研究者詳細 - 鈴木　信弘

論文 - 鈴木　信弘

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Effects of Defective Interfering RNA on Symptom Induction by, and Replication of, a Novel Partitivirus from a Phytopathogenic Fungus, Rosellinia necatrix 査読

Sotaro Chiba, Yu-Hsin Lin, Hideki Kondo, Satoko Kanematsu, Nobuhiro Suzuki

JOURNAL OF VIROLOGY 87 ( 4 ) 2330 - 2341 2013年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC MICROBIOLOGY

A novel mycovirus termed Rosellinia necatrix partitivirus 2 (RnPV2), isolated from a phytopathogenic fungus, Rosellinina necatrix strain W57, was molecularly and biologically characterized in both natural and experimental host fungi. Three double-stranded RNA (dsRNA) segments, dsRNA1, dsRNA2, and defective interfering dsRNA1 (DI-dsRNA1), whose sizes were approximately 2.0, 1.8, and 1.7 kbp, respectively, were detected in W57. While the dsRNA2 sequence, encoding the coat protein, was reported previously, dsRNA1 and DI-dsRNA1 were shown to encode competent and defective (truncated) RNA-dependent RNA polymerase, respectively. Artificial introduction of RnPV2 into an RNA silencing-defective, Dicer-like 2 knockout mutant (Delta dcl-2) of a nonnatural host, Cryphonectria parasitica (chestnut blight fungus), resulted in successful infection by the DI-dsRNA1-carrying and -free RnPV2. The DI-dsRNA1-free RnPV2 strain was characterized by a higher ratio of accumulation of the intact dsRNA1 to dsRNA2, enhanced replication and severer symptom expression, compared with the DI-carrying strain. These findings confirmed the nature of DI-dsRNA1 as a DI-RNA. Both viral strains replicated to higher levels in a Delta dcl-2 mutant than in a wild-type C. parasitica fungal strain (EP155) and induced severe symptoms in the Delta dcl-2 mutant but subtle symptoms in EP155, indicating that the host RNA silencing targets the partitivirus. No obvious phenotypic effects of infection by either virus strain were detected in the natural host fungus. These combined results represent the first example of a partitivirus with DI-RNA that alters viral symptom induction in a host-dependent manner.

DOI： 10.1128/JVI.02835-12

Evidence for negative-strand RNA virus infection in fungi 査読

Hideki Kondo, Sotaro Chiba, Kazuhiro Toyoda, Nobuhiro Suzuki

Virology 435 ( 2 ) 201 - 209 2013年1月

記述言語：英語掲載種別：研究論文（学術雑誌）

Fungal viruses comprise two groups: a major group of five families with double-stranded RNA genomes and a minor group with positive-sense single-stranded (ss)RNA genomes. Although many fungal viruses have been identified, no negative-stranded (-)ssRNA mycoviruses have been reported. Here we present two lines of evidence suggesting the presence of (-)ssRNA viruses in filamentous fungi based on an exhaustive search using extant (-)ssRNA viruses as queries. This revealed (-)ssRNA virus L protein-like sequences in the genome of a phytopathogenic obligate ascomycete, Erysiphe pisi. A similar search for (-)ssRNA viruses in fungal transcriptome shotgun assembly libraries demonstrated that two independent libraries from Sclerotinia homoeocarpa, another phytopathogenic ascomycete, contained several sequences considered to correspond to the entire mononegavirus L gene and likely originating from an infecting (-)ssRNA virus. These results provide strong evidence for both ancient and extant (-)ssRNA virus infections in fungi. © 2012 Elsevier Inc.

