillus mycovirus 1816; AbV1, Agaricus bisporus virus 1; PcV,Penicillium chrysogenum virus; Hv145SV, Helminthosporium victoriae 145S vi-rus; ACD-DV, Amasya cherry disease-associated chrysovirus; AaV1, Alternariaalternata mycovirus 1; AsV341, Aspergillus mycovirus 341; FoCV1, Fusariumoxysporum chrysovirus 1; LRV1-1, Leishmania RNA virus 1-1; LRV2-1, Leish-mania RNA virus 2-1; ScVL-A, Saccharomyces cerevisiae virus L-A; ScVL-BC,Saccharomyces cerevisiae virus L-BC; Hv190SV, Helminthosporium victoriae vi-rus 190S; SsRV1, Sphaeropsis sapinea RNA virus 1; SsRV2, Sphaeropsis sapineaRNA virus 2; GaRV-L1, Gremmeniella abietina RNA virus L1; AhPV, Atkinso-nella hypoxylon partitivirus; HmPV-V1-1, Helicobasidium mompa partitivirusV1-1; RsV717, Rhizoctonia solani virus 717; FUPO-1, Fusarium poae virus 1;EbRV, Eimeria brunetti RNA virus 1; TVV1, Trichomonas vaginalis virus 1;UmVH1, Ustilago maydis virus H1; HRV-C, Human rotavirus (group C/strainBristol); HRV-B, Human rotavirus B strain Bang373; AoV, Aspergillus ochra-ceous virus; DdV2, Discula destructiva virus 2; DdV1, Discula destructiva virus 1;CHV-EP713, Cryphonectria hypovirus 1-EP713; CHV-NB58, Cryphonectriahypovirus 2-NB58; CnV1-BS122, Cryphonectria nitschkei BS122 strain; TcV2,Tolypocladium cylindrosporum virus 2; FgV2, Fusarium graminearum virus 2;FgV-ch9, Fusarium graminearum mycovirus-China 9.

Structural Analysis of Mycoviral Proteins of MoCV1

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dsRNAs (dsRNA1, -2, -3, and -4) of MoCV1 were also found indsRNA5 (see Fig. S1 in the supplemental material). While thesequence of dsRNA3 (3,074 nucleotides [nt]) was longer than thatof dsRNA4 (3,043 nt), dsRNA3 showed higher mobility thandsRNA4 on a 5% native PAGE gel (Fig. 1A and B). On the basis ofRdRp protein sequences, MoCV1 forms a separate clade withFgV-ch9, FgV2, AsV1816, and AbV1 in the Chrysoviridae family(Fig. 1C). The FgV-ch9 and FgV2 genomes are composed of fivedsRNA segments.

MoCV1 particles consist of four major structural proteinsencoded by dsRNA1, dsRNA3, and dsRNA4. Coomassie bluestaining of SDS-PAGE gels showed that a purified fraction ofMoCV1 particles extracted from mycelia cultured for 4 weeksconsisted of four major polypeptides, P130, P70, P65, and P58(Fig. 2A). P130 was subjected to Edman degradation; however, nophenylthiohydantoin (PTH) amino acid derivatives were ob-served, indicating that the N terminus of P130 was blocked (datanot shown). Purified P130 was digested with trypsin, and the in-ternal peptides were analyzed by HPLC. A tryptic digested peptidein chromatographic peaks was subjected to Edman degradation;the sequence of the peptide (TMIDYYRQVG) matched the aminoacid sequence of ORF1 at amino acids [aa] 1,025 to 1,034 (nt 3,191

to 3,220). The ORF has 1,127 aa residues, leading to a calculatedmolecular mass of 125 kDa, consistent with the size of P130 esti-mated by SDS-PAGE (Fig. 2A). P70 was also subjected to Edmandegradation. The sequence of the peptide (RIDQG) matched theamino acid sequence of ORF4 positioned at aa 15 to 19 (nt 204 to218). The first methionine of ORF4 was positioned at nt 162 to164, indicating that 14 amino acids of the N terminus of P70would be degraded by posttranslational cleavage. P65 was con-firmed to be a derivative of P70 by Western blotting using anti-ORF4 antiserum (see below).

