Arch Virol DOI 10.1007/s00705-014-2144-x

BRIEF REPORT

Detection and sequence analysis of two novel co-infecting double-strand RNA mycoviruses in Ustilaginoidea virens

Jie Zhong • Xiang Hua Lei • Jun Zi Zhu • Ge Song • Ya Dong Zhang • Yi Chen • Bi Da Gao

Received: 3 December 2013 / Accepted: 31 May 2014 Ó Springer-Verlag Wien 2014

Abstract Four novel double-stranded RNA molecules, virens RNA 2 and Ustilaginoidea virens partitivirus 4, named dsRNA 1 (5124 bp), dsRNA 2(1711 bp), dsRNA 3 respectively. (1423 bp) and dsRNA 4 (855 bp), were detected in strain HNHS-1 of Ustilaginoidea virens, the causal agent of rice false smut disease. Sequence analysis showed that the Mycoviruses have been described in a wide range of fila- dsRNA1 contains two overlapping open reading frames mentous fungi, yeasts, and oomycetes [1]. The mycovi- (ORF) potentially encoding proteins with modest levels of ruses harboring dsRNA genomes are currently classified sequence similarity to the coat protein (CP) and putative into six families: , , , RNA-dependent RNA polymerase (RdRp), respectively, of , and , whose of the family Totiviridae. The deduced gene members have 1, 2, 4, 11 or 12, 2 and 4 genome segments, product of the ORF encoded by dsRNA2 is homologous to respectively, and are encapsidated in rigid isodiametric putative RdRp of viruses in the family Partitiviridae; the virus particles [2, 3]. The genome segments of viruses in ORF encoded by dsRNA3 shares some similarity to a the family Partitiviridae are composed of two dsRNAs, hypothetical protein with unknown function. It is note- ranging in size from 1.4 to 3.0 kb, the larger of which worthy that the dsRNA4 lacked integrated ORFs. Isomeric encodes an RNA-dependent RNA polymerase (RdRp) and viral particles of about 40 nm in diameter were observed the smaller, a capsid protein (CP), and these are encapsi- by transmission electron microscopy in a mycelium tissue dated in isometric particles with diameters of about preparation of strain HNHS-1-R1, a single-spore subculture 30–40 nm [4]. The family Partitiviridae was formerly of strain HNHS-1 containing only the dsRNA1 segment. divided into four genera, Partitivirus, Alphacryptovirus, Phylogenetic analysis and examination of the organization Betacryptovirus and Cryspovirus, which included viruses of the two putative RdRp sequences both indicated that that infect fungi, plants, plants and protozoa, respectively there are at least two novel virus species present in strain [5]. Currently, the family Partitiviridae is divided into 5 HNHS-1. We named the two novel viruses Ustilaginoidea genera: Alphapartitivirus, Betapartitivirus, Gammapartiti- virus, Deltapartitivirus, and Cryspovirus [6]. Viruses in the family Totiviridae are classified into five genera, including Totivirus, Victorivirus, Giardiavirus, Leishmaniavirus, and Trichomonasvirus. The viruses in the family Totiviridae Electronic supplementary material The online version of this have a monopartite dsRNA genome segment that encodes article (doi:10.1007/s00705-014-2144-x) contains supplementary material, which is available to authorized users. only two proteins, namely the coat protein (CP) and RdRp, or a CP/RdRp fusion protein [7, 8]. J. Zhong X. H. Lei J. Z. Zhu G. Song Ustilaginoidea virens is the causal agent of false smut Y. D. Zhang Y. Chen B. D. Gao (&) disease of rice, which was first reported in Tirunelveli Hunan Provincial Key Laboratory for Biology and Control of district of Tamil Nadu State of India [9]. As new hybrid Plant Diseases and Insect Pests, Hunan Agricultural University, Nongda Road 1, Furong District, Changsha 410128, China varieties were widely planted, the false smut became the e-mail: [email protected] most devastating grain disease, causing yield losses in the 123 J. Zhong et al. majority of the rice-growing areas of the world [10]. Fur- NCBI database [22]. Sequence analysis and alignments thermore, ustiloxin, derived