DOI： 10.1016/j.virol.2012.10.002

Viruses of the white root rot fungus, Rosellinia necatrix. 査読国際誌

Hideki Kondo, Satoko Kanematsu, Nobuhiro Suzuki

Advances in virus research 86 177 - 214 2013年

記述言語：英語掲載種別：研究論文（学術雑誌）

Rosellinia necatrix is a filamentous ascomycete that is pathogenic to a wide range of perennial plants worldwide. An extensive search for double-stranded RNA of a large collection of field isolates led to the detection of a variety of viruses. Since the first identification of a reovirus in this fungus in 2002, several novel viruses have been molecularly characterized that include members of at least five virus families. While some cause phenotypic alterations, many others show latent infections. Viruses attenuating the virulence of a host fungus to its plant hosts attract much attention as agents for virocontrol (biological control using viruses) of the fungus, one of which is currently being tested in experimental fields. Like the Cryphonectria parasitica/viruses, the R. necatrix/viruses have emerged as an amenable system for studying virus/host and virus/virus interactions. Several techniques have recently been developed that enhance the investigation of virus etiology, replication, and symptom induction in this mycovirus/fungal host system.

DOI： 10.1016/B978-0-12-394315-6.00007-6

A novel quadripartite dsRNA virus isolated from a phytopathogenic filamentous fungus, Rosellinia necatrix 査読

Yu-Hsin Lin, Sotaro Chiba, Akio Tani, Hideki Kondo, Atsuko Sasaki, Satoko Kanematsu, Nobuhiro Suzuki

VIROLOGY 426 ( 1 ) 42 - 50 2012年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

Here we report the biological and molecular attributes of a novel dsRNA virus isolated from Rosellinia necatrix, a filamentous phytopathogenic fungus. The virus, termed Rosellinia necatrix quadrivirus 1 (RnQV1), forms rigid spherical particles approximately 45 nm in diameter in infected mycelia. The particles contain 4 dsRNA segments, dsRNA1 to dsRNA4, with a size range of 4.9 to 3.7 kbp, each possessing a single large ORF. A comparison of the virus-infected and -cured isogenic fungal strains suggested that RnQV1 infection has no appreciable phenotypic effects. Phylogenetic analysis using the dsRNA3-encoded RdRp sequence revealed that RnQV1 is more distantly related to quadripartite chrysoviruses than to monopartite totiviruses, and is placed in a distinct group from other mycoviruses. No significant sequence similarities were evident between known proteins and RnQV1 structural proteins shown to be encoded by dsRNA2 or dsRNA4. These suggest that RnQV1 is a novel latent virus, belonging to a new family. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2012.01.013

Mycoreovirus genome alterations: similarities to and differences from rearrangements reported for other reoviruses 査読

Toru Tanaka, Ana Eusebio-Cope, Liying Sun, Nobuhiro Suzuki

FRONTIERS IN MICROBIOLOGY 3 186 2012年

記述言語：英語出版者・発行元：FRONTIERS RESEARCH FOUNDATION

The family Reoviridae is one of the largest virus families with genomes composed of 9-12 double-stranded RNA segments. It includes members infecting organisms from protists to humans. It is well known that reovirus genomes are prone to various types of genome alterations including intragenic rearrangement and reassortment under laboratory and natural conditions. Recently distinct genetic alterations were reported for members of the genus Mycoreovirus, Mycoreovirus 1 (MyRV1), and MyRV3 with 11 (S1-S11) and 12 genome segments (S1-S12), respectively. While MyRV3 S8 is lost during subculturing of infected host fungal strains, MyRV1 rearrangements undergo alterations spontaneously and inducibly. The inducible MyRV1 rearrangements are different from any other previous examples of reovirus rearrangements in their dependence on an unrelated virus factor, a multifunctional protein, p29, encoded by a distinct virus Cryphonectria parasitica hypovirus 1 (CHV1). A total of 5 MyRV1 variants with genome rearranged segments (S1-S3, S6 and S10) are generated in the background of a single viral strain in the presence of CHV1 p29 supplied either transgenically or by coinfection. MyRV1 S4 and S10 are rearranged, albeit very infrequently, in a CHV1 p29 independent fashion. A variant of MyRV1 with substantial deletions in both S4 and S10, generated through a combined reassortment and rearrangement approach, shows comparable replication levels to the wild-type MyRV1. In vivo and in vitro interactions of CHV1 p29 and MyRV1VP9 are implicated in the induction of MyRV1 rearrangements. However, the mechanism underlying p29-mediated rearrangements remains largely unknown. MyRV1 S4 rearrangements spontaneously occurred independently of CHV1 p29. In the absence of reverse genetics systems for mycoreoviruses, molecular and biological characterization of these MyRV1 and MyRV3 variants contribute to functional analyses of the protein products encoded by those rearranged segments.