P58 was also subjected to Edman degradation. The sequence ofthe peptide (GLTLD) matched the amino acid sequence of ORF3 ataa 2 to 6 (nt 148 to 162). An N-terminal methionine was not detectedin the P58 fragments, suggesting that the first methionine might havebeen removed posttranslationally. In addition to the main sequence,we also detected a minor peptide sequence (RIDQG) that matchedthe amino acid sequence of ORF4 at aa 15 to 19 (nt 204 to 218),indicating that the degraded P70 migrates at around 58 kDa. Thesedata indicated that P130 is encoded by dsRNA1, P70 and P65 areencoded by dsRNA4, and P58 is encoded by dsRNA3.

P58 lacks the C-terminal region of the deduced full-sizeORF3. Although the N-terminal sequence of P58 was consistent

FIG 2 Characterization of P58 of MoCV1 particles. (A) SDS-PAGE analysis of the purified MoCV1 particles stained with CBB. MoCV1 particles were purifiedfrom 28-day mycelia cultured in a flask. Arrows indicate viral structural proteins. (B) The deduced amino acid sequence of ORF3 is written in one-letter code.Partial amino acid sequences of P58 digested by trypsin were determined by peptide mass fingerprinting analysis and are boxed. The N-terminal amino acidsequence of P58 is indicated by underlining. The deduced amino acid sequence of ORF3, indicated by dashed underlining (residues 786 to 799) is the sequenceof the synthetic peptide antigen. (C) MALDI-TOF mass spectrum for purified MoCV1 particles. (D and E) MoCV1 particles, purified from 28-day flask-culturedmycelia of strain S-0412-II 1a, were separated by SDS-PAGE and immunoblotted. Antiserum raised against purified MoCV1 particles (D) or against synthesizedORF3 C-terminal peptide (shown in dashed line of panel A) (E) was used for immunoblotting. Lanes 1, MoCV1 particles; lanes 2, control preparation purifiedfrom 28-day cultures of MoCV1-free S-0412-II 1a.

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with the sequence of ORF3 encoded by dsRNA3, its apparent mo-lecular mass (58 kDa) was smaller than the deduced molecularmass of ORF3 (799 aa; 84 kDa). Then, we performed MALDI-TOFpeptide mass fingerprinting analysis of P58. Mass spectrometry oftryptic peptides of P58 identified peptides specific to the deducedamino acid sequence of ORF3, but no peptide sequence mappedto the C terminus of the deduced amino acid sequence of ORF3(M565 to L799) (Fig. 2B). MALDI-TOF MS was performed todetermine the molecular mass of P58. A resultant ion signal at m/z62,559 was observed (Fig. 2C). The molecular mass of the deducedamino acid sequence from residues G1 to V590 was 62,530 andfrom residues G1 to Q591 was 62,658. These results suggested thatthe deduced C-terminal end of P58 might be located around res-idues M565 to Q591 of ORF3. An ion signal at m/z 31,307 corre-sponding to a divalent ion was also observed.

We carried out Western analysis using anti-MoCV1 antiserumto detect full-size ORF3 protein. Signals of full-size ORF3 (84kDa) were not detected in purified MoCV1 preparations (Fig. 2D,lane 1) from mycelia cultured for 28 days in the absence of aprotease inhibitor. No specific signal was detected from prepara-tions (Fig. 2D, lane 2) of the MoCV1-free isogenic strain. Rabbitantiserum, which was raised against a synthesized peptide corre-sponding to residues S785 to L798 (CSSDGASGGSRGEEL) of thefull-size ORF3, did not detect P58 protein or the full-size ORF3 ofMoCV1 particles prepared from an MoCV1-infected strain (Fig.2E, lane 1). These data were consistent with the data of MALDITOF-based peptide mass fingerprinting (Fig. 2B), indicating thatP58 of MoCV1 particles is the C-terminal truncated form of thefull-size ORF3.

