Molecular characterization of a new hypovirus infecting a phytopathogenic fungus, Valsa ceratosperma

Virus Research 165 (2012) 143–150

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Molecular characterization of a new hypovirus infecting a phytopathogenic fungus, Valsa ceratosperma Hajime Yaegashi ∗ , Satoko Kanematsu, Tsutae Ito Apple Research Station, National Institute of Fruit Tree Science, National Agriculture and Food Research Organization (NARO), Morioka 020-0123, Japan

a r t i c l e

i n f o

Article history: Received 27 December 2011 Received in revised form 9 February 2012 Accepted 9 February 2012 Available online 17 February 2012 Keywords: dsRNA Mycovirus Hypoviridae Valsa ceratosperma VcHV1

a b s t r a c t A double-stranded (ds) RNA, approximately 9.5 kb in size; was identified in the MVC86 isolate of Valsa ceratosperma. Complete sequence of the dsRNA revealed a 9543-bp segment (excluding the 3 poly-A tail) that is predicted to encode a single large protein (P330). P330 has 63%, 49%, and 55% amino acid sequence identities to the proteins encoded by hypoviruses Cryphonectria hypovirus 3 (CHV3), CHV4, and Sclerotinia sclerotiorum hypovirus 1 (SsHV1), respectively. Like polyproteins encoded by CHV3, CHV4, and SsHV1, P330 comprises four conserved domains, including a papain-like protease, a UDP glucose/sterol glucosyltransferase (UGT), an RNA-dependent RNA polymerase (RdRp), and an RNA helicase. These molecular characteristics suggest that this dsRNA represents a new hypovirus that we tentatively designate Valsa ceratosperma hypovirus 1 (VcHV1). Phylogenetic analysis of the RdRp and RNA helicase domains of VcHV1 revealed that VcHV1, CHV3, CHV4, and SsHV1 clustered together into one clade distinct from that of CHV1 and CHV2, indicating the existence of two lineages in the family Hypoviridae. Comparison of biological properties between VcHV1-infected and VcHV1-free isogenic strains did not reveal differences in colony morphology or fungal virulence under laboratory conditions. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Viruses that infect fungi are called mycoviruses, and are found in all major fungal groups (Ghabrial and Suzuki, 2009). Most mycoviruses have double-stranded (ds) RNA genomes encapsidated within spherical virions, and are classified into four families: Totiviridae, Partitiviridae, Reoviridae, and Chrysoviridae. Recently, novel dsRNA mycoviruses that do not classify into the established viral families have been reported, e.g., Rossellinia necatrix megabirnavirus 1 (Chiba et al., 2009) and Fusarium graminearum virus 3 (Yu et al., 2009). On the other hand, some mycoviruses have single-stranded (ss) RNA genomes without true virions, and these mycoviruses are classified into the families Hypoviridae, Narnaviridae, and Endornaviridae (Ghabrial and Suzuki, 2009). Other ssRNA mycoviruses also have been reported; e.g., Botrytis virus F (Howitt et al., 2001; Gammaflexiviridae, Mycoflexivirus), Botrytis virus X (Howitt et al., 2006; Alphaflexiviridae, Botrexvirus), Sclerotinia sclerotiorum debilitation-associated RNA virus (Xie et al., 2006; Alphaflexiviridae, Sclerodarnavirus), Diaporthe RNA virus (Preisig

∗ Corresponding author at: 92 Shimokuriyagawa, Morioka, Iwate 020-0123, Japan. Fax: +81 19 641 3819. E-mail address: [email protected] (H. Yaegashi). 0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2012.02.008

et al., 2000; unassigned), Sclerotinia sclerotiorum RNA virus L (Liu et al., 2009; unassigned), and Fusarium graminearum virus 1-DK21 (Kwon et al., 2007; unassigned). In addition, a recent study discovered a novel ssDNA mycovirus in Sclerotinia sclerotiorum (Yu et al., 2010). Thus, the increasing number of reports on novel mycoviruses implies the high diversity of mycoviruses in the fungal kingdom. While most mycoviruses infect the host fungus without apparent alteration of fungal phenotype, others have clear effects on the host, including changes in pigmentation, mycelial growth, sporulation, and virulence (Ghabrial and Suzuki, 2009). Particular interest has focused on attenuation of fungal virulence by mycoviral infection, since this phenomenon suggests a possible route for biological control of phytopathogenic fungal disease (referred to as “virocontrol”; Chiba et al., 2009; Ghabrial and Suzuki, 2009). The mycovirus-fungus system, Cryphonectria hypovirus 1 (CHV1)Chestnut blight fungus Cryphonectria parasitica, is the best-studied example both in virocontrol in the field and in fundamental fungal virology (Nuss, 2005; Milgroom and Cortesi, 2004). At present, four hypoviruses from C. parasitica (CHV1, CHV2, CHV3, and CHV4) have been recognized as confirmed species of the genus Hypovirus within the family Hypoviridae (Nuss and Hillman, 2011). CHV1 has a 12.7-kb genome that contains two open reading frames (ORFs), and CHV1 infection drastically reduces fungal virulence, pigmentation, and sporulation (Hillman et al., 1990; Shapira et al., 1991).

