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The family Reoviridae is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family Reoviridae, the genera Cardoreovirus, Phytoreovirus, Seadornavirus, Mycoreovirus, and Coltivirus contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for Reoviridae viruses such as Orthoreovirus, Orbivirus, Cypovirus, and Rotavirus, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for Reoviridae viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus Coltivirus, family Reoviridae), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an N-glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus Coltivirus but also the replication machinery of the family Reoviridae.

Vertical transmission of Homalodisca vitripennis reovirus (HoVRV) from glassy-winged sharpshooter (GWSS, Homalodisca vitripennis (Germar)) females to progeny occurred in laboratory assays at frequencies too low (2.6%–15.4%) to account for HoVRV incidence (90–100%) in field populations resident in citrus. Because citrus is immune to HoVRV and no plant host is known, horizontal transmission of HoVRV from insect-to-insect was evaluated. Exposure of colony-reared, virus-free test nymphs to HoVRV-infected source adults held in the same cage for 10 days on virus-immune cowpea resulted in HoVRV transmission (13.3%–30.7%) to test nymphs. HoVRV was not transmitted when exposure was indirect and required passive movement of virions through the xylem of immune citrus seedlings. Collectively, these results demonstrate direct insect-to-insect horizontal transmission of HoVRV, providing a plausible explanation for high incidence of HoVRV in GWSS field populations in the absence of efficient vertical transmission or a plant host.

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.

Numerous viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. These viruses circulate in the vector body, enter the salivary gland, and then are released into the apical plasmalemma-lined cavities, where saliva is stored. The cavity plasmalemma of vector salivary glands thus represents the last membrane barrier for viral transmission. Here, we report a novel mechanism used by a persistent virus to overcome this essential barrier. We observed that the infection by rice gall dwarf virus (RGDV), a species of the genus Phytoreovirus in the family Reoviridae , induced the formation of virus-associated filaments constructed by viral nonstructural protein Pns11 within the salivary glands of its leafhopper vector, Recilia dorsalis . Such filaments attached to actin-based apical plasmalemma and induced an exocytosis-like process for viral release into vector salivary gland cavities, through a direct interaction of Pns11 of RGDV and actin of R. dorsalis . Failure of virus-induced filaments assembly by RNA interference with synthesized double-stranded RNA targeting the Pns11 gene inhibited the dissemination of RGDV into salivary cavities, preventing viral transmission by R. dorsalis . For the first time, we show that a virus can exploit virus-induced inclusion as a vehicle to pass through the apical plasmalemma into vector salivary gland cavities, thus overcoming the last membrane barrier for viral transmission by insect vectors. IMPORTANCE Understanding how persistent viruses overcome multiple tissue and membrane barriers within the insect vectors until final transmission is the key for viral disease control. The apical plasmalemma of the cavities where saliva is stored in the salivary glands is the last barrier for viral transmission by insect vectors; however, the mechanism is still poorly understood. Here we show that a virus has evolved to exploit virus-induced filaments to perform an exocytosis-like process that enables viral passage through the apical plasmalemma into salivary cavities. This mechanism could be extensively exploited by other persistent viruses to overcome salivary gland release barriers in insect vectors, opening new perspectives for viral control.

In the double-shelled capsid of Phytoreovirus, the outer capsid attaches firmly to the 3-fold axes of the T=1 core. It then forms a T=13 lattice via lateral interactions among the P8 trimers (Wu et al., 2000, Virology 271, 18-25). Purified P8 molecules also assemble into hexagonal monolayers as well as tubular crystals. To explore the mechanisms of formation of these structures, the configurations of P8 trimers were compared and verified in particles of Rice dwarf virus and in tubular crystals (tubes) whose structure was determined by cryoelectron microscopy using helical reconstruction technique. Remarkable variations in intertrimer contacts were observed in the tubes and in the surface lattice of Rice dwarf virus capsid. Superposition of the atomic structure of P8 trimers in the structures analyzed by cryoelectron microscopy allowed us to identify groups of specific and stable interactions, some of which were preserved in the tubes and the quasi-equivalent T=13 icosahedral lattice of the virion's shell. The flexible nature of the binding between P8 trimers, created via electrostatic interactions that hold radially inward, appears to allow the outer-capsid P8 trimers to envelop the ragged surface of the core, forming the double shell of an intact viral particle.

