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Toshihiro Omura - One of the best experts on this subject based on the ideXlab platform.
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Electron microscopic imaging revealed the flexible filamentous structure of the cell attachment protein P2 of Rice Dwarf Virus located around the icosahedral 5-fold axes.
Journal of Biochemistry, 2015Co-Authors: Naoyuki Miyazaki, Fusamichi Akita, Hiroyuki Hibino, Toshihiro Omura, Atsushi Nakagawa, Akifumi Higashiura, Tomoko Higashiura, Kenji IwasakiAbstract:The minor outer capsid protein P2 of Rice Dwarf Virus (RDV), a member of the genus PhytoreoVirus in the family Reoviridae, is essential for viral cell entry. Here, we clarified the structure of P2 and the interactions to host insect cells. Negative stain electron microscopy (EM) showed that P2 proteins are monomeric and flexible L-shaped filamentous structures of ∼20 nm in length. Cryo-EM structure revealed the spatial arrangement of P2 in the capsid, which was prescribed by the characteristic virion structure. The P2 proteins were visualized as partial rod-shaped structures of ∼10 nm in length in the cryo-EM map and accommodated in crevasses on the viral surface around icosahedral 5-fold axes with hydrophobic interactions. The remaining disordered region of P2 assumed to be extended to the radial direction towards exterior. Electron tomography clearly showed that RDV particles were away from the cellular membrane at a uniform distance and several spike-like densities, probably corresponding to P2, connecting a viral particle to the host cellular membrane during cell entry. By combining the in vitro and in vivo structural information, we could gain new insights into the detailed mechanism of the cell entry of RDV.
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nac transcription factor family genes are differentially expressed in Rice during infections with Rice Dwarf Virus Rice black streaked Dwarf Virus Rice grassy stunt Virus Rice ragged stunt Virus and Rice transitory yellowing Virus
Frontiers in Plant Science, 2015Co-Authors: Mohammed Nuruzzaman, Takumi Shimizu, Toshihiro Omura, Kouji Satoh, Takahide Sasaya, Akhter Most Sharoni, Mohammad Rezaul Karim, Jennifer Ann Harikrishna, Mohammad A Haque, Sayed Zain M HasanAbstract:Expression levels of the NAC gene family were studied in Rice infected with Rice Dwarf Virus (RDV), Rice black-streaked Dwarf Virus (RBSDV), Rice grassy stunt Virus (RGSV), Rice ragged stunt Virus (RRSV), and Rice transitory yellowing Virus (RTYV). Microarray analysis showed that 75 (68%) OsNAC genes were differentially regulated during infection with RDV, RBSDV, RGSV, and RRSV compared with the control. The number of OsNAC genes up-regulated was highest during RGSV infection, while the lowest number was found during RTYV infection. These phenomena correlate with the severity of the syndromes induced by the Virus infections. Most of the genes in the NAC subgroups NAC22, SND, ONAC2, ANAC34, and ONAC3 were down-regulated for all Virus infections. These OsNAC genes might be related to the health stage maintenance of the host plants. Interestingly, most of the genes in the subgroups TIP and SNAC were more highly expressed during RBSDV and RGSV infections. These results suggested that OsNAC genes might be related to the responses induced by the Virus infection. All of the genes assigned to the TIP subgroups were highly expressed during RGSV infection when compared with the control. For RDV infection, the number of activated genes was greatest during infection with the S-strain, followed by the D84-strain and the O-strain, with seven OsNAC genes up-regulated during infection by all three strains. The Os12g03050 and Os11g05614 genes showed higher expression during infection with four of the five Viruses, and Os11g03310, Os11g03370, and Os07g37920 genes showed high expression during at least three viral infections. We identified some duplicate genes that are classified as neofunctional and subfunctional according to their expression levels in different viral infections. A number of putative cis-elements were identified, which may help to clarify the function of these key genes in network pathways.