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CHV2 has a 12.5-kb genome that contains two ORFs; CHV2 infection drastically reduces fungal virulence, and moderately reduces pigmentation and sporulation (Hillman et al., 1992, 1994; Smart et al., 1999). The 9.8-kb CHV3 genome has a single ORF, and infection with this virus reduces fungal virulence without drastic effects on other phenotypes (Smart et al., 1999). The CHV4 genome (9.1 kb) is the smallest of the four and contains a single ORF; infection with CHV4 does not affect fungal virulence (Enebak et al., 1994; Linder-Basso et al., 2005). Thus, the size, viral genome organization, and biological impact on the host fungus all differ among these four hypoviruses. Recently, Xie et al. (2011) identified a novel mycovirus, Sclerotinia sclerotiorum hypovirus 1 (SsHV1), which is closely related to CHV3 and CHV4. Valsa canker is an important fungal disease in apple and pear, and is thought to be distributed throughout eastern Asia, including Japan, Korea, and China (Sakuma, 1990). Valsa ceratosperma, the causative agent of this disease (Kobayashi, 1970), is a fungus that invades from fruit scars, twig stubs, crotches, and sites of injury on the bark, generating cankers at infection sites. Diseased tree are killed when the canker encircles the tree’s trunk. Both V. ceratosperma and C. parasitica are classified into Ascomycota, Sordariomycetes, Diaporthales (Gryzenhout et al., 2006), and the two fungi have similar ecological niches, causing cankers on woody plants. As mentioned above, multiple C. parasitica mycoviruses have been reported, including four hypoviruses, two mycoreoviruses, and a mitovirus (Pearson et al., 2009). To our knowledge, no endogenous mycovirus has been reported for V. ceratosperma, although researchers have introduced heterologous mycoviruses by transformation of this fungus with a CHV1-infectious cDNA clone (Sasaki et al., 2002) and by protoplast-mediated infection with Rosellinia necatrix partitivirus 1 and Rosellinia necatrix mycoreovirus 3 (Kanematsu et al., 2010). In the present study, we identify and characterize a 9.5kb dsRNA element in V. ceratosperma isolate MVC86, which was obtained from an orchard apple tree. Complete nucleotide sequence of the dsRNA segment defines this as a novel mycovirus with sequence similarity to CHV3, CHV4, and SsHV1. The novel mycovirus, tentatively designated Valsa ceratosperma hypovirus 1 (VcHV1), is predicted to encode a single polyprotein with four separate enzymatic domains that are conserved among related mycoviruses. Phylogenetic analysis of the RdRp and RNA helicase domains shows that VcHV1, CHV3, CHV4, and SsHV1 cluster together into one group distinct from CHV1 and CHV2 within Hypoviridae. These results are consistent with the results of Xie et al. (2011) and confirm the existence of two evolutionary lineages in Hypoviridae, supporting establishment of a new genus within this family. Infection of V. ceratosperma with VcHV1 does not appear to affect colony morphology or fungal virulence under the conditions tested, consistent with the limited effect of CHV3, CHV4, and SsHV1 on their respective host fungi.

2. Materials and methods 2.1. Fungal isolate Valsa ceratosperma MVC86 was isolated from canker tissue on an apple tree in the orchard of the Apple Research Station, National Institute of Fruit Tree Science, Morioka, Iwate, Japan. Following removal of the surface of diseased bark tissue, small segments (1-mm-square) were excised from inside of the diseased tissues using a surgical knife, and these segments were cultured at 25 ◦ C on 1/10-strength potato-dextrose agar (PDA; Difco, 1.5% agar) containing streptomycin (200 mg/L). Resulting V. ceratosperma mycelium then was cultured on PDA without contamination, and mycelial agar discs were stored in sterilized 10% glycerol solution at −80 ◦ C.