The nucleotide sequences of segments S1 and S12 of a Chinese isolate of Rice gall dwarf virus (RGDV) were determined. This provides the first complete sequences of these segments. The complete sequence of S1, the largest genome segment of RGDV, was 4,505 nucleotides in length and was predicted to encode a large protein of 1,458 amino acids with a calculated molecular mass of nearly 166.2 kDa. The protein was related to that encoded by S1 of Rice dwarf virus (RDV; 50% identity and 67% similarity) and (to a lesser extent) to some large proteins of other reoviruses. It appears to be an RNA-dependent RNA polymerase (RdRp) and is probably present in particles as a minor core protein. S12, the smallest genome segment of RGDV, was 853 nucleotides in length, encoding a single major protein of 206 amino acids with a calculated molecular mass of nearly 23.6 kDa. This protein, though a little larger than those of RDV S11 and Wound tumor virus (WTV) S12 in size, showed some similarity to them, especially in the conserved N-terminal region and may have RNA-binding properties. Despite having a common host plant, RDV and RGDV were not more closely related to one another than either of them was to WTV. Phylogenetic analysis of the RdRp showed that members of the genus Phytoreovirus were more closely related to those of the genus Rotavirus than to any other genus within the family Reoviridae.

The 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.

An analysis, using microarrays, of gene expression in rice plants infected with Rice dwarf virus revealed significant decreases in levels of expression of genes that are involved in the formation of cell walls, reflecting the stunted growth of diseased plants. The expression of plastid-related genes also was suppressed, as anticipated from the white chlorotic appearance of infected leaves. By contrast, the expression of defense- and stress-related genes was enhanced after viral infection. These results suggest that virus-infected rice plants attempt to survive viral infection and replication by raising the levels of expression of defense- and stress-related genes while suppressing the expression of genes required for the elongation of cells and photosynthesis.

The salivary glands of the Glassy-winged sharpshooter (GWSS), Homalodisca vitripennis Germar 1821, (syn. H. coagulata, Hemiptera: Cicadellidae) were collected and used to produce a cDNA library. Examination by BLASTX analyses identified 2 viral sequences, one a 610-base pair fragment and a second 839-base pair fragment, both of which had significant homology to viruses within the genus Phytoreovirus. Resequencing of the fragments confirmed sequence validities. These sequences were used for in silico protein translation and BLASTP analysis confirming the established homology. While the GWSS is the primary vector of Pierce’s disease of grapes, this is the first report that GWSS may be a vector of a phytoreoviruses. Phylogenetic and homology comparisons with BLASTX, BLASTP, and PAUP analyses indicated that the viral sequences isolated from GWSS were closely related to the viruses in the Family Reoviridae, Genus Phytoreovirus, specifically Rice Dwarf Phytoreovirus (RDV). RDV is the only plant reoviru...

The family Reoviridae separates two subfamilies and consists of 15 genera. Fourteen viruses in three genera (Phytoreovirus, Oryzavirus, and Fijivirus) infect plants. The outbreaks of the plant-infecting reoviruses cause sometime the serious yield loss of rice and maize, and are a menace to safe and efficient food production in the Southeast Asia. The plant-infecting reoviruses are double-shelled icosahedral particles, from 50 to 80nm in diameter, and include from 10 to 12 segmented double-stranded genomic RNAs depending on the viruses. These viruses are transmitted in a persistent manner by the vector insects and replicated in both plants and in their vectors. This review provides a brief overview of the plant-infecting reoviruses and their recent research progresses including the strategy for viral controls using transgenic rice plants.

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.

The strongly conserved amino acid sequences of the P8 outer capsid proteins of Rice dwarf virus (RDV) and Rice gall dwarf virus (RGDV) and the distribution of electrostatic potential on the proteins at the interfaces between structural proteins suggested the possibility that P8-trimers of RGDV might bind to the 3-fold symmetrical axes of RDV core particles, with vertical interaction between heterologous P3 and P8 proteins and lateral binding of homologous P8 proteins, thereby allowing formation of the double-layered capsids that are characteristic of viruses that belong to the family Reoviridae. We proved this hypothesis using chimeric virus-like particles composed of the P3 core capsid protein of RDV and the P8 outer capsid protein of RGDV, which were co-expressed in a baculovirus expression system. This is the first report on the molecular biological proof of the mechanism of the assembly of the double-layered capsids with disparate icosahedral lattices.

The viral and virus-related structures of Rice dwarf virus have been visualized by cryo-electron microscopy and tomography revealing the viral infection and replication mechanisms., 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.

