Faculty Profiles - SUZUKI Nobuhiro

Papers - SUZUKI Nobuhiro

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Baculovirus expression of the 11 mycoreovirus-1 genome segments and identification of the guanylyltransferase-encoding segment

S. Supyani, Bradley I. Hillman, Nobuhiro Suzuki

JOURNAL OF GENERAL VIROLOGY 88 342 - 350 2007.1

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC GENERAL MICROBIOLOGY

The type member Mycoreovirus 1 (MyRV-1) of a newly described genus, Mycoreovirus, isolated from a hypovirulent strain 9B21 of the chestnut blight fungus, has a genome composed of 11 dsRNA segments (S1-S11). All of the segments have single ORFs on their capped, positive-sense strands. Infection of insect cells by baculovirus recombinants carrying full-length cDNAs of S1-S1 1 resulted in overexpression of protein products of the expected sizes, based on their deduced amino acid sequences. This expression system was utilized to identify the S3-encoded protein (VP3) as a guanylyltransferase by an autoguanylylation assay, in which only VP3 was radiolabelled with [alpha-(32) P]GTP. A series of progressive N-terminal and C-terminal deletion mutants was made to localize the autoguanylylation-active site of VP3 to aa residues 133-667. Within this region, a sequence stretch (aa 170-250) with relatively high sequence similarity to homologues of two other mycoreoviruses and two coltivinuses was identified. Site-directed mutagenesis of conserved aa residues revealed that H233, H242, Y243, F244 and F246, but not K172 or K202, play critical roles in guanylyltransferase activities. Together with broader sequence alignments of 'turreted' reoviruses, these results supported the a/vxxHx(8)Hyf/lvf motif, originally noted for orthoreovirus and aquareoviruses, as an active site for guanylyltransferases of viruses within the Orthoreovirus, Aquareovirus, Cypovirus, Oryzavirus, Fijivirus, Coltivirus and Mycoreovirus genera, as well as for the proposed Dinovernavirus genus.

DOI： 10.1099/vir.0.82318-0

Synergism between a mycoreovirus and a hypovirus mediated by the papain-like protease p29 of the prototypic hypovirus CHV1-EP713 Reviewed

Liying Sun, Donald L. Nuss, Nobuhiro Suzuki

JOURNAL OF GENERAL VIROLOGY 87 3703 - 3714 2006.12

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC GENERAL MICROBIOLOGY

Infection of the chestnut blight fungus, Cryphonectria parasitica, by the prototypic hypovirus Cryphonectria hypovirus 1-EP713 (CHV1-EP713) or by the type member, Mycoreovirus 1-Cp9B21 (MyRV1-Cp9B21), of a novel genus (Mycoreovirus) of the family Reoviridae results in hypovirulence, but with a different spectrum of phenotypic changes. The former virus depresses pigmentation and conidiation dramatically, whilst the latter virus has little effect on these processes. This study showed that double infection by the two viruses resulted in a phenotype similar to that of CHV1-EP713 singly infected colonies, but with further decreased levels of host conidiation and vegetative growth and increased levels of MyRV1-Cp9B21 genomic dsRNA accumulation (twofold) and vertical transmission (sixfold). In contrast, CHV1-EP713 RNA accumulation was not altered by MyRV1-Cp9B21 infection. It was also found that the papain-like cysteine protease p29, encoded by CHV1-EP713 ORF A, contributes to the phenotypic alterations and transactivation of MyRV1-Cp9B21 replication and transmission. Chromosomally expressed p29 was able to increase MyRV1-Cp9B21 vertical transmission by more than twofold and genomic RNA accumulation by 80 %. Transactivation was abolished by Cys -> Gly mutations at p29 residues 70 and 72 located within the previously identified symptom-determinant domain required for suppression of host pigmentation and sporulation and p29-mediated in trans enhancement of homologous Delta p29 mutant virus RNA replication. Transactivation was not altered by Ser substitutions at the p29 protease catalytic residue Cys(162). These results indicated a link between p29-mediated enhancement of heterologous virus accumulation and transmission and p29-mediated host symptom expression. The role of p29 as a suppressor of RNA silencing is discussed.