Expression of ORF3 and ORF4 proteins in Escherichia coli.MoCV1 ORFs were heterologously expressed in E. coli. Expressionof ORF3 in E. coli using pET-22b(�)-ORF3 or pColdI-ORF3yielded protein products of 84 kDa, which is consistent with thepredicted molecular mass of ORF3 and was confirmed by Westernanalysis with anti-MoCV1 antiserum (see Fig. S2 in the supple-mental material). Expression of the ORF4 protein in E. coli usingpET-22b(�)-ORF4 or pColdI-ORF4 yielded protein products of85 kDa, which was consistent with the predicted molecular massof ORF4 and was confirmed by Western analysis with anti-MoCV1 antiserum (see Fig. S2 in the supplemental material). Weused the ORF3 and ORF4 recombinant proteins to immunize

guinea pigs, and the resultant antisera were tested for specific re-activity with MoCV1 structural proteins by immunoblotting.ORF1 and ORF2 were also cloned into pET-22b(�) and pColdIvectors, but no protein product was detected. We also confirmedthat recombinant ORF3 and ORF4 were detected by anti-His6

antibody. Anti-ORF3 C-terminal peptide antiserum detected therecombinant ORF3. No signal was detected in cell lysates harbor-ing empty vectors using anti-MoCV1 antiserum (data notshown).

Detection of full-size ORF3 and ORF4 proteins in mycelialcrude extracts. We performed Western analysis to detect MoCV1viral proteins in crude mycelial extracts. P58 was detected by anti-MoCV1 antiserum in extracts of mycelia cultured for 28 days (seeFig. S3, lane 6, in the supplemental material) but was hardly de-tectable in mycelia cultured for 9 or 15 days (see Fig. S3, lanes 2and 4).

Using anti-ORF3 antiserum, a protein band corresponding tothe full-size ORF3 (84 kDa) was detected in the 9-, 15-, and 28-dayextracts (Fig. 3A, lanes 2, 4, and 6). P58 was also detected in 28-dayextracts to the same extent as the full-size 84-kDa ORF3 (Fig. 3A,lane 6), but a relatively small amount of P58 was detected in 9- and15-day extracts (Fig. 3A, lanes 2 and 4). Full-size ORF3 was alsodetected in mycelial extracts using anti-ORF3 C-terminal antisera,but no signal for P58 was detected (Fig. 3B, lanes 2, 4, and 6),indicating that P58 was generated through processing involvingC-terminal cleavage of the full-size ORF3. As the culture durationincreased, the amount of the P58 was gradually accumulated inthe mycelial extracts, while the full-size ORF3 (84 kDa) was re-duced.

The ORF4 protein is also associated with MoCV1 virus parti-cles. Using anti-ORF4 antiserum, full-size ORF4 was detected inmycelial extracts (Fig. 3C, lanes 2, 4, and 6). P70 and P65 were alsodetected in 28-day extracts (Fig. 3C, lane 6), but only very smallamounts of them were detected in 9- and 15-day extracts (Fig. 3C,lanes 2 and 4). No signal was detected in mycelial extracts pre-pared from the S-0412-II 1a MoCV1-free strain by any of the threeantisera (Fig. 3A, B, and C, lanes 1, 3, and 5).

Isolation of MoCV1 particles associated with full-size ORF3and ORF4. In mycelia cultured for 28 days, full-size ORF3 andORF4 proteins were detected in crude extracts (Fig. 3A, B, andC) but not in purified MoCV1 particles (Fig. 2D and E). To

FIG 3 Detection of ORF3 and ORF4 products from crude mycelial extracts. Aqueous crude extracts of mycelia of MoCV1-infected or MoCV1-free S-0412-II 1awere subjected to SDS-PAGE and immunoblot analysis. A 20-�g aliquot of protein from each extract was used. Antiserum raised against recombinant full-sizeORF3 (A), ORF3 C-terminal peptide (B), or recombinant full-size ORF4 (C) was used for immunoblotting. Lanes 1, extracts from MoCV1-free isolate culturedfor 9 days; lanes 2, extracts from MoCV1-infected isolate cultured for 9 days; lanes 3, extracts from MoCV1-free isolate cultured for 15 days; lanes 4, extracts fromMoCV1-infected isolate cultured for 15 days; lanes 5, extracts from MoCV1-free isolate cultured for 28 days; lanes 6, extracts from MoCV1-infected isolatecultured for 28 days.

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investigate whether the full-size proteins are components ofMoCV1 particles, we attempted to purify the particles fromfresh mycelia grown in a fermentation reactor. We also im-proved our purification methods for MoCV1 particles (see Ma-terials and Methods).