Rice dwarf virus (RDV) is a member of the genus Phytoreovirus , which is composed of viruses with segmented double-stranded RNA genomes. Proteins that support the intercellular movement of these viruses in the host have not been identified. Microprojectile bombardment was used to determine which open reading frames (ORFs) support intercellular movement of a heterologous virus. A plasmid containing an infectious clone of Potato virus X (PVX) defective in cell-to-cell movement and expressing either β-glucuronidase or green fluorescent protein (GFP) was used for cobombardment with plasmids containing ORFs from RDV gene segments S1 through S12 onto leaves of Nicotiana benthamiana . Cell-to-cell movement of the movement-defective PVX was restored by cobombardment with a plasmid containing S6. In the absence of S6, no other gene segment supported movement. Identical results were obtained with Nicotiana tabacum , a host that allows fewer viruses to infect and spread within its tissue. S6 supported the cell-to-cell movement of the movement-defective PVX in sink and source leaves of N. benthamiana . A mutant S6 lacking the translation start codon did not complement the cell-to-cell movement of the movement-defective PVX. An S6 protein product (Pns6)-enhanced GFP fusion was observed near or within cell walls of epidermal cells from N. tabacum . By immunocytochemistry, unfused Pns6 was localized to plasmodesmata in rice leaves infected with RDV. S6 thus encodes a protein with characteristics identical to those of other viral proteins required for the cell-to-cell movement of their genome and therefore is likely required for the cell-to-cell movement of RDV.

The nucleotide sequences of segments S1 to S6 of a Chinese isolate of Rice black-streaked dwarf virus (RBSDV) were determined. This provides the first complete sequence of a plant pathogenic member of the genus Fijivirus. The complete ten-segment genome has 29,141 nucleotides, making it the largest reovirus genome so far reported. Each of the segments S1-S6 is predicted to encode a single major protein. Protein comparisons indicated that S1 encoded an RNA dependent RNA polymerase, with similarities to that encoded by S1 of Nilaparvata lugens reovirus (NLRV). S2 and S3 appeared to be homologous to S3 and S4 respectively of both Fiji disease virus (FDV) and NLRV. The protein encoded on S4 showed some similarity to that of NLRV S2. The proteins encoded on S5 and S6, though similar in size to those of NLRV S5 and S6, had no detectable homologies to them or to any other known protein.

The structures of the double-shelled rice dwarf virus and of its single-shell core have been determined by cryoelectron microscopy and image reconstruction. The core carries a prominent density located at each of the icosahedral faces of its T = 1 lattice. These protrusions are formed by outer shell trimers, tightly inserted at the threefold positions of the core. Such configuration of the core may guide the assembly of the outer shell, aided by lateral interactions between its subunits, into a T = 13 lattice. The organization of the phytoreovirus capsid elucidates for the first time a general model for assembling two unique T numbers of quasi-equivalence.

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 [32P]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 Spodo-ptera 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.

Three forms of tobacco leaf curl (termed classes I, II, and III, based on symptomatology) recently have been described in southern Africa. Numerous attempts to isolate virus particles responsible for a nongeminivirus-induced leaf curl disease (class I) of tobacco in South Africa have been unsuccessful. Recently, 12 dsRNA segments were isolated from tobacco exhibiting class I leaf curl symptoms, suggesting a possible reovirus genome. The objective of our study was to confirm whether the dsRNA segments are associated with a reovirus. Isolation of icosahedral particles with an outer core 60 to 65 nm in diameter and an inner core 40 to 45 nm in diameter was achieved. Twelve distinct nonpolyadenylated dsRNAs were isolated from purified virions, and the total molecular masses of the dsRNAs ranged from 17.86 to 18.40 × 106 Da in polyacrylamide and agarose gels, respectively. Using hybridization analysis, dsRNAs were identified as non-homologous distinct segments. Comparisons with other known reoviruses revealed a unique banding pattern that was most similar to the wound tumor virus (WTV), the type species of the genus Phytoreovirus. Hybridizations of WTV cloned DNA probes (segments S4 and S6 to S9) and dsRNAs from infected tobacco indicated no significant sequence similarity, whereas indirect enzyme-linked immunosorbent assay with a polyclonal antiserum to WTV showed strong positive cross-reactivity to tobacco virions. Our results indicate a virus with features consistent with those of phytoreoviruses. This is the first report of a plant reovirus in tobacco, the first record in Africa, and the second example of a field-isolated dicot phytoreovirus.

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.

The function of rice dwarf virus segment 11 and the corresponding segments of other phytoreoviruses is not yet determined. The amino acid sequence of Pns11, encoded by segment 11, contains a putative zinc finger and five flanking basic regions at the C-terminus. The full-length Pns11 protein and three truncated derivatives, which lack the N-terminus, the zinc-finger, or the C-terminal five basic regions were expressed inEscherichia coliand their nucleic acid binding properties were studied. Pns11 interacts with single- and double-stranded forms of DNA and RNA in a sequence-nonspecific manner. The truncated derivative which contains both the zinc-finger and the C-terminal basic regions has the same binding properties as the full-length Pns11. However, removal of either of these domains prevents binding activity. The binding activity of Pns11 was drastically reduced when the blots were treated with a high concentration of EDTA. Moreover, Pns11 extracted from infected rice also binds to single-stranded RNA. These data suggest that RDV Pns11 binding activity is structure-dependent and it may play an important role in virus replication and/or genome assortment.