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RNA-binding Domain of the Key Structural Protein P7 for the Rice Dwarf Virus Particle Assembly
2015Co-Authors: Boxiong Zhong, Yan-wei Shen, Toshihiro OmuraAbstract:Abstract The Rice Dwarf Virus (RDV) P7 structural protein is the key protein in the RDV particle assembly. The P7 protein was digested partially or completely by Staphylococcus aureus V8 protease and/or Pseudomonas fragi Asp-N protease. The molecular mass and the N-terminal amino acid sequence of the polypeptide fragments of the P7 protein were determined by SDS-PAGE and the Edman degradation method, respectively. Then the polypeptides were located in the deduced amino acid sequence of the RDV P7 protein based on the nucleotide sequence information, with the knowledge of the specific cleavage sites of the Staphylococcus aureus V8 and Pseudomonas fragi Asp-N protease, and the two RNA-binding domains in the P7 protein were identified. Domain 1 was located in the residue 128–249 containing 122 amino acids and domain 2 was located in the residue 325–355 containing 31 amino acids. Thus, these two domains may play an important role in the Virus particle assembly by contributing to the packaging of viral dsRNAs inside the particles. The two domains may be novel RNA-binding domains, because no amino acid sequences highly similar to the conservative sequences of known dsRNA-binding domains reported so far. The similarity between the motif of domain 1 and the motif of the DNA-binding protein suggests that the DNA-binding activity of the RDV P7 protein may be due to this sequence. The similarity between the motif of domain 1 and the motif of the RNA polymerase domain suggests that the P7 protein may also play a role in RNA synthesis
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cryo electron tomography moving towards revealing the viral life cycle of Rice Dwarf Virus
Journal of Synchrotron Radiation, 2013Co-Authors: Naoyuki Miyazaki, Fusamichi Akita, Toshihiro Omura, Atsushi Nakagawa, Kazuyoshi Murata, Kenji IwasakiAbstract:It is well known that Viruses utilize the host cellular systems for their infection and replication processes. However, the molecular mechanisms underlying these processes are poorly understood for most Viruses. To understand these molecular mechanisms, it is essential to observe the viral and Virus-related structures and analyse their molecular interactions within a cellular context. Cryo-electron microscopy and tomography offer the potential to observe macromolecular structures and to analyse their molecular interactions within the cell. Here, using cryo-electron microscopy and tomography, the structures of Rice Dwarf Virus are reported within fully hydrated insect vector cells grown on electron microscopy grids towards revealing the viral infection and replication mechanisms.
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Aggregation Ability of Virus-Specific Antibodies is Correlated with their Capacity to Neutralize Rice Dwarf Virus
Japan Agricultural Research Quarterly: JARQ, 2012Co-Authors: Hongyan Chen, Takumi Shimizu, Fusamichi Akita, Hiroshi Mizuno, Taiyun Wei, Tamaki Uehara-ichiki, Ai Saotome, Kengo Sakaguchi, Yasuo Shikamoto, Toshihiro OmuraAbstract:Antibodies (immunoglobulin G (IgGs)) from antisera raised against viral particles that had been dissociated by treatment with SDS and intact particles of Rice Dwarf Virus (RDV) were studied for their ability to prevent viral infection of vector cells in monolayers in vitro. Even though IgGs raised against dissociated Virus had a higher titer than those raised against intact Viruses in an analysis of viral proteins on Western blots, they did not neutralize RDV. Conversely, IgGs raised against intact RDV effectively neutralized viral infectivity. Electron microscopic observation of the aggregation of RDV particles after incubation with IgGs raised against intact RDV, but no aggregation of RDV particles after incubation with IgGs raised against dissociated RDV suggested that IgGs raised against intact Viruses might prevent viral invasion by causing clumping of Viruses, thereby reducing the number of infectious units. Our results reveal, for the first time, a possible mechanism for the neutralization, mediated by antibodies, of plant Viruses that propagate in insect vector cells.
Taiyun Wei - One of the best experts on this subject based on the ideXlab platform.