Isometric virus particles with buoyant densities of 1.38 to 1.40g cm�3 in CsCl and a diameter of about 35 nm were observed byelectron microscopy in fractions purified from mycelia grown in afermentor for 2 days (Fig. 4A and C). SDS-PAGE analysis of thepooled fractions revealed six protein bands of 130, 85 (84), 75, 65,and 63 kDa (Fig. 4E, lane 1). Anti-ORF3 antiserum detected an84-kDa protein corresponding to full-size ORF3 in addition to75-, 70-, 67-, and 63-kDa proteins (Fig. 4F, lane 1). Anti-ORF4antiserum detected an 85-kDa protein corresponding to full-sizeORF4 as well as a 70-kDa protein (Fig. 4G, lane 1). These resultsindicated that full-size ORF3 and ORF4 might be components ofMoCV1 particles. The other protein bands smaller than 84 or 85kDa must be degraded forms of the full-size proteins.

We also isolated MoCV1 particles from mycelia grown in thefermentor for 14 days. The buoyant densities of the isometric virusparticles ranged from 1.38 to 1.40 g cm�3, which was almost thesame as the particles obtained from mycelia grown in a fermentorfor 2 days or in flasks for 28 days (Fig. 4B and D) (29). SDS-PAGEand Western analysis of the pooled fractions showed that the pro-tein band patterns observed on staining with CBB (Fig. 4E, lane 2)and the bands recognized by the anti-ORF3 (Fig. 4F, lane 2) andanti-ORF4 (Fig. 4G, lane 2) antisera were almost the same as thoseobserved for 28-day cultures, except for a 55-kDa protein detectedby anti-ORF4.

No degradation of ORF1 protein (P130) was observed. We per-formed RdRp assays using [�-32P]UTP on the purified MoCV1 par-ticles from mycelia cultured for 2 or 14 days (12). Autoradiography ofthe native PAGE gel revealed radioactive signals from both types ofMoCV1 particles (see Fig. S4, arrow, lanes 3 and 6, in the supplemen-tal material). No radioactivity was detected in preparations from theMoCV1-free strain (data not shown).

FIG 4 Isolation of MoCV1 particles associated with full-size ORF3 and ORF4. MoCV1 particles were purified from mycelia of MoCV1-infected strain S-0412-II1a cultured in fermentors for 2 days (A and C) or 14 days (B and D). (A) The density of each fraction (in g cm�3) was 1.45 (lane 1), 1.43 (lane 2), 1.41 (lane 3),1.40 (lane 4), 1.38 (lane 5), 1.37 (lane 6), and 1.35 (lane 7). (B) The density of each fraction (in g cm�3) was 1.43 (lane 1), 1.42 (lane 2), 1.41 (lane 3), 1.39 (lane4), 1.37 (lane 5), 1.35 (lane 6), and 1.33 (lane 7). Negative-contrast electron micrograph of uranyl acetate-stained virus particles of mycelia cultured for 2 days (C)or 14 days (D). Scale bar, 100 nm. Proteins of the collected virus fractions were separated by 8% SDS-PAGE (15 mA; 2 h), stained with CBB (E) andimmunoblotted with antiserum against recombinant ORF3 (F) and antiserum against recombinant ORF4 (G). Lanes 1, fractions containing MoCV1 particles inCsCl (1.38 to 1.40 g cm�3) purified from mycelia cultured for 2 days; lanes 2, particles purified from mycelia cultured for 14 days.

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Heterologous expression of ORF4 induced cytological dam-age to the yeast Saccharomyces cerevisiae. We previously re-ported that MoCV1 affects the fungal host phenotype of isolateS-0412-II 1a, causing irregular mycelial growth, unusual pigmen-tation, and abnormal vesiculation (29). We attempted to identifywhich gene products of MoCV1 are involved in these types ofcytological damage by utilizing an S. cerevisiae expression system.First, each of the MoCV1 ORFs was expressed using five types ofshuttle vector: pRSA313 (HIS3), pRSA314 (TRP1), pRSA315(LEU2), pRSA316 (URA3), and pRSA317 (LYS2). These shuttlevectors have a single-copy CEN6 replication origin, and the ex-pression of the ORFs was controlled by an ADH1 promoter, whichleads to moderate expression levels. Yeast transformed with each

of the shuttle vectors bearing ORF1, -2, -3, -4, and -5 was grownon the appropriate selective agar medium. The transformantswere grown at 25°C, 30°C, and 37°C and were investigated forgrowth and morphological changes.

No change was observed in the yeast transformants, except forthose expressing ORF4. Colonies of the transformants expressingORF4 were smaller than those of the transformants expressingORF1, ORF2, ORF3, or ORF5 on selection medium when grownat 37°C (data not shown).

