Assembly of double-shelled, virus-like particles in transgenic rice plants expressing two major structural proteins of rice dwarf virus

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.     

Three-dimensional structure of the inner core of rice dwarf virus

Rice dwarf virus (RDV) is a double-shelled icosahedral virus.Using electron cryomicroscopy and computer reconstruction techniques, we have determined a 3.3 nm resolution three-dimensional (3D) structure of the inner shell capsid without the outer shell and viral RNA. The results show that the inner shell is a thin, densely packed, smooth structure, which provides a scaffold for the full virus. A total of 120 copies of the major inner shell capsid protein P3 forms 60 dimers arranged in a T=1 icosahedral lattice. A close examination on the subunit packing of the T=1 inner core P3 with that of the T=13l outer shell P8 indicated that P8 trimers connect with P3 through completely non-equivalent, yet highly specific, intermolecular interactions.     

Three-dimensional structure of the inner core of rice dwarf virus

Rice dwarf virus (RDV) is a double-shelled icosahedral virus. Using electron cryomicro-scopy and computer reconstruction techniques, we have determined a 3.3 nm resolution three-dimensional (3D) structure of the inner shell capsid without the outer shell and viral RNA. The results show that the inner shell is a thin, densely packed, smooth structure, which provides a scaffold for the full virus. A total of 120 copies of the major inner shell capsid protein P3 forms 60 dimers arranged in a T=1 icosahedral lattice. A close examination on the subunit packing of the T=1 inner core P3 with that of the T=13/ outer shell P8 indicated that P8 trimers connect with P3 through completely non-equivalent, yet highly specific, intermolecular interactions.     

Transcapsidation and the conserved interactions of two major structural proteins of a pair of phytoreoviruses confirm the mechanism of assembly of the outer capsid layer

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.     

Crystallization and preliminary X-ray study of a double-shelled spherical virus, rice dwarf virus

Rice dwarf virus (RDV) is a double-shelled spherical plant virus consisting of 46,000 Mr capsid and 114,000 Mr core proteins and minor structural proteins, and containing 12 genome segments of double-stranded RNA. The virus has been crystallized in the cubic space group I23 with a = 789 A. There are two particles per unit cell, each positioned on a point of 23 symmetry. Packing considerations showed that the diameter of the virus particle is 693 A. The crystals diffract to at least 6.5 A resolution.     

Structure of Double-Shelled Rice Dwarf Virus

Rice dwarf virus (RDV), a member of the Reoviridae family, is a double-stranded RNA virus. Infection of rice plants with RDV reduces crop production significantly and can pose a major economic threat to Southeast Asia. A 25-Å three-dimensional structure of the 700-Å-diameter RDV capsid has been determined by 400-kV electron cryomicroscopy and computer reconstruction. The structure revealed two distinctive icosahedral shells: a T=13l outer icosahedral shell composed of 260 trimeric clusters of P8 (46 kDa) and an inner T=1 icosahedral shell of 60 dimers of P3 (114 kDa). Sequence and structural comparisons were made between the RDV outer shell trimer and the two crystal conformations (REF and HEX) of the VP7 trimer of bluetongue virus, an animal analog of RDV. The low-resolution structural match of the RDV outer shell trimer to the HEX conformation of VP7 trimer has led to the proposal that P8 consists of an upper domain of β-sandwich motif and a lower domain of α helices. The less well fit REF conformation of VP7 to the RDV trimer may be due to the differences between VP7 and P8 in the sequence of the hinge region that connects the two domains. The additional mass density and the absence of a known signaling peptide on the surface of the RDV outer shell trimer may be responsible for the different interactions between plants and animal reoviruses.     

Pleomorphic configuration of the trimeric capsid proteins of Rice dwarf virus that allows formation of both the outer capsid and tubular crystals

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.     

RNA-binding domain of the key structural protein P7 for the Rice dwarf virus particle assembly

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 Pseudomonasfragi 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 Pseudomonasfragi 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, besides its function in the assembly and subsequent packaging of viral dsRNA into core particles.     

The rice dwarf virus P2 protein interacts with ent-kaurene oxidases in vivo, leading to reduced biosynthesis of gibberellins and rice dwarf symptoms

The mechanisms of viral diseases are a major focus of biology. Despite intensive investigations, how a plant virus interacts with host factors to cause diseases remains poorly understood. The Rice dwarf virus (RDV), a member of the genus Phytoreovirus, causes dwarfed growth phenotypes in infected rice (Oryza sativa) plants. The outer capsid protein P2 is essential during RDV infection of insects and thus influences transmission of RDV by the insect vector. However, its role during RDV infection within the rice host is unknown. By yeast two-hybrid and coimmunoprecipitation assays, we report that P2 of RDV interacts with ent-kaurene oxidases, which play a key role in the biosynthesis of plant growth hormones gibberellins, in infected plants. Furthermore, the expression of ent-kaurene oxidases was reduced in the infected plants. The level of endogenous GA1 (a major active gibberellin in rice vegetative tissues) in the RDV-infected plants was lower than that in healthy plants. Exogenous application of GA3 to RDV-infected rice plants restored the normal growth phenotypes. These results provide evidence that the P2 protein of RDV interferes with the function of a cellular factor, through direct physical interactions, that is important for the biosynthesis of a growth hormone leading to symptom expression. In addition, the interaction between P2 and rice ent-kaurene oxidase-like proteins may decrease phytoalexin biosynthesis and make plants more competent for virus replication. Moreover, P2 may provide a novel tool to investigate the regulation of GA metabolism for plant growth and development.     