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Viral Release Threshold in the Salivary Gland of Leafhopper Vector Mediates the Intermittent Transmission of Rice Dwarf Virus.
Frontiers in microbiology, 2021Co-Authors: Qian Chen, Yuyan Liu, Zhirun Long, Hengsong Yang, Taiyun WeiAbstract:Numerous piercing-sucking insects can persistently transmit viral pathogens in combination with saliva to plant phloem in an intermittent pattern. Insect vectors maintain viruliferous for life. However, the reason why insect vectors discontinuously transmit the Virus remains unclear. Rice Dwarf Virus (RDV), a plant reoVirus, was found to replicate and assemble the progeny virions in salivary gland cells of the leafhopper vector. We observed that the RDV virions moved into saliva-stored cavities in the salivary glands of leafhopper vectors via an exocytosis-like mechanism, facilitating the viral horizontal transmission to plant hosts during the feeding of leafhoppers. Interestingly, the levels of viral accumulation in the salivary glands of leafhoppers during the transmitting period were significantly lower than those of viruliferous individuals during the intermittent period. A putative viral release threshold, which was close to 1.79 × 104 copies/μg RNA was proposed from the viral titers in the salivary glands of 52 leafhoppers during the intermittent period. Thus, the viral release threshold was hypothesized to mediate the intermittent release of RDV from the salivary gland cells of leafhoppers. We anticipate that viral release threshold-mediated intermittent transmission by insect vectors is the conserved strategy for the epidemic and persistence of vector-borne Viruses in nature.
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Interaction between non-structural protein Pns10 of Rice Dwarf Virus and cytoplasmic actin of leafhoppers is correlated with insect vector specificity.
The Journal of general virology, 2014Co-Authors: Qian Chen, Haitao Wang, Tangyu Ren, Lianhui Xie, Taiyun WeiAbstract:Many insect-transmissible pathogens are transmitted by specific insect species and not by others, even if the insect species are closely related. The molecular mechanisms underlying such strict pathogen-insect specificity are poorly understood. Rice Dwarf Virus (RDV), a plant reoVirus, is transmitted mainly by the leafhopper species Nephotettix cincticeps but is transmitted ineffectively by the leafhopper Recilia dorsalis. Here, we demonstrated that Virus-containing tubules composed of viral non-structural protein Pns10 of RDV associated with the intestinal microvilli of N. cincticeps but not with those of R. dorsalis. Furthermore, Pns10 of RDV specifically interacted with cytoplasmic actin, the main component of microvilli of N. cincticeps, but not with that of R. dorsalis, suggesting that the interaction of Pns10 with insect cytoplasmic actin is consistent with the transmissibility of RDV by leafhoppers. All these results suggested that the interaction of Pns10 of RDV with insect cytoplasmic actin may determine pathogen-vector specificity.
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Aggregation Ability of Virus-Specific Antibodies is Correlated with their Capacity to Neutralize Rice Dwarf Virus
Japan Agricultural Research Quarterly: JARQ, 2012Co-Authors: Hongyan Chen, Takumi Shimizu, Fusamichi Akita, Hiroshi Mizuno, Taiyun Wei, Tamaki Uehara-ichiki, Ai Saotome, Kengo Sakaguchi, Yasuo Shikamoto, Toshihiro OmuraAbstract:Antibodies (immunoglobulin G (IgGs)) from antisera raised against viral particles that had been dissociated by treatment with SDS and intact particles of Rice Dwarf Virus (RDV) were studied for their ability to prevent viral infection of vector cells in monolayers in vitro. Even though IgGs raised against dissociated Virus had a higher titer than those raised against intact Viruses in an analysis of viral proteins on Western blots, they did not neutralize RDV. Conversely, IgGs raised against intact RDV effectively neutralized viral infectivity. Electron microscopic observation of the aggregation of RDV particles after incubation with IgGs raised against intact RDV, but no aggregation of RDV particles after incubation with IgGs raised against dissociated RDV suggested that IgGs raised against intact Viruses might prevent viral invasion by causing clumping of Viruses, thereby reducing the number of infectious units. Our results reveal, for the first time, a possible mechanism for the neutralization, mediated by antibodies, of plant Viruses that propagate in insect vector cells.