DOI： 10.3389/fmicb.2012.00186

Rearrangements of mycoreovirus 1 S1, S2 and S3 induced by the multifunctional protein p29 encoded by the prototypic hypovirus Cryphonectria hypovirus 1 strain EP713 査読

Toru Tanaka, Liying Sun, Kouhei Tsutani, Nobuhiro Suzuki

JOURNAL OF GENERAL VIROLOGY 92 ( Pt 8 ) 1949 - 1959 2011年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SOC GENERAL MICROBIOLOGY

Mycoreovirus 1 (MyRV1), a member of the family Reoviridae possessing a genome consisting of 11 dsRNA segments (S1-S11), infects the chestnut blight fungus and reduces its virulence (hypovirulence). Studies have previously demonstrated reproducible induction of intragenic rearrangements of MyRV1 S6 (S6L: almost full-length duplication) and S10 (S10ss: internal deletion of three-quarters of the ORF), mediated by the multifunctional protein p29 encoded by the prototype hypovirus, Cryphonectria hypovirus 1 (CHV1) strain EP713, of the family Hypoviridae with ssRNA genomes. The current study showed that CHV1 p29 also induced rearrangements of the three largest MyRV1 segments, S1, S2 and S3, which encode structural proteins. These rearranged segments involved in-frame extensions of almost two-thirds of the ORFs (S1L, S2L and S3L, respectively), which is rare for a reovirus rearrangement. MyRV1 variants carrying S1L, S2L or S3L always contained S10ss (MyRV1/S1L+S10ss2, MyRV1/S2L+S10ss2 or MyRV1/S3L+S10ss2). Levels of mRNAs for the rearranged and co-existing unaltered genome segments in fungal colonies infected with each of the MyRV1 variants appeared to be comparable to those for the corresponding normal segments in wild-type MyRV1-infected colonies, suggesting that the rearranged segments were fully competent for packaging and transcription. Protein products of the rearranged segments were detectable in fungal colonies infected with S2L MyRV1/S2L+S10ss2 and S3L MyRV1/S3L+S10ss2, whilst S1L-encoded protein remained undetectable. S1L, S2L and S3L were associated with enhancement of the aerial hyphae growth rate. This study has provided additional examples of MyRV1 intragenic rearrangements induced by p29, and suggests that normal S1, S2 and S3 are required for the symptoms caused by MyRV1.

DOI： 10.1099/vir.0.031138-0

Widespread endogenization of genome sequences of non-retroviral rna viruses into plant genomes

Sotaro Chiba, Hideki Kondo, Akio Tani, Daisuke Saisho, Wataru Sakamoto, Satoko Kanematsu, Nobuhiro Suzuki

PLoS Pathogens 7 ( 7 ) 2011年7月

掲載種別：研究論文（学術雑誌）

Non-retroviral RNA virus sequences (NRVSs) have been found in the chromosomes of vertebrates and fungi, but not plants. Here we report similarly endogenized NRVSs derived from plus-, negative-, and double-stranded RNA viruses in plant chromosomes. These sequences were found by searching public genomic sequence databases, and, importantly, most NRVSs were subsequently detected by direct molecular analyses of plant DNAs. The most widespread NRVSs were related to the coat protein (CP) genes of the family Partitiviridae which have bisegmented dsRNA genomes, and included plant- and fungus-infecting members. The CP of a novel fungal virus (Rosellinia necatrix partitivirus 2, RnPV2) had the greatest sequence similarity to Arabidopsis thaliana ILR2, which is thought to regulate the activities of the phytohormone auxin, indole-3-acetic acid (IAA). Furthermore, partitivirus CP-like sequences much more closely related to plant partitiviruses than to RnPV2 were identified in a wide range of plant species. In addition, the nucleocapsid protein genes of cytorhabdoviruses and varicosaviruses were found in species of over 9 plant families, including Brassicaceae and Solanaceae. A replicase-like sequence of a betaflexivirus was identified in the cucumber genome. The pattern of occurrence of NRVSs and the phylogenetic analyses of NRVSs and related viruses indicate that multiple independent integrations into many plant lineages may have occurred. For example, one of the NRVSs was retained in Ar. thaliana but not in Ar. lyrata or other related Camelina species, whereas another NRVS displayed the reverse pattern. Our study has shown that single- and double-stranded RNA viral sequences are widespread in plant genomes, and shows the potential of genome integrated NRVSs to contribute to resolve unclear phylogenetic relationships of plant species. © 2011 Chiba et al.

