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P2 Protein Encoded by Genome Segment S2 of Rice Dwarf Phytoreovirus Is Essential for Virus Infection
Abstract
Loss of infectivity to insect vector cell monolayers of rice dwarf phytoreovirus (RDV) after CCl4 treatment 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 CCl4 for 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 CCl4 treatment 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.
... A cell culture from embryonic tissue of A. sanguinolenta was developed by Chiu et al. (1970). After several sub-cultures, the cell suspension monolayer was inoculated with purified potato yellow dwarf virus (PYDV; Mitsuhashi and Maramorosch 1964a, b;Chiu et al. 1966;Reddy and Black 1972;Gamez and Black 1967, 68;Gamez and Chiu 1968 Modified Kimura 1984Kimura , 1986Omura et al. 1984Omura et al. , 1988Omura et al. , 1998Yan et al. 1996;Wei et al. 2006aWei et al. , b, 2007Wei et al. , 2008Wei et al. , 2009Wei et al. , 2011Chen et al. 2012 Nephotettix aplicalis Wayadande and Fletcher (1998). These researchers followed a procedure modified from thrips cell culture (Hunter and Hsu 1996). ...
... The function of P2 protein of RDV in leafhopper vector cells was investigated using N. cincticeps cell line NC-24 (Yan et al. 1996;Omura et al. 1998). Treatment of RDV with carbon tetrachloride resulted in a reduction in P2 protein and simultaneous loss of Phytoparasitica infectivity to vector cells (Yan et al. 1996). ...
... The function of P2 protein of RDV in leafhopper vector cells was investigated using N. cincticeps cell line NC-24 (Yan et al. 1996;Omura et al. 1998). Treatment of RDV with carbon tetrachloride resulted in a reduction in P2 protein and simultaneous loss of Phytoparasitica infectivity to vector cells (Yan et al. 1996). Omura et al. (1998) further demonstrated that the infection of the vector cell monolayer by a transmission-competent RDV isolate was reduced with a reduction in the amount of P2 protein. ...
Culturing insect cells in vitro has witnessed remarkable growth in the past decades. Over 600 cell lines of dipteran, hemipteran, and lepidopteran insects have been reported so far. Besides its wide adoption for the production of recombinant proteins, viral pesticides, and vaccines, insect cell culture is increasingly being used as a tool for basic research in genetics, molecular biology, biochemistry, and virology. Since its first application in plant virus studies in 1956, insect cell culture has become an indispensable tool to study the complex and intimate interactions of plant viruses and their insect vectors. The in vitro cell cultures of several insect vectors of plant viruses including leafhoppers, planthoppers, aphids, thrips, and whiteflies have been successfully employed to understand the functions of viral proteins and receptor-mediated endocytosis into vector cells. This review summarizes the development of novel media and insect cell culture systems and their applications in plant virus research.
... The core particle of the virus contains 12 segments of double-stranded RNA and four species of protein. The core is enclosed by an outer capsid layer composed of P2 and P8 proteins (21). The P2 minor outer capsid protein was recently shown to be essential for infection (21). ...
... The core is enclosed by an outer capsid layer composed of P2 and P8 proteins (21). The P2 minor outer capsid protein was recently shown to be essential for infection (21). The P8 major outer capsid protein also appears to be associated with infection, since neutralizing antibodies can be raised against this protein (9a). ...
... The P8 major outer capsid protein also appears to be associated with infection, since neutralizing antibodies can be raised against this protein (9a). The appearances of viral particles with and without the P2 protein are indistinguishable (21), an observation that suggests that the basic morphological organization of the outer capsid of the virus depends on the arrangement of the P8 protein. The threedimensional structure of virus particles suggests that the outer capsid of RDV is composed of 260 capsomeres (trimers), which consist of 780 protein subunits (8). ...
Two-dimensional crystals were obtained from purified P8, an outer capsid protein of rice dwarf phytoreovirus. A filtered image of the two-dimensional crystal, in combination with the results of biochemical analysis, revealed the unit formation of the capsid protein, a capsomere structure, which appeared to be an approximately equilateral triangle with sides of approximately 6 nm and which was composed of a trimer of P8 protein. Details of the arrangements of the outer capsid of the virus are described.