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Sequential infection of Rice Dwarf Virus in the internal organs of its insect vector after ingestion of Virus.
Virus research, 2011Co-Authors: Hongyan Chen, Toshihiro Omura, Qian Chen, Tamaki Uehara-ichiki, Taiyun WeiAbstract:Confocal microscopy revealed that Rice Dwarf Virus (RDV) initially accumulated in epithelial cells of the filter chamber of leafhopper vector Nephotettix cincticeps 2 days after acquisition access feeding on diseased plants. Subsequently, RDV accumulation progressed to the anterior midgut, and then spread to the nervous system before infection of other organs. Furthermore, RDV accumulation progressed to the visceral muscles surrounding the anterior midgut. Later, RDV accumulation was detected in other parts of the alimentary canal, salivary glands and the follicular cells of the ovarioles in viruliferous insect vector. Our results suggest that RDV may use the muscle or neural tissues for viral dissemination from the infected vector's midgut into other tissues.
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Release of Rice Dwarf Virus from insect vector cells involves secretory exosomes derived from multivesicular bodies.
Communicative & integrative biology, 2009Co-Authors: Taiyun Wei, Hiroyuki Hibino, Toshihiro OmuraAbstract:Plant reoViruses in insect vector cells are sequestered in spherical multivesicular compartments. We demonstrated previously that the plant-infecting reoVirus Rice Dwarf Virus (RDV) exploits multivesicular compartments for the transport and release of viral particles from infected insect vector cells. These multivesicular compartments contain small vesicles and, morphologically, they resemble previously reported endosomal multivesicular bodies (MVBs) exploited by enveloped RNA Viruses during budding from the plasma membrane of infected cells. Electron microscopy revealed that, at a late stage of infection, RDV virions are released, together with small vesicles similar to secreted vesicles (exosomes), from infected cells. The incorporation of lysosomes into the multivesicular compartments raised the possibility that functions of host MVBs are required for the efficient release of RDV virions from infected insect vector cells. An actin-myosin transport system has been shown to mediate the transport of these multivesicular compartments. In this addendum, we provide evidence for the proposed model of release of RDV virions from infected insect vector cells that exploits secretory exosomes derived from MVBs.
Hongyan Chen - One of the best experts on this subject based on the ideXlab platform.
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nonstructural protein pns4 of Rice Dwarf Virus is essential for viral infection in its insect vector
Virology Journal, 2015Co-Authors: Qian Chen, Linghua Zhang, Hongyan ChenAbstract:Background Rice Dwarf Virus (RDV), a plant reoVirus, is mainly transmitted by the green Rice leafhopper, Nephotettix cincticeps, in a persistent-propagative manner. Plant reoViruses are thought to replicate and assemble within cytoplasmic structures called viroplasms. Nonstructural protein Pns4 of RDV, a phosphoprotein, is localized around the viroplasm matrix and forms minitubules in insect vector cells. However, the functional role of Pns4 minitubules during viral infection in insect vector is still unknown yet.
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nonstructural protein pns12 of Rice Dwarf Virus is a principal regulator for viral replication and infection in its insect vector
Virus Research, 2015Co-Authors: Qian Chen, Hongyan ChenAbstract:Abstract Plant reoViruses are thought to replicate and assemble within cytoplasmic structures called viroplasms. The molecular mechanisms underling the formation of the viroplasm during infection of Rice Dwarf Virus (RDV), a plant reoVirus, in its leafhopper vector cells remain poorly understood. Viral nonstructural protein Pns12 forms viroplasm-like inclusions in the absence of viral infection, suggesting that the viroplasm matrix is basically composed of Pns12. Here, we demonstrated that core capsid protein P3 and nonstructural protein Pns11 were recruited in the viroplasm by direct interaction with Pns12, whereas nonstructural protein Pns6 was recruited through interaction with Pns11. The introduction of dsRNA from Pns12 gene into cultured insect vector cells or intact insect strongly inhibited such viroplasm formation, preventing efficient viral spread in the leafhopper in vitro and in vivo . Thus, nonstructural protein Pns12 of RDV is a principal regulator for viral replication and infection in its insect vector.