DOI： 10.1371/journal.ppat.1002146

Rice Dwarf Viruses with Dysfunctional Genomes Generated in Plants Are Filtered Out in Vector Insects: Implications for the Origin of the Virus 査読

Yingying Pu, Akira Kikuchi, Yusuke Moriyasu, Masatoshi Tomaru, Yan Jin, Haruhisa Suga, Kyoji Hagiwara, Fusamichi Akita, Takumi Shimizu, Osamu Netsu, Nobuhiro Suzuki, Tamaki Uehara-Ichiki, Takahide Sasaya, Taiyun Wei, Yi Li, Toshihiro Omura

JOURNAL OF VIROLOGY 85 ( 6 ) 2975 - 2979 2011年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC MICROBIOLOGY

Rice dwarf virus (RDV), with 12 double-stranded RNA (dsRNA) genome segments (S1 to S12), replicates in and is transmitted by vector insects. The RDV-plant host-vector insect system allows us to examine the evolution, adaptation, and population genetics of a plant virus. We compared the effects of long-term maintenance of RDV on population structures in its two hosts. The maintenance of RDV in rice plants for several years resulted in gradual accumulation of nonsense mutations in S2 and S10, absence of expression of the encoded proteins, and complete loss of transmissibility. RDV maintained in cultured insect cells for 6 years retained an intact protein-encoding genome. Thus, the structural P2 protein encoded by S2 and the nonstructural Pns10 protein encoded by S10 of RDV are subject to different selective pressures in the two hosts, and mutations accumulating in the host plant are detrimental in vector insects. However, one round of propagation in insect cells or individuals purged the populations of RDV that had accumulated deleterious mutations in host plants, with exclusive survival of fully competent RDV. Our results suggest that during the course of evolution, an ancestral form of RDV, of insect virus origin, might have acquired the ability to replicate in a host plant, given its reproducible mutations in the host plant that abolish vector transmissibility and viability in nature.

DOI： 10.1128/JVI.02147-10

[Mycoviruses and virocontrol]. 査読

Chiba S, Kondo H, Kanematsu S, Suzuki N

Uirusu 60 ( 2 ) 163 - 176 2010年12月

Complexity in Influenza Virus Targeted Drug Design: Interaction with Human Sialidases 査読

Leonard M. G. Chavas, Ryuichi Kato, Nobuhiro Suzuki, Mark von Itzstein, Maretta C. Mann, Robin J. Thomson, Jeffrey C. Dyason, Jennifer McKimm-Breschkin, Paola Fusi, Cristina Tringali, Bruno Venerando, Guido Tettamanti, Eugenio Monti, Soichi Wakatsuki

JOURNAL OF MEDICINAL CHEMISTRY 53 ( 7 ) 2998 - 3002 2010年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER CHEMICAL SOC

With the global spread of the pandemic H1N1 and the ongoing pandemic potential of the H5N1 subtype, the influenza virus represents one of the most alarming viruses spreading worldwide. The influenza virus sialidase is an effective drug target, and a number of inhibitors are clinically effective against the virus (zanamivir, oseltamivir, peramivir). Here we report structural and biochemical studies of the human cytosolic sialidase Neu2 with influenza virus sialidase-targeting drugs and related compounds.