Speciation was performed using internal transcribed spacer (ITS) sequences as described previously (Suzaki, 2008). 2.2. RNA extraction MVC86 was cultured on PDA for 4–6 days at 25 ◦ C in the dark, and then 2 or 3 mycelial agar discs from the PDA plate were inoculated to 20 mL of potato-dextrose broth (PDB; Difco) and cultured for 4–6 days at 25 ◦ C in the dark. Mycelial tissue was recovered from PDB after washing with water. RNA was extracted from the mycelial tissue as described previously (Yaegashi et al., 2011) with minor modifications. In brief, mycelial tissues were frozen in liquid nitrogen and homogenized in RNA extraction buffer (0.15 M sodium acetate (pH 5.2), 0.1 M LiCl, 4% SDS, 10 mM EDTA (pH 8.0), 20 mM ␤-mercaptoethanol) using a mortar and pestle. Total nucleic acid was purified from these extracts by two rounds of extraction (phenol/chloroform followed by chloroform), precipitated in isopropanol, rinsed with 70% ethanol, dissolved in sterile nucleasefree water, and digested with DNase I and S1 nuclease. The final dsRNA fraction was electrophoresed in 1% agarose gel and nucleic acids were visualized by staining with ethidium bromide. 2.3. cDNA synthesis, sequence, and phylogenetic analysis The dsRNA fragment of 9.5 kb was purified from the agarose gel using MagExtractor (TOYOBO) according to the manufacturer’s instructions. The resulting sample was mixed with random 9-mers, heat-denatured at 94 ◦ C for 15 min, and chilled on crushed ice for 10 min. The mixture was then subjected to cDNA synthesis using the M-MLV cDNA Synthesis Kit (TAKARA) according to the manufacturer’s instructions. The resulting cDNAs were filtered using an Illustra MicroSpin S-400 HR Column (GE Healthcare) and then separated on 1% agarose by electrophoresis. All cDNAs over 1000 bp were purified from the agarose gel using Magextractor. The final blunt-end cDNA fragments were ligated to SmaI-digested pBluescript II SK (−) vector (Stratagene), and the recombinant plasmids were introduced into competent Escherichia coli. Recombinant plasmids harboring cDNA inserts of 1000 bp or larger were purified from 2-mL cultures in Luria-Bertani (LB) broth using GenElute HP Plasmid Miniprep Kits (Sigma–Aldrich). The plasmids were sequenced using a Macrogen custom sequencing service (Macrogen Japan). Reverse transcription polymerase chain reaction (RT-PCR) was conducted to determine gap sequences between cDNA clones. The mixture of purified dsRNA (template) and random 9-mers was heat-denatured at 94 ◦ C for 15 min and chilled on ice for 10 min. The denatured mixtures were then reverse transcribed using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche) at 50 ◦ C for 60 min, and the enzyme then was inactivated at 85 ◦ C for 10 min. The resulting cDNA samples were used for PCR using Expand Long Range Enzyme Mix (Roche) with specific primer pairs as follows (see supplemental Table 1): VC86L-1F(+) and VC86L-1R(−), VC86L-2F(+) and VC86L-2R(−), VC86L-3F(+) and VC86L-3R(−), VC86L-4F(+) and VC86L-4R(−). PCR products were ligated into the pGEM-Teasy vector (Promega), and resulting plasmids were recovered, purified, and sequenced as described above. We carried out 3 RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RLM-RACE) as described by Chiba et al. (2009) or 3 RACE using adaptor-linked oligo-d(T) primer (FirstChoice RLMRACE Kit; Ambion) to determine 5 - or 3 -terminal sequences of the positive strand of the dsRNA element, respectively. Specific primers for amplification of 5 -end cDNA [VC86L-5-1(−) and VC86L-5-2(−)] and of 3 -end cDNA [VC86L-3-1(+) and VC86L-3-2(+)] are listed in supplemental Table 1. The amplified cDNA fragments were ligated into the pGEM-Teasy vector; resulting plasmids were recovered, purified, and sequenced as described above.

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Sequence data were collected, assembled, and analyzed with DNASIS software (Hitachi). Homology search was carried out with the tBLASTn program available in the DNA Databank of Japan (DDBJ). Multiple alignment and phylogenetic analyses were performed using Clustal-W with the default settings; phylogenetic trees were constructed using Tree View ver. 2.01. 2.4. Hyphal tipping MVC86 was cultured on a 3% agar plate for 4–6 days at 25 ◦ C in the dark. Small agar fragments containing the tip of single hyphae were excised from the plate using a surgical knife under stereomicroscopic observation, and each small piece was cultured on PDA at 25 ◦ C for 4–7 days in the dark. After two rounds of hyphal tipping, absence of dsRNA was confirmed by agarose gel electrophoresis and RT-PCR using the primer pair VC86L-977(+) and VC86L-1485(−) (supplemental Table 1). The resulting virus-free strain was stored in sterilized 10% glycerol solution at −80 ◦ C. 2.5. Virulence assay Inoculation of V. ceratosperma to fresh and dormant apple twigs was carried out according to the procedure described by Kanematsu et al. (1999). Inoculated twigs were maintained in plastic boxes to prevent desiccation and incubated at 20 ◦ C for 11 days in the dark. The surface of each twig then was removed and canker diameters were measured.