DOI： 10.1099/vir0.82213-0

Pns4 of rice dwarf virus is a phosphoprotein, is localized around the viroplasm matrix, and forms minitubules Reviewed

T. Wei, A. Kikuchi, N. Suzuki, T. Shimizu, K. Hagiwara, H. Chen, T. Omura

Archives of Virology 151 ( 9 ) 1701 - 1712 2006.9

Language：English Publishing type：Research paper (scientific journal)

Rice dwarf virus (RDV), a member of the family Reoviridae, has a 12-segmented dsRNA genome. Seven segments, designated S1, S2, S3, S5, S7, S8, and S9, encode structural proteins, while the remainder encode nonstructural proteins. One of the nonstructural proteins, Pns4, which is encoded by S4, was characterized. Pns4 was a phosphorylatable substrate in a phosphorylation assay in vivo
it associated with large cytoplasmic fibrils and formed novel minitubules in infected cultured cells of its leafhopper insect vector, as revealed by immunofluorescence and immunoelectron microscopy. Early in infection, Pns4 was detected at the periphery of the viroplasm, and it was then observed on amorphous or fibrillar inclusions, which were identified as bundles of minitubules, at later stages of infection. Since viroplasms are believed to be the site of RDV replication, the intracellular location of Pns4 suggests that this protein might be involved in the process of assembly of the RDV virion. © Springer-Verlag 2006.

DOI： 10.1007/s00705-006-0757-4

The spread of Rice dwarf virus among cells of its insect vector exploits virus-induced tubular structures Reviewed

Taiyun Wei, Akira Kikuchi, Yusuke Moriyasu, Nobuhiro Suzuki, Takumi Shimizu, Kyoji Hagiwara, Hongyan Chen, Mami Takahashi, Tamaki Ichiki-Uehara, Toshihiro Omura

JOURNAL OF VIROLOGY 80 ( 17 ) 8593 - 8602 2006.9

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

Various cytopathological structures, known as inclusion bodies, are formed upon infection of cultured leafhopper cells by Rice dwarf virus, a member of the family Reoviridae. These structures include tubules of approximately 85 nm in diameter which are composed of the nonstructural viral protein Pns10 and contain viral particles. Such tubular structures were produced in heterologous non-host insect cells that expressed PnsIO of the virus. These tubules, when associated with actin-based filopodia, were able to protrude from the surface of cells and to penetrate neighboring cells. A binding assay in vitro revealed the specific binding of Pns10 to actin. Infection of clusters of cells was readily apparent 5 days after inoculation at a low multiplicity of infection with the virus, even in the presence of neutralizing antibodies. However, treatment of host cells with drugs that inhibited the elongation of actin filaments abolished the extension of PnsIO tubules from the surface of cells, with a significant simultaneous decrease in the extent of infection of neighboring cells. These results together revealed a previously undescribed aspect of the intercellular spread of Rice dwarf virus, wherein the virus exploits tubules composed of a nonstructural viral protein and actin-based filopodia to move into neighboring cells.

DOI： 10.1128/JVI.00537-06

Pns12 protein of Rice dwarf virus is essential for formation of viroplasms and nucleation of viral-assembly complexes Reviewed

Taiyun Wei, Takumi Shimizu, Kyoji Hagiwara, Akira Kikuchi, Yusuke Moriyasu, Nobuhiro Suzuki, Hongyan Chen, Toshihiro Omura

Journal of General Virology 87 ( 2 ) 429 - 438 2006.2

Language：English Publishing type：Research paper (scientific journal)