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Aggregation Ability of Virus-Specific Antibodies is Correlated with their Capacity to Neutralize Rice Dwarf Virus
Japan Agricultural Research Quarterly: JARQ, 2012Co-Authors: Hongyan Chen, Takumi Shimizu, Fusamichi Akita, Hiroshi Mizuno, Taiyun Wei, Tamaki Uehara-ichiki, Ai Saotome, Kengo Sakaguchi, Yasuo Shikamoto, Toshihiro OmuraAbstract:Antibodies (immunoglobulin G (IgGs)) from antisera raised against viral particles that had been dissociated by treatment with SDS and intact particles of Rice Dwarf Virus (RDV) were studied for their ability to prevent viral infection of vector cells in monolayers in vitro. Even though IgGs raised against dissociated Virus had a higher titer than those raised against intact Viruses in an analysis of viral proteins on Western blots, they did not neutralize RDV. Conversely, IgGs raised against intact RDV effectively neutralized viral infectivity. Electron microscopic observation of the aggregation of RDV particles after incubation with IgGs raised against intact RDV, but no aggregation of RDV particles after incubation with IgGs raised against dissociated RDV suggested that IgGs raised against intact Viruses might prevent viral invasion by causing clumping of Viruses, thereby reducing the number of infectious units. Our results reveal, for the first time, a possible mechanism for the neutralization, mediated by antibodies, of plant Viruses that propagate in insect vector cells.
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Sequential infection of Rice Dwarf Virus in the internal organs of its insect vector after ingestion of Virus.
Virus research, 2011Co-Authors: Hongyan Chen, Toshihiro Omura, Qian Chen, Tamaki Uehara-ichiki, Taiyun WeiAbstract:Confocal microscopy revealed that Rice Dwarf Virus (RDV) initially accumulated in epithelial cells of the filter chamber of leafhopper vector Nephotettix cincticeps 2 days after acquisition access feeding on diseased plants. Subsequently, RDV accumulation progressed to the anterior midgut, and then spread to the nervous system before infection of other organs. Furthermore, RDV accumulation progressed to the visceral muscles surrounding the anterior midgut. Later, RDV accumulation was detected in other parts of the alimentary canal, salivary glands and the follicular cells of the ovarioles in viruliferous insect vector. Our results suggest that RDV may use the muscle or neural tissues for viral dissemination from the infected vector's midgut into other tissues.
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Entry of Rice Dwarf Virus into Cultured Cells of Its Insect Vector Involves Clathrin-Mediated Endocytosis
Journal of Virology, 2007Co-Authors: Hongyan Chen, Tamaki Ichiki-uehara, Hiroyuki Hibino, Toshihiro OmuraAbstract:Electron microscopy revealed that the entry of Rice Dwarf Virus (RDV) into insect vector cells involved endocytosis via coated pits. The treatment of cells with drugs that block receptor-mediated or clathrin-mediated endocytosis significantly reduced RDV infectivity. However, the drug that blocks caveola-mediated endocytosis had a negligible effect on such infection. Infection was also inhibited when cells had been pretreated with bafilomycin A1, which interferes with acidification of endosomes. Moreover, immunofluorescence staining indicated that the Virus is internalized into early endosomes. Together, our data indicate that RDV enters insect vector cells through receptor-mediated, clathrin-dependent endocytosis and is sequestered in early endosomes.
Kyoji Hagiwara - One of the best experts on this subject based on the ideXlab platform.