Fig. 1. Agarose gel electrophoresis of dsRNA from MVC86 isolate of Valsa ceratosperma. Lane M, Lambda-HindIII DNA marker; lane RnMyRV3, dsRNA of Rosellinia necatrix mycoreovirus 3 (4.1–0.9 kb); lane RnMBV1, dsRNA of Rosellinia necatrix meganirnavirus 1 (8.9 kb and 7.2 kb); lane MVC78, dsRNA from V. ceratosperma MVC78 isolate (10 kb); lane MVC86, dsRNA from V. ceratosperma MVC86 isolate (9.5 kb). Arrows indicate position of 9.5 kb dsRNA in MVC86.

3. Results 3.1. dsRNA in V. ceratosperma isolate MVC86 Fungal isolate MVC86 was isolated from an apple tree exhibiting a large valsa canker in an orchard of the Apple Research Station, National Institute of Fruit Tree Science (Morioka, Japan). The isolate MVC86 was speciated as V. ceratosperma by analysis of internal transcribed spacer (ITS) sequences (data not shown). Presence of dsRNA in fungi is a hallmark of mycovirus infection; most mycoviruses have dsRNA genome(s) or (for ssRNA mycoviruses) produce dsRNA as a replication intermediate (Ghabrial and Suzuki, 2009). In order to assess mycovirus infection in V. ceratosperma isolate MVC86, dsRNA was extracted from mycelial tissue of the isolate. As shown in Fig. 1, electrophoresis of the dsRNA fraction from MVC86 detected a single dsRNA element (designated VC86L); the electrophoretic mobility of this element was slightly slower than that of the dsRNA-1 of Rosellinia necatrix megabirnavirus (approximately 9 kb; Chiba et al., 2009) but faster than that of the large dsRNA element identified in MVC78, another V. ceratosperma isolate (approximately 10 kb; Yaegashi et al., unpublished data). No other V. ceratosperma isolates from our laboratory harbored a dsRNA element of the same size (data not shown). 3.2. Molecular characterization of a novel mycovirus from V. ceratosperma isolate MVC86 Complete nucleotide sequence of VC86L was determined by conventional random priming cDNA synthesis, RT-PCR, and 3 RLM-RACE. 5 -terminal sequence of the positive strand of VC86L was determined by the 3 RLM-RACE method, performed as described by Chiba et al. (2009), but specific cDNA containing the 3 -terminal sequence of the plus-strand of VC86L was not amplified by this method. To determine the 3 -terminal sequence of the positive-strand RNA, we utilized an alternative 3 RACE method using adaptor-linked oligo-dT primer. The amplification of a specific PCR product using an oligo-dT primer demonstrated that VC86L contains a poly-A tail at the 3 terminus of its plus strand.

The size of the full-length cDNA was 9543 bp, excluding the polyA tail; this value agreed with size of VC86L estimated by agarose gel electrophoresis. The positive strand of VC86L consisted of a 378-nt 5 -untranslated region (5 -UTR; nt 1–378); a single large 8823-nt putative ORF (nt 379–9201) capable of encoding a 2941residue protein (with a predicted size of approximately 330 kDa; P330); and a 342-nt 3 -UTR (nt 9202–9543), excluding the polyA tail (Fig. 2A). A homology search with P330 using the tBLASTn program in the DDBJ revealed significant sequence similarities (evalues of <0.01) with the polyproteins of Cryphonectria hypovirus 3 (CHV3-GH2), CHV4-SR2, and Sclerotinia screlotiorum hypovirus 1 (SsHV1-SZ150) (Table 1). The nucleotide identities between the full-length sequence of VC86L and those of CHV3-GH2 (9799 bp), CHV4-SR2 (9149 bp), and SsHV1-SZ150 (10398 bp) were 64%, 59%, and 60%, respectively (Table 2). Notably, the first 100 nt of the 5 -UTR and the last 100 nt the 3 -UTR of VC86L have high identities with those of CHV3-GH2 (88% in 5 -UTR, 72% in 3 -UTR), CHV4-SR2 (69% in 5 -UTR, 60% in 3 -UTR), and SsHV1-SZ150 (70% in 5 -UTR, 69% in 3 -UTR) (Fig. 2B and C, Table 2), indicating high conservation of the 5 - and 3 -UTRs among these hypoviruses. The amino acid identities between P330 (2941 amino acids, aa) and the polyproteins of CHV3-GH2 (2875 aa), CHV4-SR2 (2849 aa), and SsHV1-SZ150 (2949 aa) were 63%, 49%, and 55%, respectively (Table 2). The four domains conserved among the polyproteins of CHV3, CHV4, and SsHV1 were also found in P330, including papain-like proteinase (nt 1267–1494 in full-length sequence, aa 297–372 in P330), UDP-glucose/sterol glucosyltransferase (UGT; nt 1873–3153 in full-length sequence, aa 499–925 in P330), RNAdependent RNA polymerase (RdRp; nt 6049–6816 in full-length sequence, aa 1891–2146 in P330), and RNA helicase (nt 7966–8733 in full-length sequence, aa 2530–2785 in P330) (Fig. 2A). Sequence similarities among these hypoviruses suggest that VC86L is a novel mycovirus closely related to CHV3, CHV4, and SsHV1; therefore, we have tentatively designated VC86L as Valsa ceratosperma hypovirus 1 (VcHV1). The complete nucleotide sequence of VcHV1 has been deposited in the DDBJ (accession number AB690372).