Cytoplasmic inclusion bodies, known as viroplasms or viral factories, are assumed to be the sites of replication of members of the family Reoviridae. Immunocytochemical and biochemical analyses were carried out to characterize the poorly understood viroplasms of the phytoreovirus Rice dwarf virus (RDV). Within 6 h of inoculation of cells, viroplasms, namely discrete cytoplasmic inclusions, were formed that contained the non-structural proteins Pns6, Pns11 and Pns12 of RDV, which appeared to be the constituents of the inclusions. Formation of similar inclusions in non-host insect cells upon expression of Pns12 in a baculovirus system and the association of molecules of Pns12 in vitro suggested that the inclusions observed in RDV-infected cells were composed basically of Pns12. Core proteins P1, P3, P5 and P7 and core virus particles were identified in the interior region of the inclusions. In contrast accumulation of the outer capsid proteins P2, P8 and P9 and of intact virus particles was evident in the peripheral regions of the inclusions. These observations suggest that core particles were constructed inside the inclusions, whereas outer capsid proteins were assembled at the periphery of the inclusions. Viral inclusions were shown to be the sites of viral RNA synthesis by labelling infected cells with 5-bromouridine 5′-tri phosphate. The number of viroplasms decreased with time post-inoculation as their sizes increased, suggesting that inclusions might fuse with one another during the virus-propagation process. Our results are consistent with a model, proposed for vertebrate reoviruses, in which viroplasms play a pivotal role in virus assembly. © 2006 SGM.

DOI： 10.1099/vir.0.81425-0

Complete genome sequence of Mycoreovirus-1/Cp9B21, a member of a novel genus within the family Reoviridae, isolated from the chestnut blight fungus Cryphonectria parasitica Reviewed

N Suzuki, S Supyani, K Maruyama, BI Hillman

JOURNAL OF GENERAL VIROLOGY 85 3437 - 3448 2004.11

Language：English Publishing type：Research paper (scientific journal) Publisher：SOC GENERAL MICROBIOLOGY

Mycoreovirus 1 (MYRV-1) is the type species of the newly described genus Mycoreovirus of the large virus family Reoviridae. The virus was isolated from a hypovirulent strain (9B21) of the chestnut blight fungus, Cryphonectria parasitica. A previous study showed that double-shelled particles introduced to fungal spheroplasts resulted in stably infected colonies. Of the 11 double-stranded RNA genomic segments (S1-S11), the three largest (S1-S3) were sequenced previously and shown to have moderate levels of similarity to the homologous segments of mammal-pathogenic coltiviruses (Eyach virus and Colorado tick fever virus) and another fungus-infecting reovirus, Mycoreovirus 3 of Rosellinia necatrix strain W370 (MYRV-3/RnW370). The sequences of the remaining segments (S4-S11) are reported here. All of the segments have single ORFs on their positive strands and the terminal sequences 5'-GAUCA----GCAGUCA-3' are conserved among currently and previously sequenced segments. Oligo-cap analysis showed that the positive strands of the genomic segments are capped, whereas the negative strands are not. Similarities among the four evolutionarily related viruses include low or moderate levels of amino acid sequence identity (14(.)7-34(.)2%) and isoelectric points among equivalent polypeptides, e.g. proteins encoded by segments S4 and S5 of the four viruses. Phylogenetic analysis indicated that MYRV-1/Cp9B21 is related more closely to MYRV-3/RnW370 than to the coltiviruses. An interesting dissimilarity is found in codon-choice pattern among the four viruses, i.e. MYRV-1/Cp9B21 segments have a lower frequency of [XYG + XYC] than corresponding segments of the other viruses, suggesting a possible adjustment of virus codon usage to their host environments.