Summary Symptom variability of tobacco leaf curl disease has been reported by numerous researchers worldwide. It has been suggested that the different types of leaf curl may be caused by more than one geminivirus strain (McClean, 1940; Osaki & Inouye, 1981), although no evidence exists to support this hypothesis. Previously, several tobacco plants exhibiting a range of leaf curl symptoms were collected from South Africa and Zimbabwe and grouped according to symptomology (Paximadis, Dusterwald, Duyver & Rey, 1997). Three classes (I, II & III) of leaf curl were distinguished, and class I leaf curl from South Africa was shown not to be associated with a geminivirus infection, but was consistently associated with 12 double-stranded RNA species, now believed to be of phytoreovirus origin. In this study, symptom descriptions of class II and III leaf curl have been reported, and, triple-antibody sandwich ELISAs, dot-blot hybridisation analysis, PCR, double-stranded RNA analysis and seed-transmission experiments were employed to screen class II and III tobacco plants for geminiviruses or other possible causal agents. Class II leaf curl symptoms were shown not to be associated with a geminivirus infection or with any double-stranded RNA species. Class II leaf curl symptoms were however transmitted through seed. Class III leaf curl plants tested negative for double-stranded RNA, but tested positive in PCR, dot-blot hybridisation and ELISAs for geminiviruses. It is suggested that ‘tobacco leaf curl’ is not a single disease but rather a wide collection of symptoms incited by different agents, and should therefore be described as a disease complex or syndrome.

The nucleotide sequence of the genome segment 2 (S2) of rice gall dwarf virus (RGDV), a phytoreovirus, when compared with the amino acid sequence of a component protein of the virus, showed that S2 potentially encoded a 127K minor outer capsid protein. This 127K protein designated as P2 and the 127K minor outer capsid protein (also termed P2) of rice dwarf virus (RDV) are similar in size, located in the outer capsid, and have well-conserved predicted polypeptide sequences, suggesting similar functions. Infectivity to insect vector cell monolayers of RGDV was maintained and the P2 protein was retained irrespective of carbon tetrachloride (CCl4) treatment. This is in contrast to the infectivity of RDV which is removed along with P2 protein following CCl4 treatment. RGDV with P2 was acquired by vector insects and transmitted to host plants, although RDV lacking P2 could not be transmitted to plants as previously published. These results imply that RDV and RGDV require P2 proteins for virus infectivity to vector insects.

Although nucleotide sequences of different isolates of rice dwarf phytoreovirus are highly conserved, genome variants can be identified from mobility differences detected in polyacrylamide electrophoresis gels. These differences may be due to base substitutions, deletions, or rearrangements of the dsRNAs. Using known variants, segment reassortment has been experimentally demonstrated for the first time with plant reoviruses, although with some other members of the Reoviridae, it is well documented. The technique for making genetic resassortants could be used to identify genes involved in certain characters such as insect transmissibility, types of symptom expression, and host specificity

The nucleotide sequence of the S3 genome segment of rice gall dwarf virus, a phytoreovirus, consisted of 3224 bp and sequence analysis showed that the segment potentially encoded a 116K major core capsid protein of the virus. This 116K protein and the 114K major core capsid protein of rice dwarf virus are similar in size and were found to have amino acid sequence homology as high as that between the major outer capsid proteins of the two viruses. Core particles and outer capsids of these viruses were interchangeable, suggesting conformational similarity in the three-dimensional architecture of their core and outer capsid proteins.

The nonstructural 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. An RNA interference construct was designed to target the gene for Pns9 of RGDV, namely Trigger_G9. The resultant transgenic plants accumulated short interfering RNAs specific for the construct. All progenies from self-fertilized transgenic plants had strong and heritable resistance to RGDV infection and did not allow the propagation of RGDV. By contrast, our transgenic plants remained susceptible to Rice dwarf virus, another phytoreovirus. There were no significant changes in the morphology of our transgenic plants compared with non-inoculated wild-type rice plants, suggesting that genes critical for the growth of rice plants were unaffected. Our results demonstrate that the resistance to RGDV of our transgenic rice plants is not due to resistance to the vector insects but to specific inhibition of RGDV replication and that the designed trigger sequence is functioning normally. Thus, our strategy to target a gene for viroplasm matrix protein should be applicable to plant viruses that belong to the family Reoviridae.