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Fig. 2. Molecular characteristics of Valsa ceratosperma hypovirus 1 (VcHV1). (A) Genome organization of VcHV1. The four domains of the predicted polyprotein (shown as an open box) include papain-like protease (Pro), UDP glucose/sterol glucosyltransferase (UGT), RNA-dependent RNA polymerase (RdRp), and RNA helicase (Hel) (shown as closed, dotted, shaded, or checked boxes, respectively). Nucleotide (nt) numbers are shown at the top of the graphic. (B and C) Multiple alignment among VcHV1, Cryphonectria hypovirus 3 (CHV3)-GH2, CHV4-SR2, and Sclerotinia sclerotiorum hypovirus 1 (SsHV1)-SZ150, for the 5 -terminal 100 nt of the 5 -untranslated region (5 -UTR; panel B), and for the 3 -terminal 100 nt (excluding the poly-A tail) of the 3 -UTR (panel C). Asterisks indicate nucleotides that are identical in all four viruses.

Table 1 Results from tBLASTn homology search with P330. Virusa

Taxon (family/genus)

Accession no.

Protein (aa size)

Overlap (aa identities %)

Bit score/e-value

CHV3-GH2 SsHV1-Sz150 CHV4-SR2 FgV1-DK21 CHV1-EP713

Hypoviridae/Hypovirus Tentative hypovirus Hypoviridae/Hypovirus Unclassified Hypoviridae/Hypovirus

AF188514 JF781304 AY307099 AY533037 M57938

Polyprotein (2875) Polyprotein (2949) Polyprotein (2849) ORF1 (1537) ORFB polyprotein (3165)

1763/2555 (69) 1545/2567 (60) 1392/2848 (48) 117/432 (24) 109/488 (22)

3720/0.0 3239/0.0 2657/0.0 144/7e-31 83.6/2e-12

a The top five distinct viruses returned by tBLASTn are shown. For each virus, a representative isolate was selected for use in this table to avoid repetition of same virus species.

3.3. Phylogenetic relationships among the novel mycovirus and other hypoviruses In order to define taxonomic relationships among VcHV1 and other hypoviruses, phylogenetic analysis was conducted for the four domains encoded by VcHV1, CHV1-EP713, CHV2-NB58, CHV3-GH2, CHV4-SR2, SsHV1-SZ150, unassigned ssRNA mycovirus Fusarium graminearum virus 1-DK21 (FgV1), and plant potyvirus Plum pox virus (PPV).

A papain-like protease domain is located at the N-terminal end of the hypoviral polyprotein and includes two strictly conserved aa residues (Cys and His) that are required for the autoproteolytic catalytic activity (Shapira and Nuss, 1991; Hillman et al., 1994; Smart et al., 1999). Multiple alignments among the examined viral protease domains showed that both of these key residues were conserved in the protease domain of VcHV1 (Cys259 and His309 ; Fig. 3A). In the protease domain of CHV3-GH2, the Gly297 residue was demonstrated to be a cleavage site (Yuan and Hillman, 2001);

Table 2 Nucleotide and amino acid identities between VcHV1 and other hypoviruses. Virus

Identities to VcHV1 Complete sequence (nt %)

5 UTR (nt %)a

3 UTR (nt %)a

Polyprotein (aa %)