Recovery of transgenic rice plants expressing the rice dwarf virus outer coat protein gene (S8)

 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. Received: 1 March 1996 / Accepted: 2 August 1996     

Rice dwarf viruses with dysfunctional genomes generated in plants are filtered out in vector insects: implications for the origin of the virus

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.     

烟草果胶甲基酯酶（PME）基因的克隆及功能分析(英)

为了研究果胶甲基酯酶（pectin methyl-esterase,PME）（EC 3.1.1.11）与植物病毒运动蛋白之间的相互作用,应用RT-PCR方法从烟草(Nicotiana benthamiana)中克隆了PME基因,并测定了全序列(GenBank登录号AY238968).序列分析显示该基因由两个保守的结构域组成(PMEI和pectinesterase). DNA印迹结果表明,该基因在基因组中存在多个拷贝,蛋白质印迹表明,植物总蛋白中存在两种形式的PME蛋白,但RNA印迹结果显示,在烟草细胞中只检测到全长的PME转录产物.酵母双杂交结果表明,PME与水稻矮缩病毒Pns11(具有非特异的核酸结合活性)之间存在相互作用,而没有检测到PME与已知的运动蛋白Pns6之间的相互作用,推测Pns11蛋白可能参与了水稻矮缩病毒粒子的运动.     

Electron cryomicroscopy and bioinformatics suggest protein fold models for rice dwarf virus

The three-dimensional structure of rice dwarf virus was determined to 6.8 A resolution by single particle electron cryomicroscopy. By integrating the structural analysis with bioinformatics, the folds of the proteins in the double-shelled capsid were derived. In the outer shell protein, the uniquely orientated upper and lower domains are composed of similar secondary structure elements but have different relative orientations from that of bluetongue virus in the same Reoviridae family. Differences in both sequence and structure between these proteins may be important in defining virus-host interactions. The inner shell protein adopts a conformation similar to other members of Reoviridae, suggesting a common ancestor that has evolved to infect hosts ranging from plants to animals. Symmetry mismatch between the two shells results in nonequivalent, yet specific, interactions that contribute to the stability of this large macromolecular machine.     

The amino-terminal region of major capsid protein P3 is essential for self-assembly of single-shelled core-like particles of Rice dwarf virus

The core protein P3 of Rice dwarf virus constructs asymmetric dimers, one of which is inserted by the amino-terminal region of another P3 protein. The P3 proteins with serial amino-terminal deletions, expressed in a baculovirus system, formed particles with gradually decreasing stability. The capacity for self-assembly disappeared when 52 of the amino-terminal amino acids had been deleted. These results demonstrated that insertion of the amino-terminal arm of one P3 protein into another appears to play an important role in stabilizing the core particles.     

Interaction of rice dwarf virus outer capsid P8 protein with rice glycolate oxidase mediates relocalization of P8

Yeast two-hybrid and coimmunoprecipitation assays indicated that P8, an outer capsid protein of Rice dwarf phytoreovirus (RDV), interacts with rice glycolate oxidase (GOX), a typical enzyme of peroxisomes. Confocal immunofluorescence microscopy revealed that P8 was colocalized with GOX in peroxisomes. Time course analysis demonstrated that the localization of P8 in Spodoptera frugiperda cells changed from diffuse to discrete, punctuate inclusions during expression from 24 to 48 h post inoculation. Coexpression of GOX with P8 may target P8 into peroxisomes, which serve as replication sites for a number of viruses. Therefore, we conclude that the interaction of P8 with the GOX of host cells leads to translocation of P8 into peroxisomes and we further propose that the interaction between P8 and GOX may play important roles in RDV targeting into the replication site of host cells. Our findings have broad significance in studying the mechanisms whereby viruses target appropriate replication sites and begin their replication.     

Transformation of rice plants by plant reovirus genes

Rice dwarf phytoreovirus (RDV), and rice ragged stunt oryzairus (RRSV) genes were introduced into rice protoplasts by using the cauliflower mosaic virus 35S promoter, tissue culture techniques and electroporation. The translation products of cDNA to RDV segment 8 were detected in transformed rice. Plants transgenic for RRSV S9 also expressed an mRNA of appropriate size but the protein was not apparently expressed. These latter plants did not show any resistance when inoculated with RRSV; on the contrary, symptom expression was intensified. Since most plant reoviruses are phloem-limited, an alternative promoter could be that of rice tungro bacilliform virus (RTBV), which is itself phloem-limited. When the β-glucuronidase (GUS) gene was coupled to this promoter and introduced into rice, GUS activity was successfully expressed only in the phloem, so the system could be of interest in the reovirus context.     

Entry of Rice dwarf virus into cultured cells of its insect vector involves clathrin-mediated endocytosis

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.