from false smut balls, is an were performed using DNAMAN (Lynnan Corp, Pointe- inhibitor of microtubules [11] and makes the contaminated Claire, QC, Canada) and CLUSTALX [23]. Phylogenetic rice panicles poisonous to animals [12, 13]. Mycoviruses in trees were constructed using the neighbor-joining method the mycelia of U. virens have been reported previously, in MEGA 4 with a bootstrap test of 1000 re-samplings [24]. including Ustilaginoidea virens RNA virus 1 (UvRV1) and The complete nucleotide sequences of the four dsRNA Ustilaginoidea virens partitivirus 1 (UvPV1), which occur segments were submitted to the GenBank database with simultaneously in a single strain of U. virens strain JYH- accession numbers KC433710, KC469948, KC469949, and ZT [14, 15]. In addition, Ustilaginoidea virens partitivirus KC469950. 2-Uv0901 [16] and the uncharacterized mycovirus reported When electrophoresed on an agarose gel, except for the by Jiang et al. [17] are also present in this species. In this fungal genomic DNA, no additional DNA bands were study, we initially screened and isolated dsRNAs present in found in the preparation of RNase-A-digested genomic the pathogenic U. virens strain HNHS-1. The complete DNA of HNHS-1, suggesting that there was no extra- nucleotide sequences of the dsRNAs present in the strain chromosomal DNA genetic element in HNHS-1. On the have been determined and compared with other dsRNA other hand, dsRNA fragments were found after CF-11 viruses. Based on sequence comparisons and phylogenetic cellulose extraction and digestion with RNase-free DNase I analysis, our results support the conclusion that co-infec- and S1 nuclease. tion of a single isolate by two mycovirus was present. The complete nucleotide sequence of dsRNA1 was The U. virens strain HNHS-1 used in this study was 5,142 bp in length after sequencing and showed the exis- isolated from rice with false smut disease in Hanshou tence of two overlapping ORFs, designated ORF1 and county in China in 2009 and verified by amplification and ORF2. The two ORFs overlapped by five nucleotides, sequencing of the ITS sequence (accession number UAAUG, where the start codon of ORF2 partially over- JX427552.1). After maintaining them on potato sucrose lapped with the stop codon of ORF1. This type of genome agar (PSA) at 27 °C for 14 days, mycelia were cultured in organization is a characteristic commonly found in mem- PS broth in an orbital shaker at 110 rpm for an additional bers of the family Totiviridae. Thus, the RdRp is probably 7 days at 27 °C. DNA was extracted in SDS lysis buffer synthesized using a termination reinitiation mechanism and subjected to RNase A (Fermentas) digestion as [25, 26]. The 5’ untranslated region (UTR) and 30-UTR described by the manufacturer. The dsRNAs were extrac- were 273 nt and 67 nt long, respectively. The molecular ted using the CF11 cellulose affinity chromatography masses of the two ORF-encoded proteins were calculated method as described by Morris and Dodds [18] with several to be 82 kDa and 91 kDa, respectively. Homology search modifications. The dsRNA preparations, after being treated results showed that the predicted amino acid sequence of with RNase-free DNaseI (TAKARA, Dalian, China) and ORF1 in dsRNA1 had significant similarity to those of coat S1 nuclease (TAKARA Dalian, China) at 37 °C for proteins. The viruses sharing significant sequence identity 30 min, were separated on agarose gels and then visualized to the ORF1 CP included Tolypocladium cylindrosporum using a gel imaging system. The dsRNAs, designated as virus 1 (TcV1), Magnaporthe oryzae virus 1 (MoV1) and dsRNA1 to dsRNA4, in order of decreasing molecular Gremmeniella abietina RNA virus L2 (GaRVL2). Homol- weight (Fig. 1a, and b), were purified individually and used ogy search results of the ORF2-encoded protein showed as temple for further cloning of the full cDNA segments. that it was related to the RdRps