Domain (aa %) Proteaseb

CHV1-EP713 CHV2-NB58 CHV3-GH2 CHV4-SR2 SsHV1-SZ150

51 52 64 59 60

53 57 88 69 70

49 51 72 60 69

d

28 28d 63 49 55

e

27/25 29 28 42 25

UGTc f

– –f 77 60 59

RdRpb

RNA helicaseb

27 26 82 76 74

32 32 70 57 67

The terminal 100-nt at the 5 - and 3 -UTR were compared to the respective UTR of each virus. The amino acid sequence of the protease, RdRp, or RNA helicase domain of each hypovirus shown in Fig. 3 was used for comparison. c The amino acid sequence of the UGT domain of VcHV1 (aa 499–952 in P330) was compared to that of CHV3-GH2, CHV4-SR2, or SsHV1-SZ150 as reported in Xie et al. (2011). d The ORFB polyprotein of CHV1 or CHV2 was used for this comparison. e Identity to the protease domain of ORFA or ORFB of CHV1 is shown to the left or right of the slash, respectively. f No UGT domain was detected in the polyproteins of CHV1 or CHV2. a

b

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Fig. 3. Multiple alignments and phylogenetic analyses of three protein domains conserved among hypoviruses. (A, C, and E): Multiple alignments of papain-like protease (Pro), RNA-dependent RNA polymerase (RdRp), and RNA helicase (Hel) domains, respectively. Abbreviated virus names: VcHV1, Valsa ceratosperma hypovirus 1-MVC86 (accession no. AB690372); CHV1, Cryphonectria hypovirus 1-EP713 (accession no. M57938); CHV2, Cryphonectria hypovirus 2-NB58 (accession no. L29010); CHV3, Cryphonectria hypovirus 3-GH2 (accession no. AF188514); CHV4, Cryphonectria hypovirus 4-SR2 (accession no. AY307099); SsHV1, Sclerotinia sclerotiorum hypovirus 1-SZ150 (accession no. JF781304); FgV1, Fusarium graminearum virus 1-DK21 (accession no. AY533037); PPV, Plum pox virus-NAT (accession no. D13751). Asterisks, colons, and dots indicate identical, conserved, or semi-conserved amino acid residues, respectively. (B, D, and F): Unrooted phylogenetic trees based on Pro, RdRp, and Hel domains, respectively. Free-form dotted circle indicates the viruses belonging to Hypoviridae. Grey ellipse indicates the virus group containing VcHV1.

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Fig. 4. Biological impact of VcHV1 infection on V. ceratosperma. (A) dsRNA electrophoresis and RT-PCR analysis of VcHV1-infected strain (MVC86) and VcHV1-free strain (MVC86-18(VF)) derived from MVC86 by hyphal tipping. Arrows indicate the positions of VcHV1 dsRNA or specific DNA fragment, respectively. (B) Colony morphology of MVC86 and MVC86-18(VF) after 4 days culture on potato dextrose agar (PDA). (C) Canker size on dormant apple twig at 11 days post-inoculation. Bars indicate standard deviation.

by analogy, the last amino acid in the protease domain of VcHV1 (Gly331 ) is predicted to be the P330 cleavage site (Fig. 3A). As shown in Table 2, the protease domain of P330 shows the highest homology (42% aa identity) with the protease domain of CHV-SR4. A phylogenetic tree based on the protease domains indicated that the protease domain of VcHV1 is more closely related to CHV4-SR2 and SsHV1-SZ150 than those of other viruses (Fig. 3B). The UGT domain of VcHV1 (499–925 aa) is located C-terminal to the protease domain, as in the polyproteins of CHV3, CHV4, and SsHV1 (Linder-Basso et al., 2005; Xie et al., 2011); no UGT domain was found in CHV1 or CHV2. Multiple alignments showed that several aa regions were conserved among VcHV1, CHV3, CHV4, and SsHV1 (data not shown). Amino acid identities of UGT domains between VcHV1 and CHV3-GH2 (polyprotein aa 433-857), CHV4SR2 (polyprotein aa 421-840), or SsHV1-SZ150 (polyprotein aa 500–925) were 77%, 60%, or 59%, respectively (Table 2), indicating that the UGT domains of these hypoviruses were highly conserved. The UGT domain of VcHV1 was most similar to that of CHV3 (Table 2). The RdRp and RNA helicase domains of VcHV1 (RdRp, 1891–2146 aa; RNA helicase, 2530–2785 aa) are located C-terminal to the protease and UGT domains, and the RdRp and RNA helicase domains are highly conserved among hypoviruses. Multiple alignments of the RdRp domain consisting of eight motifs (Koonin et al., 1991) among VcHV1 and other viruses indicated that several amino acid residues are conserved (Fig. 3C). Notably, an SDD tripeptide (aa 2074–2076 in P330) is highly conserved among the hypoviruses (Fig. 3C); in contrast, the first amino acid of the