DOI： 10.1099/vir.0.80293-0

A reovirus of the fungus Cryphonectria parasitica that is infectious as particles and related to the Coltivirus genus of animal pathogens Reviewed

BI Hillman, S Supyani, H Kondo, N Suzuki

JOURNAL OF VIROLOGY 78 ( 2 ) 892 - 898 2004.1

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

RNA viruses of filamentous fungi fall into two broad categories, those that contain double-stranded RNA (dsRNA) genomes in rigid particles and those that are more closely related to positive-sense, single-stranded RNA viruses with dsRNA replicative intermediates found within lipid vesicles. Effective infectivity systems have been described for the latter, using RNA transcripts, but not for the former. We report the characterization of a reovirus from Cryphonectria parasitica, the filamentous fungus that causes chestnut blight disease. The virus substantially reduces the virulence of the fungus and results in dramatically altered colony morphology, as well as changes in other associated fungal traits, relative to the virus-free isogenic strain. Virus particles from infected mycelium contained 11 segments of dsRNA and showed characteristics typical of the family Reoviridae. Sequences of the largest three segments revealed that the virus is closely related to the Coltivirus genus of animal pathogens, which includes the human pathogen Colorado tick fever virus. The introduction of purified virus particles into protoplasts from virus-free isolates of the fungus resulted in a newly infected mycelium with the same morphology and virus composition as the original virus-infected isolate. This represents the completion of Koch's postulates for a true dsRNA virus from a filamentous fungus and the description of a definitive fungal member of the family Reoviridae.

DOI： 10.1128/JVI.78.2.892-898.2004

Viruses of the Chestnut Blight Fungus, Cryphonectria parasitica

Bradley I. Hillman, Nobuhiro Suzuki

Advances in Virus Research 63 423 - 472 2004

Hypovirus papain-like protease p29 functions in trans to enhance viral double-stranded RNA accumulation and vertical transmission

N Suzuki, K Maruyama, M Moriyama, DL Nuss

JOURNAL OF VIROLOGY 77 ( 21 ) 11697 - 11707 2003.11

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

The prototypic hypovirus CHV1-EP713 attenuates virulence (hypovirulence) and alters several physiological processes of the chestnut blight fungus Cryphonectria parasitica. The papain-like protease, p29, and the highly basic protein, p40, derived, respectively, from the N-terminal and C-terminal portions of the CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, both contribute to reduced pigmentation and sporulation. The p29 coding region was shown to suppress pigmentation and asexual sporulation in the absence of virus infection in transformed C. parasitica, whereas transformants containing the p40-coding domain exhibited a wild-type, untransformed phenotype. Deletion of either p29 or p40 from the viral genome also results in reduced accumulation of viral RNA. We now show that p29, but not p40, functions in trans to enhance genomic RNA accumulation and vertical transmission of p29 deletion mutant viruses. The frequency of virus transmission through conidia was found to decrease with reduced accumulation of viral genomic double-stranded RNA (dsRNA): from almost 100% for wild-type virus to similar to50% for Deltap29, and 10 to 20% for Deltap69. When expressed from a chromosomally integrated cDNA copy, p29 elevated viral dsRNA accumulation and transmission for Deltap29 mutant virus to the level shown by wild-type virus. Increased viral RNA accumulation levels were also observed for a Deltap69 mutant lacking almost the entire ORF A sequence. Such enhancements were not detected in transgenic fungal colonies expressing p40. Mutation of p29 residues Cys(70) or Cys(72), strictly conserved in hypovirus p29 and potyvirus HC-Pro, resulted in the loss of both p29-mediated suppressive activity in virus-free transgenic C. parasitica and in trans enhancement of RNA accumulation and transmission, suggesting a linkage between these functional activities. These results suggest that p29 is an enhancer of viral dsRNA accumulation and vertical virus transmission through asexual spores.