encoded by the viruses in Full-length cDNA clones were obtained using a modified the family Totiviridae, particularly to that of Magnaporthe single-primer amplification technique (M-SPAT) [19, 20], oryzae virus 2 (36 % identity). Furthermore, ORF2 con- using the 30-amino-blocked adaptor A for terminal ligation tains eight conserved motifs characteristic of RdRPs of and a complementary primer B for reverse transcription. dsRNA viruses infecting lower eukaryotes [27]. A multiple Every cDNA clone was sequenced independently at least alignment of the amino acid sequences of the putative four times. In addition, virus-like particle (VLP) fractions RdRp encoded by dsRNA1 and five other selected viruses were purified from the mycelia preparations, using the is shown in Fig. 2a. The results suggest that the dsRNA1 method described previously with modifications [20, 21]. belongs to a mycovirus, tentatively designated as Ustilag- The VLPs, with a diameter of about 40 nm, were nega- inoidea virens RNA virus 2 (UvRV2). Phylogenetic ana- tively stained with uranyl acetate and examined by electron lysis of the putative amino acid sequences of the RdRps in microscopy, and the purified virus preparations were UvRV2 and other selected viruses in the family Totiviridae electrophoresed in a sodium dodecyl sulfate (SDS)-poly- was performed (Fig. 2b). UvRV2 clustered closely with acrylamide (10 %) gel (SDS-PAGE) and stained with members of the genus Victorivirus in the family Totiviridae Coomassie brilliant blue. Sequence similarity and open but formed a distinct clade and had highest similarity to reading frame (ORF) searches were conducted on the Eimeria brunetti RNA virus 1. In addition, a CP-based 123 Detection and sequence analysis

Fig. 1 Agarose gel electrophoresis of dsRNA, genomic organization VLPs showing the 82-kDa and 40-kDa protein components electro- of UvRV2 and UvPV4, TEM and SDS-PAGE analysis of the VLPs phoresed in a 10 % polyacrylamide gel. (e) Agarose gel electropho- purified from strain HNHS-1 or HNHS-1-R1. (a) Confirmation of four resis of dsRNA extracted from strain HNHS-1-R1, the subcultures of dsRNA segments extracted from strain HNHS-1 after staining with strain HNHS-1. (f) SDS-PAGE analysis of purified virus particles ethidium bromide and observation under UV light. (b) Diagrammatic from strain HNHS-1-R1 showing only the corresponding 82-kDa representation of the four dsRNAs. (c) TEM images of uranyl-acetate- protein band electrophoresed in a 10 % polyacrylamide gel. (g) TEM stained VLPs from HNHS-1. (d) SDS-PAGE analysis of purified observation of the VLPs extracted from strain HNHS-1-R1 phylogenetic tree was also constructed, and its topology 1,627 encoding a protein of 522 amino acids with a was similar to that of the RdRp-based tree, further sup- deduced molecular weight of 60 kDa. The 50 UTR and 30 porting the phylogenetic status of UvRV2 (Fig. S1b). UTR of the ORF were 58 and 84 bp long, respectively. Coincidentally, after subculturing of strain HNHS-1, a A BLAST search performed on the NCBI database [22] single-spore isolate containing only the dsRNA1 segment showed that the predicted protein product of the ORF of was obtained and named HNHS-1-R1 (Fig. 1e). Particles dsRNA2 was similar to RdRps of the viruses in the family of strain HNHS-1-R1 and HNHS-1 were purified and Partitiviridae, particularly Ustilaginoidea virens partitivi- examined by transmission electron microscopy (TEM) and rus 2-Uv0901 (UvPV2-Uv0901), and Colletotrichum then analyzed by sodium dodecyl sulfate polyacrylamide acutatum RNA virus 1 (CaPV1), with 63 % and 53 % gel electrophoresis (SDS-PAGE). TEM observation of the protein sequence similarity, respectively. As shown in two strains revealed the presence of isomeric virus-like Fig. 3a, a multiple alignment of the amino acid sequences particles (VLPs) with an approximate diameter of 40 nm of the putative RdRp encoded by dsRNA2 with that