corresponding tripeptide is a Gly residue in most ssRNA viruses, including potyviruses (Koonin et al., 1991). Comparison among RNA helicase domains revealed that motifs that are characteristic of the helicase superfamily 2 (Kadare and Haenni, 1997), including a GKST box (aa 2537–2540 in P330) and a DExH box (aa 2610–2613 in P330), were shared among all the viruses used in our sequence comparisons (Fig. 3E). Amino acid identities of RdRp or RNA helicase domain between VcHV1 and each virus were 26–82% or 32–70%, respectively (Table 2). In both domains, VcHV1 had the highest identity with CHV3-GH2 (RdRp/RNA helicase; 82%/70%), CHV4-SR2 (76%/57%), and SsHV1-SZ-150 (74%/67%). Phylogenetic trees based on the RdRp or RNA helicase domain showed similar results, i.e., VcHV1, CHV3-GH2, CHV4-SR2, and SsHV1-SZ-150 clustered together, forming a clade within the family Hypoviridae that was distinct from CHV1-EP713 and CHV2-NB58 (Fig. 3D and F). 3.4. Biological effect of VcHV1 on V. ceratosperma Most hypoviruses, including CHV1, CHV2, and CHV3, reduce fungal virulence and/or other phenotypes, including pigmentation, sporulation, and laccase activity (Smart et al., 1999). To assess the effect of VcHV1 infection on the biological properties of V. ceratosperma, hyphal tipping from V. ceratosperma MVC86 was used to generate a VcHV1-free strain. Electrophoretic inspection (of total mycelial RNA) and RT-PCR (using VcHV1-specific primers) confirmed that one strain (MVC86-18(VF)) out of twenty was cured for VcHV1 (Fig. 4A). MVC86-18(VF) was indistinguishable from the

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parent strain in vitro (grown on PDA at 25 ◦ C in the dark), as judged by both colony morphology and mycelial growth (Fig. 4B). A virulence assay of MVC86 and MVC86-18(VF) on dormant apple twigs (cultivar “Fuji”) revealed the development of characteristic valsa cankers on twigs inoculated with either strain (data not shown); there was no significant difference in mean canker diameter (n = 9) between the two strains (Fig. 4C). In a similar assay on fresh apple twigs (cultivar “Fuji”), canker sizes again did not differ significantly between the two strains (data not shown). Thus, under the tested conditions, VcHV1 infection does not appear to affect the morphology or virulence of V. ceratosperma.

4. Discussion In the present study, we describe the molecular and biological characterization of a 9.5-Kb mycovirus-like dsRNA from V. ceratosperma isolate MVC86. Complete nucleotide sequence of the dsRNA reveals the presence of a 378-nt 5 -UTR, a 8823-nt ORF encoding a putative polyprotein of approximately 330 kDa (P330), and a 342-nt 3 -UTR (excluding the poly-A tail). A homology search revealed that P330 has significant sequence similarity to the polyproteins of hypoviruses, including CHV3, CHV4, and SsHV1, spanning four conserved protein domains. Moreover, both the 5 - and 3 -UTRs are highly conserved among the V. ceratosperma dsRNA (this study), CHV3, CHV4, and SsHV1. Based on these results, we propose that this dsRNA represents a novel mycovirus closely related to hypoviruses; we therefore have tentatively named the new hypovirus as Valsa ceratosperma hypovirus 1 (VcHV1). The VcHV1 polyprotein did not exhibit apparent sequence similarity to any viral coat proteins, indicating that the naked positive-strand ssRNA would be the VcHV1 genome, with the dsRNA representing the replicative form, as seen in other hypoviruses. The family Hypoviridae contains one genus (Hypovirus), to which the four hypovirus species from C. parasitica (CHV1–4) currently are assigned (Nuss and Hillman, 2011). Following the recent identification of SsHV1 (in S. sclerotiorum; Xie et al., 2011), VcHV1 from V. ceratosperma is the second example of a new hypovirus isolated from a non-Cryphonectria fungal host. These direct evidences strongly suggest that hypoviruses are widely distributed among filamentous fungi. Nonetheless, we note that VcHV1 was detected in only a single strain within a collection of over 100 isolates obtained from several different orchards (data not shown), indicating that VcHV1 has limited distribution within the V. ceratosperma population. The virus might represent a recent infection of this fungal host, or the spread of this virus within V. ceratosperma populations may be limited by some factor(s). Among VcHV1, CHV3, CHV4, and SsHV1, homology among the protease domains was lower than those of the UGT, RdRp, and RNA helicase domains, suggesting higher divergence among protease sequences. Phylogenetic analysis of protease domains among hypoviruses showed that VcHV1, CHV4, and SsHV1 cluster into the same group, while the protease domain of CHV3 was more similar to P29 of CHV1. Several papers have demonstrated that the papain-like proteases of prototypic hypovirus CHV1 (P29 and P48) are multifunctional proteins. P29 (encoded by CHV1-ORFA) is involved in reduction of pigmentation and sporulation of C. parasitica (Craven et al., 1993), enhancement of viral accumulation and transmission (Suzuki et al., 2003; Sun et al., 2006), genome rearrangements of mycoreovirus 1 (Sun and Suzuki, 2008; Tanaka et al., 2011), and suppression of RNA silencing (Segers et al., 2006), which is a counter-defense mechanism of fungi against viral infection (Segers et al., 2007). P48 (encoded by CHV1-ORFB) is involved in a slight reduction of sporulation and pigmentation, initiation of virus propagation, and vertical transmission, but does not contribute to the attenuation of virulence (Deng and Nuss, 2008).