DOI： 10.1128/JVI.77.21.11697-11707.2003

Extending chestnut blight hypovirus host range within diaporthales by biolistic delivery of viral cDNA Reviewed

A Sasaki, M Onoue, S Kanematsu, K Suzaki, M Miyanishi, N Suzuki, DL Nuss, K Yoshida

MOLECULAR PLANT-MICROBE INTERACTIONS 15 ( 8 ) 780 - 789 2002.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER PHYTOPATHOLOGICAL SOC

Biolistic bombardment was used to successfully transform three phytopathogenic fungal species with an infectious cDNA clone of the prototypic hypovirus, CHV1-EP713, a genetic element responsible for the virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica. The fungal species included two strains each of C. parasitica and Valsa ceratosperma, as well as one strain of Phomopsis G-type (teleomorph Diaporthe Nitschke); all are members of the order Diaporthales but classified into three different genera. A subset of transformants for each of the fungal species contained CHV1EP713 dsRNA derived from chromosomally integrated viral cDNA. As has been reported for CHV1-EP713 infection of the natural host C. parasitica, biolistic introduction of CHV1-EP713 into the new fungal hosts V. ceratosperma and Phomopsis G-type resulted in altered colony morphology and, more importantly, reduced virulence. These results suggest a potential for hypoviruses as biological control agents in plant-infecting fungal pathogens other than the chestnut blight fungus and closely related species. In addition, the particle delivery technique offers a convenient means of transmitting hypoviruses to potential host fungi that provides new avenues for fundamental mycovirus research and may have practical applications for conferring hypovirulence directly on infected plants in the field.

DOI： 10.1094/MPMI.2002.15.8.780

Contribution of protein p40 to hypovirus-mediated modulation of fungal host phenotype and viral RNA accumulation

N Suzuki, DL Nuss

JOURNAL OF VIROLOGY 76 ( 15 ) 7747 - 7759 2002.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

The papain-like protease p29, derived from the N-terminal portion of the hypovirus CHV1-EP713-encoded open reading frame (ORF) A polyprotein, p69, was previously shown to contribute to reduced pigmentation and sporulation by the infected host, the chestnut blight fungus Cryphonectria parasitica, while being dispensable for virus replication and attenuation of fungal virulence (hypovirulence). We now report that deletion of the C-terminal portion of p69, which encodes the highly basic protein p40, resulted in replication-competent mutant viruses that were, however, significantly reduced in RNA accumulation. While the Deltap40 mutants retained the ability to confer hypovirulence, Deltap40-infected fungal strains produced more asexual spores than strains infected with either wild-type CHV1-EP713 or a Deltap29 mutant virus. As observed for Deltap29-infected colonies, pigment production was significantly increased in Deltap40-infected fungal strains relative to that in CHV1-EP713-infected strains. Virus-mediated suppression of laccase production was not affected by p40 deletion. A gain-of-function analysis was employed to map the p40 symptom determinant to the N-terminal domain, encompassing p69 amino acid residues Thr(288) to Arg(312). Evidence that the gain of function was due to the encoded protein rather than the corresponding RNA sequence element was provided by introducing frameshift mutations on either side of the activity determinant domain. Moreover, restoration of symptoms correlated with increased accumulation of viral RNA. These results suggest that p40 indirectly contributes to virus-mediated suppression of fungal pigmentation and conidiation by providing an accessory function in hypovirus RNA amplification. A possible role for p40 in facilitating ORF B expression and the relationship between hypovirus RNA accumulation and symptom expression are discussed.

DOI： 10.1128/JVI.76.15.7747-7759.2002

Engineering hypoviruses for fundamental and practical applications

Donald L. Nuss, Baoshan Chen, Lynn M. Geletka, Todd B. Parsley, Nobuhiro Suzuki

dsRNA Genetic Elements: Concepts and Applications in Agriculture, Forestry, and Medicine 145 - 163 2001.1