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The functional organization of the internal components of Rice Dwarf Virus.
Journal of biochemistry, 2010Co-Authors: Naoyuki Miyazaki, Tomitake Tsukihara, Atsushi Nakagawa, Lena Marmstål Hammar, Akifumi Higashiura, Kyoji Hagiwara, Che Yen Wang, Li Xing, Toshihiro OmuraAbstract:The capsid structures of particles of Rice Dwarf Virus that consisted of different components, namely, intact particles, empty particles lacking the 12 segments of double-stranded RNA (dsRNA), and Virus-like particles composed of only the P3 core and P8 outer capsid proteins, generated with a baculoVirus gene-expression system, were determined by cryo-electron microscopy. Combining the results with those of biochemical analysis, we assigned proteins of the transcriptional machinery and dsRNA to density clusters around the 5-fold axes and along the radial concentric layers, respectively. P7 protein, a component of the transcriptional machinery, was assigned to the outermost region of the density clusters. The density connecting the transcription complex to the outermost RNA densities implied interactions between the dsRNA and the P7 protein. Our structural analysis and the non-specific nucleic acid-binding activity of P7 explain the spiral organization of dsRNA around the 5-fold axis.
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Retention of Rice Dwarf Virus by Descendants of Pairs of Viruliferous Vector Insects After Rearing for 6 Years.
Phytopathology, 2007Co-Authors: Kazuto Honda, Taiyun Wei, Takahiko Higashi, Kyoji Hagiwara, Ikuo Kimura, Katsumi Akutsu, Toshihiro OmuraAbstract:ABSTRACT Rice Dwarf Virus (RDV) is characterized by its unusual ability to multiply in both plants and leafhopper vector insects and by its transovarial mode of transmission. Colonies of Nephotettix cincticeps, derived originally from pairs of leafhoppers infected with an ordinary strain of RDV, were maintained for 6 years in the laboratory and were found, at the end of this time, still to harbor RDV. Moreover, the isolate of RDV, designated RDV-I, obtained from these colonies retained the ability to infect Rice plants. When we raised leafhoppers separately from eggs that had been placed individually on pieces of water-soaked filter paper and reared them in the presence of healthy Rice seedlings, we found that all of these leafhoppers harbored RDV. This observation suggested that RDV-I had been maintained in the leafhoppers by transovarial transmission. Two further observations, namely, the low rate of acquisition of RDV by Virus-free insect nymphs on symptomless plants on which viruliferous insects had been reared, and the fact that only 2 to 5% of plants had symptoms when Rice seedlings were inoculated via RDV-I-viruliferous insects, confirmed that the maintenance of RDV-I by any other mode of transmission through plants and insects was unlikely. This efficient and long-term maintenance of RDV in a population of viruliferous insects might explain the prolonged duration of Rice Dwarf disease in the field, once there has been a serious outbreak.
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The Spread of Rice Dwarf Virus among Cells of Its Insect Vector Exploits Virus-Induced Tubular Structures
Journal of virology, 2006Co-Authors: Taiyun Wei, Takumi Shimizu, Tamaki Ichiki-uehara, Hongyan Chen, Yusuke Moriyasu, Akira Kikuchi, Nobuhiro Suzuki, Kyoji Hagiwara, Mami Takahashi, Toshihiro OmuraAbstract: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 Pns10 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 Pns10 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.