DOI： 10.1021/jm100078r

Mycoreovirus 1 S4-coded protein is dispensable for viral replication but necessary for efficient vertical transmission and normal symptom induction 査読

Ana Eusebio-Cope, Liying Sun, Bradley I. Hillman, Nobuhiro Suzuki

VIROLOGY 397 ( 2 ) 399 - 408 2010年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

Rearrangements of two segments, S6 and S10, of Mycoreovirus 1 (MyRV1), a member of the family Reoviridae, were previously shown to be induced at a high rate by the multifunctional protein p29 encoded by a distinct ssRNA virus, the prototype hypovirus CHV1-EP713 (Sun and Suzuki, RNA 14, 2557-2571, 2008). Here we report the occurrence of rearrangements of MyRV1 S4. albeit at a very low frequency, in the absence of CHV1 p29, resulting in internal 80-90% deletions of the open reading frame (ORF) in S4. Comparative analyses of fungal strains infected by wild-type MyRV1 and its variants carrying rearrangements of S4, S4 Plus S10 and S10 indicated that S4-encoded VP4, like VP10, is non-essential for virus replication but required for efficient vertical transmission and symptom expression caused by MyRV1. This is the first example of a reovirus variant that carries deletions of over 75% of the ORFs in two genome segments and is still replication-competent. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2009.11.035

Overexpression of microRNA395c or 395e affects differently the seed germination of arabidopsis thaliana under stress conditions 査読

Joo Yeol Kim, Hwa Jung Lee, Hyun Ju Jung, Kazuyuki Maruyama, Nobuhiro Suzuki, Hunseung Kang

Planta 232 ( 6 ) 1447 - 1454 2010年

記述言語：英語掲載種別：研究論文（学術雑誌）

The Arabidopsis genome encodes six members of microRNA395 (miR395) family previously determined to regulate the expression of ATP sulfurylase (APS) and the sulfate transporter SULTR2
1. However, the mRNA targets for the individual miR395 family members and the biological consequences produced by target gene regulation of each miR395 remain to be identified. In this study, a transgenic approach was employed to determine the mRNA targets for each miR395 family member as well as the role each member plays in plant growth under abiotic stress conditions. Over-expression of miR395c or miR395e retarded and accelerated, respectively, the seed germination of Arabidopsis under high salt or dehydration stress conditions. Despite a single nucleotide difference between miR395c and miR395e, the cleavage of mRNA targets,APS1,APS3, APS4 and SULTR2
1, was not same in miR395c- and miR395e-overexpressing plants. These results demonstrate that a given miRNA family containing a single nucleotide difference can guide the cleavage of various mRNA targets, thereby acting as a positive or negative regulator of seed germination under stress. © Springer-Verlag 2010.

DOI： 10.1007/s00425-010-1267-x

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 査読

Sotaro Chiba, Lakha Salaipeth, Yu-Hsin Lin, Atsuko Sasaki, Satoko Kanematsu, Nobuhiro Suzuki

JOURNAL OF VIROLOGY 83 ( 24 ) 12801 - 12812 2009年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC MICROBIOLOGY

White root rot, caused by the ascomycete Rosellinia necatrix, is a devastating disease worldwide, particularly in fruit trees in Japan. Here we report on the biological and molecular properties of a novel bipartite double-stranded RNA (dsRNA) virus encompassing dsRNA-1 (8,931 bp) and dsRNA-2 (7,180 bp), which was isolated from a field strain of R. necatrix, W779. Besides the strictly conserved 5' (24 nt) and 3' (8 nt) terminal sequences, both segments show high levels of sequence similarity in the long 5' untranslated region of approximately 1.6 kbp. dsRNA-1 and -2 each possess two open reading frames (ORFs) named ORF1 to -4. Although the protein encoded by 3'-proximal ORF2 on dsRNA-1 shows sequence identities of 22 to 32% with RNA-dependent RNA polymerases from members of the families Totiviridae and Chrysoviridae, the remaining three virus-encoded proteins lack sequence similarities with any reported mycovirus proteins. Phylogenetic analysis showed that the W779 virus belongs to a separate clade distinct from those of other known mycoviruses. Purified virions similar to 50 nm in diameter consisted of dsRNA-1 and -2 and a single major capsid protein of 135 kDa, which was shown by peptide mass fingerprinting to be encoded by dsRNA-1 ORF1. We developed a transfection protocol using purified virions to show that the virus was responsible for reduction of virulence and mycelial growth in several host strains. These combined results indicate that the W779 virus is a novel bipartite dsRNA virus with potential for biological control (virocontrol), named Rosellinia necatrix megabirna-virus 1 (RnMBV1), that possibly belongs to a new virus family.