To test the effects of overproduction of ORF4 protein on yeastgrowth, we introduced single-copy (CEN6 and TDH3-ORF4) orhigh-copy-number (2�m and TDH3-ORF4) plasmids into strainW303-1A (L-A-o), in which the expression of ORF4 was con-

FIG 5 Heterologous expression of ORF3 and ORF4 in S. cerevisiae. (A) Growth tests of yeast strain W303-1A (L-A-o) harboring pRSA316-ORF4, pRST316-ORF4, and pRST426-ORF4 shuttle vectors at 30°C or 37°C on minimal medium (SC) without uracil. After incubation for 3 days at 30°C or 37°C, cellstransformed with the pRST426-ORF4 plasmids showed few and very small colonies. (B and C) Western blot analysis. After growth in SC-Ura liquid medium,proteins were extracted, separated by SDS-PAGE (8% acrylamide), transferred to membranes, and probed with antiserum against recombinant ORF4 (B) andrecombinant ORF3 (C). A 20-�g aliquot of protein from each extract was used. (D) Individual cells harboring empty pRST426 or pRST426-ORF4 grown onSC-Ura plates at 30°C for 3 days were resuspended in water and photographed using a 1,000� differential interference contrast objective.

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trolled by the strong promoter of TDH3 (22). As shown in Fig. 5A,overexpression of ORF4 protein by pRST426-ORF4 caused severegrowth inhibition at both 30°C and 37°C. Confirmation thatORF4 protein was overproduced was obtained by Western blot-ting using antiserum against ORF4 protein (Fig. 5B).

Morphological changes were also observed with a light micro-scope. The cells overexpressing ORF4 protein showed extremelyenlarged vacuoles that occupied the majority of the cell volume(Fig. 5D, right panel), while few vesicles were observed in cellscarrying pRST426 empty vector (Fig. 5D, left panel).

DAPI staining of W303-1A (L-A-o) harboring pRST426-ORF4showed strong chromatin condensation with fragments or severalrandomly distributed nuclear fragments (Fig. 6A). Enlarged cellswere easily stained without detergent permeabilization. These ob-servations suggested that heterologous overproduction of theORF4 protein in S. cerevisiae resulted in cytological changes andnuclear fragmentation, and the phenotypic severity depended onthe ORF4 expression level. Overexpression of ORF3 by pRSA315-

ORF3, pRST315-ORF3, or pRST426-ORF3 conferred no recog-nizable phenotypic changes in yeast cells (Fig. 5C and 6B).

DISCUSSION

MoCV1 particles consist of several major polypeptides, P130, P70,P65, and P58 (Fig. 2A), that are encoded by dsRNA1, -3, and -4(Fig. 2, 4, and data not shown). We demonstrated that P58, en-coded by dsRNA3, lacks the C-terminal region of the deducedamino acid sequence (approximately 200 aa) of ORF3 (Fig. 2),while P70 and P65, encoded by dsRNA4, lack the N-terminal 15amino acids in addition to their C-terminal regions. The appear-ance of the P70 and P65 protein bands might be caused by post-translational modifications. A similar observation was reportedfor structural proteins of FgV-ch9, one of the mycoviruses mostclosely related to MoCV1 (5).

Processing of MoCV1 proteins was investigated in crude ex-tracts of mycelia grown in flasks; the full-size ORF3 (84 kDa) andORF4 (85 kDa) proteins were detected as major components ofMoCV1 particles until 15 days of culturing, while the processedORF3 (58 kDa) and ORF4 (70 and 65 kDa) proteins graduallyaccumulated after 15 days (Fig. 3A and C). The processed ORF3and ORF4 proteins were hardly detected in 9-day cultures, sug-gesting that the processing of these viral proteins is not essentialfor the initial viral assembly. The full-size ORF3 and ORF4 beingassembled as viral structural proteins might be degraded by cellu-lar proteases in senescent cells. Although full-size ORF3 (84 kDa)and ORF4 (85 kDa) proteins were detected in crude mycelial ex-tracts, we could not purify MoCV1 particles consisting of onlyfull-length ORF3 and ORF4 proteins, suggesting that proteolyticdegradation of intact MoCV1 particles might occur rapidly duringpurification as well as intracellularly (Fig. 4E, F, and G). The pro-cessed MoCV1 particles, on the other hand, were relatively stableduring purification or storage in a refrigerator (for at least 3weeks), suggesting that the processed particles are relatively resis-tant to cellular proteases.