Examination of cultured insect vector cells that had been infected with Rice gall dwarf virus (RGDV), using transmission electron microscopy and confocal microscopy, revealed the presence of clusters of virus-coated mitochondria around viroplasms in which replication and assembly of RGDV occurred, suggesting a role for mitochondria in supplying the energy required for viral morphogenetic processes. Electron tomography revealed that RGDV particles on the surface of mitochondria are arrayed in an orderly but loose manner, unlike tightly packaged particles in vesicular compartments, suggesting the presence of counterpart molecules on the surface of mitochondria. The viral particles in close proximity to mitochondria were aligned along intermediate filaments, which might serve as scaffolds for the anchorage of these particles. RGDV has a putative mitochondrion-targeting sequence on the outer surface of the outer-capsid protein P8. The arrangement of RGDV particles around mitochondria suggests that the region of the P8 protein containing the mitochondrion-targeting sequence might attach to a molecule like a receptor on the outer mitochondrial membrane. Our analysis demonstrates the three-dimensional arrangement and molecular basis for the mitochondrial proximity of RGDV particles during viral replication.

The individual synthetic plus-strand transcripts from full-length cDNAs of rice dwarf phytoreovirus genome segments S4 to S10 directed the in vitro synthesis of predominant polypeptides, whose migration positions in SDS-polyacrylamide gel indicated molecular masses of 83 k (P4), 89 k (P5), 56 k (P6), 58 k (P7), 43 k (P8), 49 k (P9), and 35 k (P10), respectively. P7 and P8 had the same mobilities as those of the structural proteins, supporting the previous observations. The other proteins (P4, P6, P10) as well as P9, which had been considered an outer capsid protein, are probably nonstructural proteins, since no polypeptides with similar migration behavior were found in a purified RDV preparation. It remains to be confirmed biochemically whether S5 encodes the outer layer protein.

Phytoreoviruses are composed of two concentric capsid layers that surround a viral genome. The capsids are formed mainly by the inner-capsid P3 protein and the outer-capsid P8 protein. During the infection of insect-vector cells, these play important roles in packaging the viral genome and the enzymes required for its transcription. P3 and P8 proteins, when co-expressed in Spodoptera frugiperda cells, co-localized in cells and were released as spherical clusters. In contrast P3 proteins expressed in the absence of P8 protein were associated with the cells when they were examined by confocal microscopy. Cryo-electron microscopy revealed that the secreted clusters, composed of P3 and P8 proteins, were double-layered virus-like particles that were indistinguishable from intact viral particles. Our results indicate that P8 proteins mediate the secretion of assembled virus-like particles from S. frugiperda insect cells and, therefore, most probably from insect-vector cells also.

As RNA viruses evolve rapidly, we hypothesized that a virus could serve as a surrogate to discriminate recently separated populations of an invasive insect species. Homalodisca vitripennis reovirus (HoVRV) was used as a surrogate to assess population structure of glassy-winged sharpshooter (GWSS), an invasive species detected in California ~ 20 years ago. HoVRV nucleotide sequence polymorphism revealed a bottleneck in the introduced population, yielded population age estimates consistent with timing of GWSS discovery in California, suggested gene flow within the native range but not among native and introduced populations, and could potentially pinpoint source of the introduced population. Collectively, the data support use of a virus surrogate to define critical attributes of invasive species populations, with the caveat that life history of the surrogate must be closely linked to that of the host.

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.

Sequence analysis revealed that rice dwarf phytoreovirus segment S12 is 1066 nucleotides long with a small out-of-phase, overlapping open reading frame (ORF) as well as a major ORF. The large ORF (positions 42 to 980) encodes 312 amino acids, while the small one (bases 313 to 591) encodes 92 amino acids with an additional in-frame AUG codon (positions 337-339) 24 nucleotides downstream from the first one. Transcripts from a full-length cDNA directed the in vitro synthesis of three polypeptides of 33 (considered to be translated from the long ORF), 8, and 7 kDa. Alteration of each of the two ATG codons on the small ORF demonstrated their involvement in the generation of the 8- and 7-kDa polypeptides. Although it is still unknown whether these proteins are expressed in vivo, the small ORF is shown to be conserved in S9s of two other members of the genus Phytoreovirus, rice gall dwarf virus and wound tumor virus, suggesting its common, important function.

The 5'- and 3'-terminal nucleotide sequences of the dsRNA genome segments of rice ragged stunt virus (RRSV), a member of the plant Reoviridae, were determined and compared with those published for other viruses in this family. The 5'- and 3'-terminal regions of the RRSV plus strand RNA from all genome segments were found to have the same conserved hexanucleotide (5' GAUAAA---) and tetranucleotide (---GUGC 3') sequences, respectively. These conserved terminal sequences were different from those found in viruses in the Phytoreovirus and Fijivirus genera. This result confirms that, as already suggested, RRSV should be placed in a third genus.