DOI： 10.1128/JVI.01830-09

Characteristics of Hemophagocytic Lymphohistiocytosis in Neonates: A Nationwide Survey in Japan 査読

Nobuhiro Suzuki, Akira Morimoto, Shouichi Ohga, Kazuko Kudo, Yasushi Ishida, Eiichi Ishii

JOURNAL OF PEDIATRICS 155 ( 2 ) 235 - 238 2009年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：MOSBY-ELSEVIER

Objective To assess the etiology, prognosis, and appropriate treatment of hemophagocytic lymphohistiocytosis (HLH) in neonates.
Study design We collected information on neonates in whom HLH was diagnosed between 1997 and 2007 from participating members of the Japanese Society of Pediatric Hematology.
Results HLH was diagnosed in 20 patients within 4 weeks after birth. Of the diagnostic criteria for HLH-2004, the incidence of fever was quite low in preterm infants, and hypertriglyceridemia and neutropenia were uncommon. Familial HLH (n = 6) or severe combined immunodeficiency-associated HLH (n = 1) was diagnosed in 7 patients, and 2 of them have survived. Herpes simplex virus-associated HLH was diagnosed in 6 patients, and 2 of them have survived. The overall survival rate for the 20 patients was 40%.
Conclusions HLH is rare in neonates and has a poor prognosis. Early diagnosis and immediate treatment are required when considering the possibility of herpes simplex virus-associated or familial HLH. (J Pediatr 2009; 155:235-8).

DOI： 10.1016/j.jpeds.2009.02.050

Occurrence of the African subgroup (Ia) of BK polyomavirus in younger Japanese children 査読

Kaori Tanaka, Tsukasa Hori, Naoki Hatakeyama, Masaki Yamamoto, Rumiko Takayama, Yuko Yoto, Nobuhiro Suzuki, Toshiaki Hayashi, Yukiho Ikeda, Hiroshi Ikeda, Tadao Ishida, Hiroyuki Tsutsumi

MICROBIOLOGY AND IMMUNOLOGY 53 ( 6 ) 319 - 322 2009年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：WILEY-BLACKWELL PUBLISHING, INC

BK polyomavirus (BKV) is ubiquitous among humans, usually infecting them asymptomatically during childhood. BKV persists in renal tissue of individuals and their progeny are excreted in urine, particularly in immunocompromised patients. JC virus, another human polyomavirus, has been considered to be transmitted from parents to children during prolonged cohabitation. However, BKV has been supposed to be transmitted not only within but also outside the family. In the present study, to clarify this possibility, we analyzed phylogenetically 35 BKV which were excreted in the urine by Japanese children and adults undergoing stem cell transplantation. Subtypes I, III and IV were detected in 15, two and one children and in 15, one and one adults, respectively. Among 15 subtype I isolates from children, three, four and eight belonged to subgroups Ia, Ib-1 and Ic, respectively. All the three children from whom Ia was detected were less than 9 years old. In contrast in the adults, three subtype I belonged to Ib-1 and the other 12 to Ic. These findings may reflect the recent transmission of BKV Ia strains to Japanese children.