© 2002 by CRC Press LLC. The concept of using a parasite of a parasite for disease control is appealing from both an intellectual and an ecological perspective. The phenomenon of transmissible hypovirulence, in which virulence of the chestnut blight fungus, Cryphonectria parasitica, is attenuated by double-stranded RNA viruses in the family Hypoviridae, is often cited as a classic example of this approach to biological disease control. Progress in the development of an infectious cDNA-based reverse genetics system for hypoviruses has provided the means for engineering these viral agents for both fundamental and practical applications, e.g., as a tool for probing signal transduction processes underlying fungal pathogenesis and for enhanced biocontrol potential. This chapter describes the basic techniques used for manipulating hypovirus genomes and provides specific examples of how they are being applied to identify virus-encoded determinants responsible for altering fungal host phenotype, to gain insights into dispensable and essential elements of viral replication, and to fine tune the interaction between a fungal pathogen and its plant host.

DOI： 10.1201/9781420039122

Essential and dispensable virus-encoded replication elements revealed by efforts to develop hypoviruses as gene expression vectors

N Suzuki, LM Geletka, DL Nuss

JOURNAL OF VIROLOGY 74 ( 16 ) 7568 - 7577 2000.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

We have investigated whether hypoviruses, viral agents responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica, could serve as gene expression vectors, The infectious cDNA clone of the prototypic hypovirus CHV1-EP713 was modified to generate 20 different vector candidates. Although transient expression was achieved for a subset of vectors that contained the green fluorescent protein gene from Aequorea victoria, long-term expression (past day 8) was not observed for any vector construct. analysis of viral RNAs recovered from transfected fungal colonies revealed that the foreign genes were readily deleted from the replicating virus, although small portions of foreign sequences were retained by some vectors after months of replication, However, the results of vector viability and progeny characterization provided unexpected new insights into essential and dispensable elements of hypovirus replication. The N-terminal portion (codons 1 to 24) of the 5'-proximal open reading frame (ORF), ORF ii was found to be required for virus replication, while the remaining 598 codons of this ORF were completely dispensable. Substantial alterations were tolerated in the pentanucleotide UAAUG that contains the ORF A termination codon and the overlapping putative initiation codon of the second of the two hypovirus ORFs, ORF B, Replication competence was maintained following either a frameshift mutation that caused a two-codon extension of ORF A or a modification that produced a single-ORF genomic organization, These results are discussed in terms of determinants of hypovirus replication, the potential utility of hypoviruses as gene expression vectors, and possible mechanisms by which hypoviruses recognize and delete foreign sequences.

DOI： 10.1128/JVI.74.16.7568-7577.2000

Similarity and divergence among viruses in the genus Furovirus

Y Shirako, N Suzuki, RC French

VIROLOGY 270 ( 1 ) 201 - 207 2000.4

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC

Nucleotide sequences of RNAs 1 and 2 of a Japanese strain of soil-borne wheat mosaic virus (sBWMV), the type species of the genus Furovirus, and sorghum chlorotic spot virus (SCSV) were determined from cloned cDNA. The relationship among the Japanese and US strains of SBWMV, SCSV, oat golden stripe virus (OGSV), and recently proposed Chinese wheat mosaic and European wheat mosaic viruses (CWMV and EWMV) were examined at the nucleotide and amino acid levels. Pairwise comparisons of genome-encoded proteins among the six viruses showed that the US strains of SBWMV and CWMV were the most closely related pair in RNA 1 and the Japanese strains of SBWMV and EWMV were most closely related in RNA 2. SCSV was most distantly related to the other five viruses. Phylogenetic analysis indicated that there may have been an ancient reassortment between RNAs 1 and 2 of the four wheat-infecting viruses and OGSV, while SCSV was shown to have separated from the rest before the other five viruses diverged. The fact that CWMV and EWMV have almost identical biological properties as well as the sequence similarities to the two strains of SBWMV suggests that they be regarded as strains of SBWMV, considering that SBWMV consists of generically diverged strains. OGSV and SCSV are distinct in biological properties in addition to genetic divergence in the genus Furovirus. (C) 2000 Academic Press.