The 5′- and 3′-terminal nucleotide sequences of the dsRNA genome segments of rice dwarf virus (RDV) and rice gall dwarf virus (RGDV), members of the Phytoreovirus genus of the Reoviridae family, were determined and compared with those of wound tumour virus (WTV). The 3′ tetranucleotides of the plus strand of all genome segments of RDV and RGDV were found to be the same (---UGAU 3′), except for segment 9 of RDV which had the 3′-terminal sequence ---CGAU 3′. The conserved 3′-terminal sequence (---UGAU 3′) was the same as that found in the genome segments of WTV, another member of the Phytoreovirus genus. On the other hand, the 5′ termini of the plus strands of RDV and RGDV were found to have two or three types of common sequence. RDV had either 5′ GGCAAA--- or 5′ GGUAAA---, whereas RGDV had 5′ GGCAUUUU---, 5′ GGUAUUUU--- or 5′ GGUAAUUU---. These conserved sequences were similar to the conserved 5′-terminal sequence of WTV (5′ GGUAUU---). Although the three viruses differ in plant host range, tissue specificity, vector specificity and disease symptom expression, these results suggest that they have a common ancestral origin.

The 46K outer capsid protein encoded by RNA segment S8 and the 42K polypeptide, previously thought to be the segment S9-encoded structural protein, were isolated from a rice dwarf phytoreovirus purified preparation, and then analysed by peptide mapping and electroblot-ELISA. Staphylococcus aureus V8 protease peptide mapping patterns of the 42K and 46K proteins were similar. Two monoclonal antibodies (MAbs), obtained after immunization with virus particles dissociated by 0.1% SDS, were each specific for both the 42K and 46K proteins. Furthermore, the MAbs bound common peptide fragments which were generated by digestion of the 42K and 46K proteins with V8 protease or proteinase K. These results strongly suggest that the 42K protein is not a gene product of S9 but a product overlapping with the 46K outer capsid protein. Whether the two proteins are functionally distinct remains to be determined.

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.

The non-structural protein Pns12 of Rice dwarf virus is one of the early proteins expressed in cultured insect cells, and it is one of 12 proteins that initiate the formation of the viroplasm, the putative site of viral replication. Pns4 is also a non-structural protein, visible as minitubules after nucleation of the viroplasm. We introduced Pns12- and Pns4-specific RNA interference (RNAi) constructs into rice plants. The resultant transgenic plants accumulated short interfering RNAs specific to the constructs. The progeny of rice plants with Pns12-specific RNAi constructs, after self-fertilization, were strongly resistant to viral infection. By contrast, resistance was less apparent in the case of rice plants with Pns4-specific RNAi constructs, and delayed symptoms appeared in some plants of each line. Our results suggest that interference with the expression of a protein that is critical for viral replication, such as the viroplasm matrix protein Pns12, might be a practical and effective way to control viral infection in crop plants.

Tobacco leaf enation virus (TLEV) is a putative member of the genus Phytoreovirus within the family Reoviridae. Previous western blot analysis of structural viral proteins (apparent molecular weights of 93 kDa; 58 kDa; 48 kDa; 39 kDa and 36 kDa) associated with TLEV, isolated from infected tobacco in South Africa, suggested that these proteins may correspond to structural Wound tumor virus (WTV) proteins. To further establish the nature of this novel virus disease phenotype in tobacco, molecular characterization of six dsRNA components was undertaken. Full-length cDNA clones were obtained by an optimized modified single-primer amplification sequence-independent dsRNA cloning method. Results of this study revealed the conserved terminal sequence: 5′GG(U/C)...UGAU 3′ of segments S6–S12, while adjacent to these conserved terminal sequences are imperfect inverted repeats (7–15 bp in length), both features being common to reoviruses. The complete nucleotide sequences of segments S5 (2,610 bp), S7 (1,740 bp), S8 (1,439 bp), S10 (1,252 bp), S11 (1,187 bp) and S12 (836 bp) were determined. Comparison of full-length nucleotide sequences with corresponding segments of other phytoreoviruses, Rice gall dwarf virus (RGDV), Rice dwarf virus (RDV) and WTV has shown nucleotide and predicted amino acid identities within the range of 30–60%. TLEV consistently shows a higher identity to WTV than to other phytoreovirus species where sequence data is available. Each segment had a single predicted open reading frame encoding proteins with calculated molecular weights of S5 (90.6 kDa); S7 (58.1 kDa); S8 (47.7 kDa); S10 (39.8 kDa); S11 (35 kDa) and S12 (19.5 kDa). The relatively low nucleotide and amino acid identity to other members of the genus demonstrates that TLEV is a novel phytoreovirus, distinct from the only other reported dicotyledenous-infecting WTV and is the first phytoreovirus reported to emerge in Africa.