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In vitro but not in planta encapsidation of Rice gall Dwarf Virus core particles by the outer capsid P8 protein of Rice Dwarf Virus expressed in transgenic Rice plants
Journal of General Plant Pathology, 2006Co-Authors: Kyoji Hagiwara, Naoyuki Miyazaki, Yafeng Zhu, Takahiko Higashi, Kazunari Takahashi, Naho Hara, Hideyuki Aoki, Qing-yu Wang, Osamu Yatou, Hiroshi TanakaAbstract:Transencapsidation of the Rice gall Dwarf Virus (RGDV) inner core by the Rice Dwarf Virus (RDV) outer capsid P8 protein was examined in vitro and in planta. When RGDV core particles were incubated with an extract from RDV P8-transgenic Rice leaf tissue, RDV P8 encapsidated the RGDV core particles to form double-shelled Virus-like particles in vitro. In contrast, when RDV P8-transgenic Rice plants were inoculated with RGDV, progeny RGDV particles contained RGDV P8 but RDV P8 was not detectable in the virions. No significant differences were found in acquisition by the vector insects and subsequent transmission rates between RGDV infecting nontransgenic Rice plants and those infecting RDV P8-transgenic Rice plants. These results indicate that mechanisms of and/or requirements for interactions between P8 and the inner core particles of phytoreoViruses differ between in vitro and in planta.
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Pns12 protein of Rice Dwarf Virus is essential for formation of viroplasms and nucleation of viral-assembly complexes.
The Journal of general virology, 2006Co-Authors: Taiyun Wei, Takumi Shimizu, Hongyan Chen, Yusuke Moriyasu, Akira Kikuchi, Nobuhiro Suzuki, Kyoji Hagiwara, Toshihiro OmuraAbstract: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'-triphosphate. 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.
Zhangliang Chen - One of the best experts on this subject based on the ideXlab platform.
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Assembly of Double-Shelled, Virus-Like Particles in Transgenic Rice Plants Expressing Two Major Structural Proteins of Rice Dwarf Virus
Journal of virology, 2000Co-Authors: Honghong Zheng, Chunhong Wei, Yunping Shen, Zhangliang ChenAbstract:Rice Dwarf Virus (RDV) is a double-shelled particle that contains a major capsid protein (P8), a major core protein (P3), several minor core proteins, and viral genomic double-stranded RNA. Coexpression of P8 and P3 in transgenic Rice plants resulted in formation of double-shelled, Virus-like particles (VLPs) similar to the authentic RDV particles. The VLPs were not detected in transgenic Rice plant cells expressing P8 alone. This in vivo result suggests that P8 interacted with P3 and that these two proteins provide the structural integrity required for the formation of VLPs in Rice cells independently of other structural proteins, nonstructural proteins, or viral genomic double-stranded RNAs.
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The 42K protein of Rice Dwarf Virus is a post-translational cleavage product of the 46K outer capsid protein
Archives of virology, 1998Co-Authors: Z. J. Mao, H. H. Zheng, J. Schiemann, R. Casper, Zhangliang ChenAbstract:The outer capsid protein (P8) heterogeneity of Rice Dwarf Virus (RDV) exists not only in purified Virus particles, but also in RDV-infected Rice, transgenic Rice expressing P8, E. coli expression of P8 product and the in vitro translation products of S8. N-terminal amino acid sequencing revealed that P8 is a cleavage product of P8′. The cleavage occurs specifically at the residues of Asp362 and Pro363. The function of the proteolytic processing is unknown.
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Recovery of transgenic Rice plants expressing the Rice Dwarf Virus outer coat protein gene (S8)
Theoretical and Applied Genetics, 1997Co-Authors: H. H. Zheng, R. Casper, M. Y. Chen, X. T. Ming, Zhangliang ChenAbstract:The coding region of the eighth largest segment (S8) of the Rice Dwarf Virus (RDV) was obtained from a RDV Fujian isolate. It was then cloned into pTrcHisA for expression in E. coli and into vector pE3 for plant transformation. By using callus derived from mature Rice embryos as the target tissue, we obtained regenerated Rice plants after bombardment of the former with plasmid pE3R8 containing the RDV S8 gene and the marker gene neomycin phosphotransferase (NPT II). Southern blotting confirmed the integration of the RDV S8 gene into the Rice genome. The expression of the outer coat protein in both E. coli and Rice plants was confirmed by western blotting. The recovery of transgenic Rice plants expressing S8 gene is an important step towards studying the function of the RDV genes and obtaining RDV-resistant Rice plants.
