Takumi Shimizu

The Spread of Rice Dwarf Virus among Cells of Its Insect Vector Exploits Virus-Induced Tubular Structures

ABSTRACT 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.

Disruption of a novel gene for a NAC‐domain protein in rice confers resistance to Rice dwarf virus

Rice dwarf virus (RDV) is a serious viral pest that is transmitted to rice plants (Oryza sativa L.) by leafhoppers and causes a dwarfism in infected plants. To identify host factors involved in the multiplication of RDV, we screened Tos17 insertion mutant lines of rice for mutants with reduced susceptibility to RDV. One mutant, designated rim1-1, did not show typical disease symptoms upon infection with RDV. The accumulation of RDV capsid proteins was also drastically reduced in inoculated rim1-1 mutant plants. Co-segregation and complementation analyses revealed that the rim1-1 mutation had been caused by insertion of Tos17 in an intron of a novel NAC gene. The rim1-1 mutant remained susceptible to the two other viruses tested, one of which is also transmitted by leafhoppers, suggesting that the multiplication rather than transmission of RDV is specifically impaired in this mutant. We propose that RIM1 functions as a host factor that is required for multiplication of RDV in rice.

Crystallographic Analysis Reveals Octamerization of Viroplasm Matrix Protein P9-1 of Rice Black Streaked Dwarf Virus

ABSTRACT The P9-1 protein of Rice black streaked dwarf virus accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in viruses in the family Reoviridae. Crystallographic analysis of P9-1 revealed structural features that allow the protein to form dimers via hydrophobic interactions. Each dimer has carboxy-terminal regions, resembling arms, that extend to neighboring dimers, thereby uniting sets of four dimers via lateral hydrophobic interactions, to yield cylindrical octamers. The importance of these regions for the formation of viroplasm-like inclusions was confirmed by the absence of such inclusions when P9-1 was expressed without its carboxy-terminal arm. The octamers are vertically elongated cylinders resembling the structures formed by NSP2 of rotavirus, even though there are no significant similarities between the respective primary and secondary structures of the two proteins. Our results suggest that an octameric structure with an internal pore might be important for the functioning of the respective proteins in the events that occur in the viroplasm, which might include viral morphogenesis.

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

ABSTRACT 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.

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

Summary.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.

The nonstructural protein pC6 of rice grassy stunt virus trans-complements the cell-to-cell spread of a movement-defective tomato mosaic virus

The nonstructural protein pC6 encoded by rice grassy stunt virus is thought to correspond functionally to the nonstructural protein pC4 of rice stripe virus, which can support viral cell-to-cell movement. In a trans-complementation experiment with a movement-defective tomato mosaic virus, pC6 and pC4 facilitated intercellular transport of the virus. Transient expression of pC6, fused with green fluorescent protein, in epidermal cells was predominantly observed close to the cell wall as well as in a few punctate structures, presumably associated with plasmodesmata. These results suggest that pC6 has a role similar to that of pC4 in viral cell-to-cell movement.

Molecular analysis of the genome segments S1, S4, S6, S7 and S12 of a Rice gall dwarf virus isolate from Thailand; completion of the genomic sequence

SummaryThe complete nucleotide sequences of the double-stranded RNA segments S1, S4, S6, S7 and S12 of the genome of a Rice gall dwarf virus (RGDV) isolate from Thailand were determined. The segments consisted of 4505, 2622, 1648, 1652 and 853 nucleotides, encoding putative proteins of 1458, 725, 489, 511 and 206 amino acids with molecular masses of approximately 166, 80, 53, 59 and 24 kDa, respectively. Homology searches indicated that each of the putative proteins has a counterpart in isolates of Rice dwarf virus (RDV) and Wound tumor virus, two other species in the genus Phytoreovirus. However, no similarities were found to other registered sequences, including those of other viruses that belong to the family Reoviridae. The identities between homologous structural proteins of RGDV and RDV ranged from 34 to 51% and were thus higher than those between homologous non-structural proteins of RGDV and RDV (16–37%). Among the nonstructural proteins, the highest amino acid sequence identity (37%) was observed for RGDV Pns11 and RDV Pns10, a constituent of tubular inclusions. This observation suggests that a specific amino acid backbone might be required for maintaining not only the three-dimensional structure of virions but also that of inclusions. The entire sequence of the RGDV genome is now available.

Viroplasm matrix protein Pns9 from rice gall dwarf virus forms an octameric cylindrical structure

The non-structural Pns9 protein of rice gall dwarf virus (RGDV) accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in host cells infected by viruses in the family Reoviridae. Immunofluorescence and immunoelectron microscopy of RGDV-infected vector cells in monolayers, using antibodies against Pns9 of RGDV and expression of Pns9 in Spodoptera frugiperda cells, demonstrated that Pns9 is the minimal viral factor necessary for formation of viroplasm inclusion during infection by RGDV. When Pns9 in solution was observed under a conventional electron microscope, it appeared as ring-like aggregates of approximately 100 Å in diameter. Cryo-electron microscopic analysis of these aggregates revealed cylinders of octameric Pns9, whose dimensions were similar to those observed under the conventional electron microscope. Octamerization of Pns9 in solution was confirmed by the results of size-exclusion chromatography. Among proteins of viruses that belong to the family Reoviridae whose three-dimensional structures are available, a matrix protein of the viroplasm of rotavirus, NSP2, forms similar octamers, an observation that suggests similar roles for Pns9 and NSP2 in morphogenesis in animal-infecting and in plant-infecting reoviruses.

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

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.

The movement protein encoded by gene 3 of rice transitory yellowing virus is associated with virus particles.

Gene 3 in the genomes of several plant-infecting rhabdoviruses, including rice transitory yellowing virus (RTYV), has been postulated to encode a cell-to-cell movement protein (MP). Trans-complementation experiments using a movement-defective tomato mosaic virus and the P3 protein of RTYV, encoded by gene 3, facilitated intercellular transport of the mutant virus. In transient-expression experiments with the GFP-fused P3 protein in epidermal leaf cells of Nicotiana benthamiana, the P3 protein was associated with the nucleus and plasmodesmata. Immunogold-labelling studies of thin sections of RTYV-infected rice plants using an antiserum against Escherichia coli-expressed His(6)-tagged P3 protein indicated that the P3 protein was located in cell walls and on virus particles. In Western blots using antisera against E. coli-expressed P3 protein and purified RTYV, the P3 protein was detected in purified RTYV, whilst antiserum against purified RTYV reacted with the E. coli-expressed P3 protein. After immunogold labelling of crude sap from RTYV-infected rice leaves, the P3 protein, as well as the N protein, was detected on the ribonucleocapsid core that emerged from partially disrupted virus particles. These results provide evidence that the P3 protein of RTYV, which functions as a viral MP, is a viral structural protein and seems to be associated with the ribonucleocapsid core of virus particles.