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Thus, the divergence among hypoviral proteases may reflect functional differences. Our phylogenetic trees based on RdRp or RNA helicase domains indicate that VcHV1, CHV3, CHV4, and SsHV1 form a distinct clade within the Hypoviridae family, suggesting the existence of two lineages in the family and that these four viruses diverged from a common ancestor. These four hypoviruses additionally share three molecular features that distinguish the four from CHV1 and CHV2. First, the genomes of VcHV1, CHV3, CHV4, and SsHV1 are smaller (9.1–10.4 kb) than those of CHV1 (12.7 kb) and CHV2 (12.5 kb). Second, VcHV1, CHV3, CHV4, and SsHV1 encode a single large polyprotein; in contrast, CHV1 and CHV2 each consist of two ORFs. Third, VcHV1, CHV3, CHV4, and SsHV1 code for proteins with UGT domains; no homologous domain is found in CHV1 or CHV2. While UGT proteins are known to direct the biosynthesis of sterol glucosides, membrane-bound lipids typical of many eukaryotes (Warnecke et al., 1999), the biological role of the UGT domains of hypoviral polyproteins remains unclear. Collectively, the phylogenetic relationships and the genome features shared among VcHV1, CHV3, CHV4, and SsHV1 clearly distinguish them from CHV1 and CHV2. Therefore, we propose the establishment, within the family Hypoviridae, of a new genus that would consist of VcHV1, CHV3, CHV4, and SsHV1. We suggest that the potential new genus tentatively be named “Betahypovirus”, while the original genus Hypovirus be re-named to “Alphahypovirus”, which will consist of CHV1 and CHV2. VcHV1 infection did not affect fungal virulence or colony morphology of V. ceratsperma. As summarized in Smart et al. (1999), CHV1, CHV2, and CHV3 attenuate virulence, but impacts of these viruses on other fungal phenotypes (including pigmentation, sporulation, and laccase activity) are varied. Notably, CHV3 infection has no or mild effects on other fungal phenotypes altered by CHV1 or CHV2 infection. On the other hand, CHV4 does not affect fungal virulence (Enebak et al., 1994). SsHV1 with its satellite RNA affects colony morphology and virulence of S. sclerotiorum, but SsHV1 without the satellite RNA does not (Xie et al., 2011), indicating that the satellite RNA (but not SsHV1) is a key player in conferring hypovirulence to the fungus. These mild effects of VcHV1, CHV3, CHV4, and SsHV1 on fungal phenotypes are expected to be important for co-existence with the host fungus, consistent with the apparently cryptic nature of many mycoviruses, e.g., partitivirus, and may be an evolutionary trait of the lineage containing betahypoviruses. Acknowledgments We are grateful to the Program for Promotion of Basic and Applied Researches for Innovation in Bio-Oriented Industries for financial support during this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2012.02.008. References Chiba, S., Salaipeth, L., Lin, Y., Sasaki, A., Kanematsu, S., Suzuki, N., 2009. A novel bipartite double-stranded RNA mycovirus from the white root rot fungus Rosellinia necatrix: molecular and biological characterization, taxonomic considerations, and potential for biological control. J. Virol. 83, 12801–12812. Craven, M.G., Pawlyk, D.M., Choi, G.H., Nuss, D.L., 1993. Papain-like protease p29 as a symptom determinant encoded by a hypovirulence-associated virus of the chestnut blight fungus. J. Virol. 67, 6513–6521. Deng, F., Nuss, D.L., 2008. Hypovirus papain-like protease p48 is required for initiation but not for maintenance of virus RNA propagation in the chestnut blight fungus Cryphonectria parasitica. J. Virol. 82, 6369–6378.

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