of (Fig. 1c and g). A single major protein band with a UvPV2-Uv0901 and five other selected partitivirus was molecular weight of 82 kDa (Fig. 1f), which was specu- performed, and several conserved motifs characteristic of lated to be the viral coat protein of UvRV2, was detected. RdRps of dsRNA mycoviruses were found in the RdRp However, two major protein bands were found in the VLPs segment of dsRNA2. Therefore, we concluded that of strain HNHS-1 by SDS-PAGE analysis (Fig. 1d), sug- dsRNA2 represents the genomic segment of another my- gesting the presence of a second coat protein belonging to a covirus distinct from the one to which dsRNA1 belongs, different virus. and we designated it as Ustilaginoidea virens partitivirus 4 The cDNA of dsRNA2 was found to be 1,711 bp long, (UvPV4). A phylogenetic analysis was conducted based on and it contained one major ORF from nucleotides 59 to the RdRp amino acid sequences of members of the family

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Partitiviridae, using neighbor-joining clustering with including UvPV2-Uv0901 and CaPV1, closely related to robust bootstrapping values from 1000 replicates. The members of the genus Gammapartitivirus. phylogenetic analysis, together with pairwise sequence The nucleotide sequence of dsRNA3 was 1,423 bp comparisons indicated that UvPV4 grouped with viruses in long. Sequence analysis showed that dsRNA3 contains a the family Partitiviridae (Fig. 3b) but formed a new cluster single putative ORF, from nucleotides 154 to 1,269,

123 Detection and sequence analysis b Fig. 2 Multiple alignment and phylogenetic analysis of amino acid A multiple alignment of the 50 UTRs of the dsRNA2 and sequences of the RNA-dependent RNA polymerase (RdRp) genes of dsRNA3 showed regions of low similarity, whereas a viruses of the family Totiviridae. (a) Sequence alignment of the RdRp 0 of UvRV2 and five selected viruses of the family Totiviridae. The higher level of sequence similarity was found in their 3 0 alignment was carried out with the program Clustal X and highlighted UTR regions, although the 3 UTR of dsRNA3 is much using the GeneDoc application. EfV1, Epichloe festucae virus 1; longer than that of dsRNA2 (Fig. S1a). The sequence motif CmRV, Coniothyrium minitans RNA virus; MoV2, Magnaporthe ‘CATTATT’, although distinct from the identical nucleo- oryzae virus 2; BbRV1, Beauveria bassiana RNA virus 1 (Beauveria bassiana victorivirus 1); HvV190-S, Helminthosporium victoriae tide stretches of the terminal motif in most partitiviruses 0 virus 190S. (b) The phylogenetic tree was analyzed using the [28, 29], was found in the 5 UTR, indicating a common neighbor-joining method with 1000 bootstrap replicates showing the viral origin of the dsRNA2 and dsRNA3. relationship of the UvRV2 to other viruses. The numbers near the In conclusion, we report here the co-existence of four branches indicate the percentage of bootstrap replicates supporting the branch. The names and their respective GenBank accession dsRNA segments in U. virens strain HNHS-1. Since dsRNA1 numbers are as follows: Beauveria bassiana RNA virus 1 and dsRNA2 code for distinct RdRp molecules with char- (CCC42235.1), Helicobasidium mompa No.17 dsRNA virus acteristic conserved RdRp motifs, the two dsRNAs are likely (NP_898833.1), Magnaporthe oryzae virus 1 (YP_122352.1), Toly- to replicate independently in HNHS-1. Based on the phylo- pocladium cylindrosporum virus 1 (FR750562), Botryotinia fuckeli- ana totivirus 1 (AM491608), Helminthosporium victoriae virus 190S genetic analysis results and genome organization, we pro- (U41345), Sphaeropsis sapinea RNA virus 2 (AF039080), Coniothy- pose the dsRNAs to be from two mycoviruses, tentatively rium minitans RNA virus (AF527633), Epichloe festucae virus 1 named UvRV2 and UvPV4, providing evidence for co- (CAK02788.1), Magnaporthe oryzae virus 2 (AB300379), Gremmen- infection of the U. virens strain by two distinct mycoviruses. iella abietina RNA virus L1 (NP_624332.2), Eimeria brunetti RNA virus 1 (NP_108651.1), Leishmania RNA virus 2-1 (U32108), However, it is not known whether there is an interaction Leishmania RNA virus 1-4 (NP_619653.1), Leishmania RNA virus between the co-infecting mycovirus. 