Rice gall dwarf virus (RGDV) is a member of the genus Phytoreovirus within the family Reovirdae. Its genome has 12 segments of double-stranded RNA (dsRNA), of which the nucleotide sequences of segments S4, S6, and S7 were determined, providing the first complete genome sequence of RGDV. Each of the segments S4, S6, and S7 contained conserved terminal sequences conforming to the RGDV consensus, 5'-GGXA ... UGAU-3' (X = U or C). Each segment had a single predicted open reading frame encoding proteins with calculated molecular weights of 79.8, 58.6, and 53.3 kDa. These proteins appeared to be homologous to those encoded by the corresponding segments of rice dwarf virus and wound tumor virus, the other known members of the same genus, having about 20-30% amino acid identity to them. It is therefore likely that RGDV S4 and S6 encode non-structural proteins and S7 an inner core protein. Probable homologies between the segments of all known phytoreoviruses are summarized. Beyond these similarities, the RGDV proteins displayed no significant similarity to any other reported viral proteins.

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.

Gene rearrangements in a head-to-tail fashion have been described several times for gene segments of the rota-, phytoreo-, and orbiviruses. Several mechanisms have been proposed to explain the occurrence of partial duplications, however, none of these models has been fully satisfactory to explain the occurrence of all the observed duplicated genes. Based on recently available structural data about the λ3 RNA-dependent-RNA-polymerase of reoviruses, we propose the ‘loop model’ as a plausible explanation for the occurrence of partial gene duplications in dsRNA viruses.

Loss of infectivity to insect vector cell monolayers of rice dwarf phytoreovirus (RDV) after CCl4treatment was associated with the removal of one of the viral proteins from the virus particles. This protein, encoded by genome segment S2 and thus named P2 protein, was located at the outer capsid of the virus particle. When RDV was treated with CCl4for various times, the reduction in the amount of P2 protein and the loss of viral infectivity to vector cell monolayers were proportional to treatment time. RDV purified using CCl4treatment, thus lacking P2 protein, lost the ability to infect vector insects through feeding and consequently failed to be transmitted to plants. These results suggest that P2 protein is essential for RDV infection of the insect cells and may be related to transmission of the virus by the vector insect.

The Phytoreovirus rice dwarf virus (RDV) has a complex nucleocapsid architecture composed of multiple proteins and RNAs. However, specific RNA-protein and protein-protein interactions involved in virion packaging have not been entirely elucidated. In order to define mechanisms governing RDV particle assembly, interactions between individual components were analyzed both in vivo and in vitro. The P7 core protein binds specifically and with high affinity to all 12 genomic RDV dsRNAs. P1, a putative RNA polymerase, P5, a putative guanyltransferase and P7 are encapsidated within the virion and also bind viral transcripts based upon in vitro binding assays. P1, P5, P7 and genomic dsRNAs were lacking in empty particles purified from infected tissues that also yielded fractions containing intact, infectious particles. In addition, P7 forms complexes with P1 and P3, a core capsid protein, in viral particles. These results indicate the possibility that core proteins and dsRNAs interact as one unit suggesting a mechanism for assortment of viral RNAs and subsequent packaging into core particles.