DOI： 10.1006/viro.2000.0251

Mapping of a hypovirus p29 protease symptom determinant domain with sequence similarity to potyvirus HC-Pro protease

Nobuhiro Suzuki, Baoshan Chen, Donald L. Nuss

Journal of Virology 73 9478 - 9484 1999.10

Hypovirus infection of the chestnut blight fungus Cryphonectria parasitica results in a spectrum of phenotypic changes that can include alterations in colony morphology and significant reductions in pigmentation, asexual sporulation, and virulence (hypovirulence). Deletion of 88% [Phe(25) to Pro(243)] of the virus-encoded papain-like protease, p29, in the context of an infectious cDNA clone of the prototypic hypovirus CHV1-EP713 (recombinant virus Ap29) partially relieved virus-mediated suppression of pigmentation and sporulation without altering the level of hypovirulence. We now report mapping of the p29 symptom determinant domain to a region extending from Phe(25) through Gln(73) by a gain-of-function analysis following progressive repair of the Δp29 deletion mutant. This domain was previously shown to share sequence similarity [including conserved cysteine residues Cys(38), Cys(48), Cys(70), and Cys(72)] with the N-terminal portion of the potyvirus-encoded helper component-proteinase (HC-Pro), a multifunctional protein implicated in aphid-mediated transmission, genome amplification, polyprotein processing, long-distance movement, and suppression of posttranscriptional silencing. Substitution of a glycine residue for either Cys(38) or Cys(48) resulted in no qualitative or quantitative changes in virus-mediated symptoms. Unexpectedly, mutation of Cys(70) resulted in a very severe phenotype that included significantly reduced mycelial growth and profoundly altered colony morphology. In contrast, substitution for Cys(72) resulted in a less severe symptom phenotype approaching that observed for Δp29. The finding that p29-mediated symptom expression is influenced by two cysteine residues that are conserved in the potyvirus-encoded HC-Pro raises the possibility that these related viral-papain-like proteases function in their respective fungal and plant hosts by impacting ancestrally related regulatory pathways.

In vivo and in vitro phosphorylation of rice dwarf phytoreovirus Pns12 cytoplasmic nonstructural protein

N Suzuki, D Hosokawa, Y Matsuura, A Kikuchi, T Omura

ARCHIVES OF VIROLOGY 144 ( 7 ) 1371 - 1380 1999

Language：English Publishing type：Research paper (scientific journal) Publisher：SPRINGER-VERLAG WIEN

In vivo and in vitro phosphorylation and intracellular location of rice dwarf phytoreovirus Pns12, which is encoded by one of the twelve dsRNA genome segments, S12, and comprises 312 amino acids, was investigated. When [P-32]phosphoric acid was incorporated into RDV-infected leafhopper cultured cells, labelled Pns12 was immunoprecipitated from the cells by a monospecific anti-Pns12 polyclonal antibody. Recombinant Pns12 was purified from Spodoptera frugiperda cells infected with AcRS12, a baculovirus recombinant carrying a full-length cDNA of RDV S12. Purified Pns12 was also demonstrated to be phosphorylated in vitro by a kinase activity present in host (rice, barley, wheat, leafhopper) cells and non-host (tobacco, spinach, white clover, S. frugiperda, mosquito, mammals) cells as well. Immunocytochemical studies showed Pns12 accumulated in the cytoplasm of infected cells, and frequently localized in a slightly electron-dense patch. These results demonstrated that RDV Pns12 was a cytoplasmic nonstructural phosphoprotein.