MoCV1 particles were also found in culture medium during aprolonged liquid culture period of 28 days (29). The MoCV1 par-ticles released into liquid medium also showed a 58-kDa band as amajor coat protein (29). These results indicated that the proteins,which consist of MoCV1 particles, tend to be degraded duringpurification or prolonged liquid culture, leading to truncatedORF3 (P58) and ORF4 (P70 and P65) proteins.

As described above, we obtained two types of particles, par-tially processed MoCV1 and fully processed MoCV1. This meansthat at least two types of viral particles might exist in infectedfungal cells. We could not observe any differences in viral struc-ture, physicochemical properties, or RdRp activity between thesetwo MoCV1 particles (Fig. 4A, B, C, and D and Fig. S4 in thesupplemental material). In the Totiviridae, two types of particlesshowing different protein compositions were separated from Hel-minthosporium victoriae mycelium (10). Ghabrial et al. demon-strated that the ratio of the amount of structural proteins was notinfluenced by the age of the culture (8). Very recently, such post-translational modification has also been found in a novel quadri-partite dsRNA virus in Rosellinia necatrix (20) and a novel bipar-tite dsRNA virus in Botrytis porri (34).

We did not observe the fifth dsRNA segment in our previousstudy (29); most cDNA clones, made by using random primers,mapped to dsRNA1 through dsRNA4. Very recently, however, werealized that MoCV1 could have five dsRNA segments. Based on

FIG 6 DIC and DAPI-stained fluorescence micrographs of S. cerevisiae cellsharboring shuttle vectors. Cells were grown on SC-Ura (A) or SC-Leu (B)plates at 30°C for 3 days. DIC panels show differential interference light mi-croscopy data. DAPI panels show DNA staining. Merged panels show super-imposition of DIC and DAPI fluorescence data. Scale bar, 10 �m. (A) Yeastcells were transformed with empty vectors (pRSA316, pRST316, or pRST426)or ORF4-expressing vectors (pRSA316-ORF4, pRST316-ORF4, or pRST426-ORF4). (B) Yeast cells were transformed with empty vectors (pRSA315,pRST315, or pRST425) or ORF3-expressing vectors (pRSA315-ORF3,pRST315-ORF3, or pRST425-ORF3).

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RdRp protein sequences, MoCV1 forms a separate clade withFgV-ch9, FgV2, AsV1816, and AbV1 in the Chrysoviridae family(Fig. 1C). The FgV-ch9 and FgV2 genomes are composed of fivedsRNA segments (5, 37).

We detected several dsRNA segments ranging from 3.0 to 3.5kb in eight isolates collected in Vietnam (29). Now, we have con-firmed that some of these mycoviruses, including MoCV1, havefive dsRNA segments. We also realized that one of these mycovi-ruses, which was detected in isolate S-0412-II 2a, has four or fivedsRNA segments. During cultivation of the host fungus, we suc-ceeded in separating a mycovirus bearing five dsRNA segmentsand a mycovirus bearing four dsRNA segments, lacking the fifthdsRNA segment. Therefore, the fifth dsRNA segment might besatellite RNA and dispensable for maintenance of the viruses.

In this study, a yeast expression system was used to evaluate theeffects of the MoCV1 gene products on host cells. Overexpressionof the ORF4 protein in S. cerevisiae caused remarkable growthinhibition (Fig. 5) and led to abnormally enlarged cells and vacu-oles with signs of membrane damage. Nuclei in the ORF4-over-expressing cells showed condensation and fragmentation (Fig. 6),suggesting that heterologous expression of ORF4 induces apopto-sis-like cell death, with the severity depending on expression level.To observe the effect of ORF4 in its natural host, ORF4 expressionin M. grisea is being studied.

ACKNOWLEDGMENTS

This research was supported in part by a Grant-in-Aid for Scientific Re-search (number 20580045) from the Ministry of Education, Culture,Sports, Science and Technology of Japan, by a grant from the programLinking Mechanism of Research Results to Practical Application fromJapan Science and Technology Agency (number 859100012), and by agrant from the New Energy and Industrial Technology Development Or-ganization (number 08C46503c).

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