The family Reoviridae of double-stranded RNA viruses is recognized to comprise nine genera (31), with at least two others recently proposed (1, 24). Members of five recognized genera—Aquareovirus, Cypovirus, Fijivirus, Orthoreovirus, and Oryzavirus—are distinctive in having pentameric turrets that sit atop the capsid around each fivefold axis in the “inner-capsid particle” or “core” (13, 29, 36). In aquareoviruses, cypoviruses, and orthoreoviruses, these turrets are known or strongly suggested to mediate the guanylyltransferase and methyltransferase reactions in 5′ capping of the viral plus-strand RNA transcripts (6, 10, 27, 29). The cores of these viruses are also distinctive in having either 120 or 150 copies of a nodule protein that sit atop the capsid and contribute to its stability (13, 17, 29, 34, 36). In light of these features, Hill et al. (13) have proposed that the turreted viruses constitute an evolutionarily related subgroup. We now report that the recognized genera of turreted Reoviridae have another distinctive feature: conserved motifs for nucleoside triphosphate (NTP) binding in proteins of similar size to the μ2 protein of mammalian orthoreoviruses (18, 23) (Fig. ​(Fig.1).1). The motifs are related to ones in other NTP-binding proteins (19, 20, 32) but have distinguishing elements. In addition to five other positions occupied by hydrophobic residues, the motifs can be summarized as KgsgKs and dSDxyG, where uppercase letters indicate wholly conserved residues and lowercase letters indicate partially conserved residues (Fig. ​(Fig.1).1). We have recently shown that one or both lysines in motif A are essential for the triphosphatase activities of μ2 (18) (see below). Although previous authors have noted similarities to NTP-binding motifs in many of these sequences (8, 12, 15, 18), none have observed that the specific motifs shown in Fig. ​Fig.11 are conserved among all genera of turreted Reoviridae. Upon comparing the full-length protein sequences, we found that similarities outside the motif regions are less striking, with pairwise identities of ≤25% between even the two most closely related genera, Orthoreovirus and Aquareovirus (2). FIG. 1. Conserved NTP-binding motifs in 60- to 85-kDa proteins from Reoviridae members. Genpept, accession number from the translated GenBank database. Motifs: uppercase red, conserved in all of the sequences; lowercase, conserved in at least half of the sequences; ... The μ2 protein is a minor component of orthoreovirus cores (∼20 copies per particle) (7). It resides inside the core in association with both capsid protein λ1 and RNA-dependent RNA polymerase (RdRp) protein λ3, constituting the transcriptase complexes (9, 37). Genetically, μ2 determines strain differences in the transcriptase and nucleoside triphosphatase (NTPase) activities of cores (23, 35), and purified μ2 functions as both an NTPase and an RNA 5′ triphosphatase (RTPase) (18). The μ2 protein also has RNA- and microtubule-binding activities (4, 25). Less is known about the proteins with μ2-like NTP-binding motifs from other turreted Reoviridae. Like μ2, cypovirus VSP4/VP4 and fijivirus P-S8/P9/73.5KD are minor components of their respective cores (12, 22). Based on homologies to μ2 over the lengths of both proteins, aquareovirus VP5 has been proposed to be a minor core component as well (2). Oryzavirus Pns7 is reported to be a nonstructural protein (30), but given the limited work on this genus, we consider this assignment to be tentative. Instead, we postulate that the proteins with μ2-like NTP-binding motifs reside inside the cores of all turreted Reoviridae members and mediate NTPase-related functions similar to those of μ2. Upon searching the protein databases with a consensus defined by the μ2-like NTP-binding motifs from turreted Reoviridae, we found that Colorado tick fever and Eyach viruses, from the Coltivirus genus of nonturreted Reoviridae (3), also contain these motifs in a protein of similar size to that of μ2 (Fig. ​(Fig.1).1). However, viruses from the other three recognized genera of nonturreted Reoviridae—Orbivirus, Phytoreovirus, and Rotavirus—do not. In fact, using a search pattern with up to 500 residues between the two motifs, we identified only the proteins shown in Fig. ​Fig.1,1, i.e., no other viral or cellular proteins. Attoui et al. (3) have noted that VP10 contains a nucleotide-binding motif as well as broader similarities to protein kinases, the fijivirus 73.5KD protein, and oryzavirus Pns7. Whether coltivirus VP10 is a core protein has not been reported, but we now predict that it is. Considering the new findings, we propose, similarly to Hill et al. (13), that Reoviridae members exhibit at least two distinct organizational strategies for their RNA synthesis components in particles. For the genera shown in Fig. ​Fig.1,1, we propose a conserved class of transcriptase complex, including an RdRp and a μ2-like NTPase anchored beneath the capsid near each fivefold axis. This type of transcriptase is usually accompanied atop the capsid by pentameric turrets that mediate the guanylyltransferase and methyltransferase reactions in RNA capping. The μ2-like NTPase may be the RTPase that mediates the first reaction in capping or may perform another function in RNA synthesis. This organization contrasts with that of the nonturreted rotaviruses and orbiviruses, which have a guanylyltransferase protein that associates with the RdRp beneath the capsid near each fivefold axis (11, 26). In orbiviruses, this protein has been shown to mediate the RTPase reaction as well (28), but the rotavirus equivalent has not (5, 21). Orbiviruses contain a third internal protein, which mediates NTPase and RNA helicase activities in vitro (16) and contains NTP-binding motifs that do not match the Fig. ​Fig.11 consensus. However, no equivalent to this protein has been found in rotavirus particles, suggesting that the nonturreted viruses may be a more diversified group than the turreted ones. The findings for coltiviruses spark additional interest and suggest several explanations. The simplest may be that coltiviruses are turreted and that this feature has simply been missed in the limited studies to date. This idea is supported by recent findings for putative Reoviridae members that infect fungi. These agents, Rosellinia necatrix antirot virus (RArV) and Cryphonectria parasitica 9B21 virus, show strong sequence similarities to coltiviruses (14, 24, 33), and our inspections of the available RArV sequences revealed μ2-like NTP-binding motifs in protein P6 (Fig. ​(Fig.1).1). Furthermore, we were impressed to see that RArV cores contain turrets (33). Another interesting result is that the RdRp sequences of coltiviruses cluster with those of the turreted viruses in phylogenetic comparisons (2). Thus, we speculate that the coltiviruses are also turreted and have transcriptase components and strategies closely related to those of the other turreted Reoviridae.