DOI： 10.1007/s007050050593

The loss of outer capsid protein P2 results in nontransmissibility by the insect vector of rice dwarf phytoreovirus

Masatoshi Tomaru, Wakako Maruyama, Akira Kikuchi, Jin Yan, Yafeng Zhu, Nobuhiro Suzuki, Masamichi Isogai, Yuzuru Oguma, Ikuo Kimura, Toshihiro Omura

Journal of Virology 71 ( 10 ) 8019 - 8023 1997

Language：English Publishing type：Research paper (scientific journal)

A transmission-defective (TD) isolate of rice dwarf phytoreovirus lacked the ability to infect cells when derived from the virus-free insect vector Nephotettix cincticeps. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified virus showed that among six structural proteins, the P2 outer capsid protein (encoded by genome segment S2) was absent from the TD isolate, whereas all six proteins were present in the transmission-competent (TC) isolate. P2 was not detected on immunoblots of rice plants infected with the TD isolate. Genome segment S2 and its transcript were detected in both TD and TC isolates. Sequence analysis of the S2 segment of the TD isolate revealed the presence of a termination codon due to a point mutation in the open reading frame, which might explain the absence of P2 in the TD isolate. These results demonstrate that the P2 protein is one of the factors essential for infection by the virus of vector cells and, thus, influences transmissibility by vector insects.

Polycistronic (tri- or bicistronic) phytoreoviral segments translatable in both plant and insect cells

N Suzuki, M Sugawara, DL Nuss, Y Matsuura

JOURNAL OF VIROLOGY 70 ( 11 ) 8155 - 8159 1996.11

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER SOC MICROBIOLOGY

Genomic segment S12 of rice dwarf virus and segment S9 of wound tumor virus, both members of the genus Phytoreovirus, have small out-of-phase overlapping open reading frames (ORFs). Western blot (immunoblot) analysis revealed that rice dwarf virus S12 mRNA specified translation products from the large ORF and two overlapping small ORFs both in rice plant hosts and in Spodoptera frugiperda insect cells. These results provide the first example of a tricistronic mRNA for a segmented double-stranded RNA virus. Similarly, wound tumor virus S9 mRNA was found to direct the synthesis of protein products from both the large ORF and small out-of-frame ORF in S. frugiperda cells. Results of site-specific and deletion mutagenesis studies were consistent with a leaky scanning translation mechanism for the synthesis of the small ORFs.

Novel NTP binding property of rice dwarf phytoreovirus minor core protein P5

N Suzuki, T Kusano, Y Matsuura, T Omura

VIROLOGY 219 ( 2 ) 471 - 474 1996.5

Language：English Publishing type：Research paper (scientific journal) Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS

Rice dwarf phytoreovirus (RDV) mRNA synthesized from purified virion has a cap structure, m(7)GpppAm-, which suggests the presence of guanylyltransferase activity in the virion. We attempted to identify the enzyme involved in the cap formation by using a nucleoside triphosphate binding assay. Incubation of virion with [alpha-P-32]GTP resulted in labeling of an 89-kDa protein that had not previously been identified in purified virus preparations. Interestingly this protein also covalently bound UTP and ATP, which is not a property of the known guanylyltransferases. RDV particles catalyzed GTP-PPi, dGTP-PPi, ATP-PPi, and UTP-PPi exchange reactions. In SDS-polyacrylamide gel electrophoresis, the 89-kDa protein comigrated with the SS-coded protein, P5, which had been expressed by a baculovirus vector. Moreover, the labeled 89-kDa protein was precipitated by an antiserum against this recombinant RDV P5. Careful reinvestigation of purified virus particles by SDS-polyacrylamide gel electrophoresis and Western blotting analyses showed that they contained a small amount of P5 (<0.5% of the total protein) within the core. These results may suggest that the minor core protein of RDV, which is coded by S5, is a candidate guanylyltransferase, although the biological significance of its ATP and UTP binding activities remains largely unknown. (C) 1996 Academic Press, Inc.

DOI： 10.1006/viro.1996.0273

DNA cloning, sequence analysis of segment 7 of rice dwarf virus genome and its high expression in E. coli.(共著) Reviewed

Qu, L, Li, Y, Quan, S, Ding, S, Suzuki, N, Chen, Z

Acta Microbiologica Sinica 36 335 - 343 1996

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