1-1 (M92355), Saccharomyces cerevisiae virus L-A (AAA50321.1), The dsRNA2 and dsRNA3 patterns seem to be consistent Saccharomyces cerevisiae virus L-BC (La) (NP_042581.1), Tricho- with the typical genome composition of partitiviruses, such monas vaginalis virus 2 (AAF29445.1), virus 1 (ABC86751.1), Trichomonas vaginalis virus 3 (NP_659390.1), as the number of segments, segment sizes, and the homology Giardia lamblia virus (NP_620070.1), Trichomonas vaginalis virus 4 results showing sequence similarities of the dsRNA2-enco- (AED99798.1), Ustilago maydis virus H1 (NP_620728.1), Tuber ded RdRp to those of viruses in the family Partitiviridae.In aestivum virus 1 (ADQ54106.1) Ustilaginoidea virens RNA virus 1 contrast, there is no typical CP encoding ORF in dsRNA3, (AGO04407.1) and the protein encoded by dsRNA3 showed no significant similarities to any know capsid protein in the GeneBank coding for a protein with a calculated molecular weight database. However, the dsRNA2 and dsRNA3 showed of 41 kDa. The 50 and 30 UTR flanking the ORF were identical nucleotide stretches at the 50 ends of their UTRs, a determined to be 153 and 154 bp long, respectively. fact considered to be important for virus replication. The A BLAST search of the deduced amino acid sequence of dsRNA3 and the dsRNA1 showed less sequence identity to dsRNA3 showed no significant similarity to any known each other, excluding the possibility that the dsRNA3 is a CP protein in the databases, with the exception of the defective RNA of dsRNA1. Furthermore, a single-spore hypothetical proteins encoded by the viruses UvPV2- isolate was obtained that only contained dsRNA1, and Uv0901 (YP_008327313.1) and CaPV1 (AGL42313.1), dsRNA2 and dsRNA3 could be co-transmitted via conidial which had amino acid sequence identities of 49 and spores. Thus, we suggest the dsRNA2 and dsRNA3 consti- 26 %, respectively, which was similar to the RdRp-based tute the partitivirus genome, although the terminal identical homology search results. stretch was different from the characteristic ones of many The smallest segment, dsRNA4, was 855 nt long and did previously described partitiviruses [28, 29]. A number of not contain an ORF. A BLAST search of dsRNA4 showed previous studies have shown that viruses spanning the three no significant similarities to any nucleotide sequence in the domains of life shared similar capsid protein structures and GenBank database. Pairwise sequence alignment between capsid architecture, which may indicate a common ancestor dsRNA4 and the other three dsRNAs segments also of viral capsids; nevertheless, such similarity is not always showed few similarities, suggesting that this dsRNA was detectable in sequence comparisons [30–32]. Based on these not derived from the other three dsRNA segments. In findings, we could not exclude the possibility that dsRNA3 addition, the genome segment dsRNA4, in some cases, codes for the CP of UvPV4. Nevertheless, in view of the disappeared during subculture, and it was never found argument that CPs in members of the family Partitiviridae independently in any single-spore isolate of strain are fairly conserved and can be recognized as CP, it is pos- HNZJ001. These features are consistent with the possibility sible that the CP-encoding segment is missing. However, the that dsRNA4 might be a satellite RNA. However, future SDS-PAGE and Coomassie stain analysis of the VPLs research to confirm the nature of dsRNA4, using full-length purified from the original strain HNHS-1, which contained infectious transcripts, is needed. all four dsRNA segments, showed the presence of two major 123 J. Zhong et al.