DOI： 10.1111/j.1348-0421.2009.00129.x

Coupled termination/reinitiation for translation of the downstream open reading frame B of the prototypic hypovirus CHV1-EP713 査読

Li-hua Guo, Liying Sun, Sotaro Chiba, Hiroyuki Araki, Nobuhiro Suzuki

NUCLEIC ACIDS RESEARCH 37 ( 11 ) 3645 - 3659 2009年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：OXFORD UNIV PRESS

Cryphonectria hypovirus 1 (CHV1), associated with the picorna-like superfamily, infects the chestnut blight fungus and attenuates the virulence of the host fungus. The genomic RNA of the virus has two continuous open reading frames, A and B, separated by the pentanucleotide UAAUG. We present here evidence suggesting that ORF B is translated from genome-sized virus mRNA by a coupled termination/reinitiation mechanism mediated by the pentamer. In the coupled translation, the overlapping UAA and AUG triplets serve as the stop codon of ORF A and the initiator of ORF B, respectively. This was established by the use of a luciferase assay with a basic construct containing the ORF A sequence and the firefly luciferase gene while retaining the pentamer between the two coding sequences. The proportion of ribosomes reinitiating translation after terminating was determined to be 2.54.4 by three independent assay systems in fungal and insect cells. Use of a series of mutant constructs identified two sequence elements, the pentamer and the p40 sequence, that affect the efficiency of coupled translation and virus replication. Together, these results provide the first example of coupled translation facilitated by the pentanucleotide UAAUG in the kingdom Fungi. The mechanism by which the preceding p40-coding sequence promotes reinitiation is discussed.

DOI： 10.1093/nar/gkp224

Cytological and electrophoretic karyotyping of the chestnut blight fungus Cryphonectria parasitica 査読

Ana Eusebio-Cope, Nobuhiro Suzuki, Hamid Sadeghi-Garmaroodi, Masatoki Taga

FUNGAL GENETICS AND BIOLOGY 46 ( 4 ) 342 - 351 2009年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

The karyotypes of nine strains including three transformants of the chestnut blight fungus Cryphonectria parasitica were analyzed by pulsed-field gel electrophoresis (PFGE) and cytology using a fluorescence microscope. Cytology of the mitotic metaphase showed n = 9 for both standard strain EP155 and field strain GH2 infected by Cryphonectria hypovirus 3. Chromosomes were morphologically characterized by size, heterochromatic segment, and constriction. PFGE resolved 5 or 6 chromosomal DNA bands ranging from 3.3 Mbp to 9.7 Mbp, but accurate determination of the chromosome number was hampered by clumping of some bands. Banding profiles in PFGE were similar among the strains except for GH2, in which a chromosome translocation was detected by Southern blot analysis. By integrating the data from cytology and PFGE, the genome size of C parasitica was estimated to be ca. 50 Mbp. This is the first report of a cytological karyotype in the order Diaporthales. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.fgb.2009.01.005

Intragenic rearrangements of a mycoreovirus induced by the multifunctional protein p29 encoded by the prototypic hypovirus CHV1-EP713 査読

Liying Sun, Nobuhiro Suzuki

RNA-A PUBLICATION OF THE RNA SOCIETY 14 ( 12 ) 2557 - 2571 2008年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT

Mycoreovirus 1 (MyRV1), a member of the Reoviridae family possessing a genome consisting of 11 dsRNA segments (S1-S11), and the prototype hypovirus (CHV1-EP713) of the Hypoviridae family, which is closely related to the monopartite picorna-like superfamily with a ssRNA genome, infect the chestnut blight fungus and cause virulence attenuation and distinct phenotypic alterations in the host. Here, we present evidence for reproducible induction of intragenic rearrangements of MyRV1 S6 and S10, mediated by the multifunctional protein p29 encoded by CHV1. S6 and S10 underwent an almost full-length ORF duplication (S6L) and an internal deletion of three-fourths of the ORF (S10ss). No significant influence on symptom induction in the fungal host was associated with the S6L rearrangement. In contrast, S10-encoded VP10, while nonessential for MyRV1 replication, was shown to contribute to virulence reduction and reduced growth of aerial mycelia. Furthermore, p29 was found to copurify with MyRV1 genomic RNA and bind to VP9 in vitro and in vivo, suggesting direct interactions of p29 with the MyRV1 replication machinery. This study provides the first example of a viral factor involved in RNA genome rearrangements of a different virus and shows its usefulness as a probe into the mechanism of replication and symptom expression of a heterologous virus.