protein bands corresponding to about 80 and 40 kDa the VLPs of strain HNHS-1-R1 containing only the totivirus (Fig. 1d), respectively. In contrast, there was only one pro- UvRV2. The minor-band protein might be derived from the tein band with a molecular weight of about 80 kDa found in deduced protein of dsRNA3 via some events in processing

123 Detection and sequence analysis b Fig. 3 Multiple sequence alignment analysis of the RdRp encoded by 2. Pearson MN, Beever RE, Boine B, Arthur K (2009) Mycoviruses UvPV4, and phylogenetic analysis of UvPV4. (a) Sequence alignment of filamentous fungi and their relevance to plant pathology. Mol of the RdRps of UvPV4 and five selected viruses. The alignment was Plant Pathol 10:115–128 carried out with the program Clustal X and highlighted using the 3. Kozlakidis Z, Herrero N, Ozkan S, Kanhayuwa L, Jamal A, GeneDoc application. These virus names are as follows: PSV-S, Bhatti MF, Coutts RH (2013) Sequence determination of a Penicillium stoloniferum virus S; OPV1, Ophiostoma partitivirus 1; quadripartite dsRNA virus isolated from Aspergillus foetidus. FuSOV, Fusarium solani virus 1; UvPV2 (UvPV2-Uv0901), Ustilag- Arch Virol 158:267–272 inoidea virens partitivirus 2-Uv0901 (Ustilaginoidea virens partitivi- 4. Ghabrial SA, Nibert ML, Maiss E, Lesker T, Baker TS, Tao YJ rus 2-Uv0901); CaPV1, Colletotrichum acutatum RNA virus 1. (2011) Family partitiviridae. In: King AMQ, Adams MJ, Carstens (b) Phylogenetic tree of the translated RdRp gene of UvPV3 and other EB, Lefkowitz EJ (eds) Virus taxonomy: ninth report of the selected viruses of the families Partitiviridae and Totiviridae. The international committee on taxonomy of viruses. Elsevier, USA, phylogenetic trees were analyzed by the neighbor-joining method pp 523–534 with a 1,000-replicate bootstrap search, and the bootstrap values are 5. Ghabrial SA, Nibert ML, Maiss E, Lesker T, Baker TS, Tao YJ indicated at the branch points. GenBank accession numbers of each (2012) Family partitiviridae. In: King AMQ, Adams MJ, Carstens virus are shown in the caption of Fig. 2 and are as follows: EB, Lefkowits EJ (eds) Virus taxonomy: ninth report of the Verticillium dahliae partitivirus 1 (AGI52210.1), Aspergillus fumig- International Committee on Taxonomy of Viruses. Elsevier, atus partitivirus 1 (CAY25801.2), beet cryptic virus 1 (EU489061.1), Academic Press Inc., New York, pp 523–534 carrot cryptic virus (FJ550604.1), Sclerotinia sclerotiorum partitivirus 6. Nibert ML, Ghabrial SA, Maiss E, Lesker T, Vainio EJ, Jiang D, S (GQ280377.1), white clover cryptic virus 1 (AY705784.1), Suzuki N (2014) Taxonomic reorganization of family Partitivir- Rosellinia necatrix partitivirus 2 (AB569997.1), Atkinsonella hypox- idae and other recent progress in partitivirus research. Virus Res ylon virus (L39125.1), Ceratocystis resinifera partitivirus 188:128–141 (AY603052.1), Fusarium poae virus 1 (AF047013.1), Pleurotus 7. Ghabrial S (2008) Totiviruses. 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