DOI： 10.1261/rna.1125408

Characterization of mutants of the chestnut blight fungus (Cryphonectria parasitica) with unusual hypovirus symptoms 査読

Md. Iqbal Faruk, Masatoshi Izumimoto, Nobuhiro Suzuki

JOURNAL OF GENERAL PLANT PATHOLOGY 74 ( 6 ) 425 - 433 2008年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER TOKYO

The type virus of the family Hypoviridae, Cryphonectria hypovirus 1 strain EP713 (CHV1-EP713), infects Cryphonectria parasitica, the filamentous causal fungus of chestnut blight, and reduces its virulence. This pathosystem serves as a model to study fungus-mycovirus interactions. We previously developed a genetic screening protocol for host factors associated with symptom induction by CHV1-EP713 and its mutants. In the procedure the standard field fungal isolate EP155 was transformed by cDNA from a mild hypovirus mutant Cys(72), launching virus infection, and mutagenized by random plasmid insertion with pHygR conferring hygromycin resistance. We now report an extension of the study to characterize different mutant strains, with different phenotypes than their parental strain TCys(72)-1. TCys(72)-1 is moderately reduced in pigmentation and sporulation compared to the uninfected wild-type strain EP155. Mutants sfb1, sfb2 and k202 were characterized biologically and molecularly in comparison to the previously isolated mutant (namA) and the parental strain. These mutants harbored one (sfb1) or more copies (sfb2 and k202) of the mutagenic plasmid, pHygR. The three mutants had similar biological attributes; that is, vegetative growth rate, conidiation and virulence (assay on apples) was reduced on potato dextrose agar media, relative to the parental strain and pigmentation was the same or slightly increased. Interestingly, viral dsRNA accumulation levels were apparently unaltered in these mutants. The screening method was efficient for mining fungal mutants with unusual hypovirus symptoms. Further, characterization of the mutants provides interesting insights into symptom induction by the hypovirus.

DOI： 10.1007/s10327-008-0121-5

A host factor involved in hypovirus symptom expression in the chestnut blight fungus, Cryphonectria parasitica 査読

M. Iqbal Faruk, Ana Eusebio-Cope, Nobuhiro Suzuki

JOURNAL OF VIROLOGY 82 ( 2 ) 740 - 754 2008年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC MICROBIOLOGY

The prototype hypovirus CRV1-EP713 causes virulence attenuation and severe suppression of asexual sporulation and pigmentation in its host, the chestnut blight fungus, Cryphonectria parasitica. We identified a factor associated with symptom induction in C. parasitica using a transformation of C. parasitica strain EP155 with a full-length cDNA clone from a mild mutant virus strain, Cys(72). This was accomplished by using mutagenesis of the transformant fungal strain TCys(72)-1 by random integration of plasmid pHygR, conferring hygromycin resistance. The mutant, nam4 (after nami-gata, meaning wave shaped), showed an irregular fungal morphology with reduced conidiation and pigmentation while retaining similar levels of virulence and virus accumulation relative to TCys(72)-1- or Cys (72)-infected strain EP155. However, the colony morphology of virus-cured namA (VC-namA) was indistinguishable from those of EP155 and virus-cured TCys(72)-1 [VC-TCys(72)-1]. The phenotypic difference between VC-namA and VC-TCys(72)-1 was found only when these strains infected with the wild type or certain mutant CHV1-EP713 strains but not when infected with Mycoreoviras 1. Sequence analysis of inverse-PCR-amplified genomic DNA fragments and cDNA identified the insertion site of the mutagenic plasmid in exon 8 of the nam-1 gene. NAM-1, comprising 1,257 amino acids, shows sequence similarities to counterparts from other filamentous fungi and possesses the CorA domain that is conserved in a class of Mg2+ transporters from prokaryotes and eukaryotes. Complementation assays using the wild-type and mutant alleles and targeted disruption of nam-1 showed that nam-1 with an extension of the pHygR-derived sequence contributed to the altered phenotype in the namA mutant. The molecular mechanism underlying virus-specific fungal symptom modulation in VC-namA is discussed.

DOI： 10.1128/JVI.02015-07

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