... Many virus-encoding proteins involving in the replication cycle have been determined. For example, RDV P2, protruding from the surface of the outer shell of virions, is essential for virions entry into insect vector cells in a receptor-mediated, clathrin-dependent endocytosis manner [151][152][153][154]. Nonstructural proteins such as RBSDV P9-1 [155,156], RDV Pns12 [157], RGDV Pns9 [158], RRSV Pns10 [77,159], and RYSV P6 [160] contribute to the assembly of viroplasm for viral genome replication and progeny virion assembly. ...
Rice (Oryza sativa L.) is one of the major staple foods for global consumption. A major roadblock to global rice production is persistent loss of crops caused by plant diseases, including rice blast, sheath blight, bacterial blight, and particularly various vector-borne rice viral diseases. Since the late 19th century, 19 species of rice viruses have been recorded in rice-producing areas worldwide and cause varying degrees of damage on the rice production. Among them, southern rice black-streaked dwarf virus (SRBSDV) and rice black-streaked dwarf virus (RBSDV) in Asia, rice yellow mottle virus (RYMV) in Africa, and rice stripe necrosis virus (RSNV) in America currently pose serious threats to rice yields. This review systematizes the emergence and damage of rice viral diseases, the symptomatology and transmission biology of rice viruses, the arm races between viruses and rice plants as well as their insect vectors, and the strategies for the prevention and control of rice viral diseases.
... In addition, Kimura (1984) succeeded in establishing cultured cells of the RDV insect vector and creating inoculation methods using the cells with RDV. Using the cultured cells, Yan et al. (1996) and Tomaru et al. (1997) clearly demonstrated that the P2 protein encoded by the genome segment S2 is crucial for infection of RDV in insect cells and transmission of the virus by the insect vector. ...
An increasing number of plant viruses and viroids have been reported from all over the world due largely to metavirogenomics approaches with technological innovation. Herein, the official changes of virus taxonomy, including the establishment of megataxonomy and amendments of the codes of virus classification and nomenclature, recently made by the International Committee on Taxonomy of Viruses were summarized. The continued efforts of the plant virology community of Japan to index all plant viruses and viroids occurring in Japan, which represent 407 viruses, including 303 virus species and 104 unclassified viruses, and 25 viroids, including 20 species and 5 unclassified viroids, as of October 2021, were also introduced. These viruses and viroids are collectively classified into 81 genera within 26 families of 3 kingdoms ( Shotokuvirae , Orthornavirae , Pararnavirae ) across 2 realms ( Monodnaviria and Riboviria ). This review also overviewed how Japan’s plant virus/viroid studies have contributed to advance virus/viroid taxonomy.
... Plant-infecting reoviruses that invade both the mesophyll and phloem tissues generally take shorter acquisition and inoculation times than other persistent-propagative viruses (Nault and Ammar, 1989). Several studies seeking to identify the role of the RDV capsid proteins (P2, P8, and P3), which enclose the virus genome, found that P2 is necessary for efficient viral infection of the insect vector (Kimura, 1986;Omura et al., 1998;Yan et al., 1996). Overall, reoviruses follow a transmission path that starts with virus entry into the epithelial cells of the foregut and/or filter chamber from where they replicate and disseminate to the anterior midgut before traversing the basal lamina to infect the muscle cells (Chen et al., 2011a,b). ...
Arthropod vectors, including insects and mites, play a significant role in the transmission of several viruses to their shared plant hosts. Four classical modes of transmission as well as two less conventional ones are used by arthropod vectors with either piercing-sucking or chewing mouthparts to transmit specific plant viruses in distinct ways. In the past few decades, significant progress has been made in the understanding of biological and molecular bases for key groups of vector–virus combinations. These findings set the stage for future studies to better comprehend vector–virus interactions and to use that knowledge to devise new and sustainable strategies for vector and viral disease control to ensure food security for a growing worldwide population.
... P2 is the minor outer capsid protein and is encoded by viral genome segment 2. This protein is a multifunctional protein that is essential for virus infection of insect vectors and contributes to the development of the dwarf phenotype in rice plants infected with RDV by interfering with the gibberellic acid synthesis (Omura et al. 1998 ). Intact RDV virions infect insect VCMs but lose viral infectivity when the P2 protein is removed by chemical treatment (Yan et al. 1996 ). Virions without P2 failed to infect insects and consequently failed to be transmitted to plants suggesting that P2 may have a role in recognition and attachment of the virus to the insect vector. ...
Plant-infecting viruses that are transmitted in a persistent-propagative manner must persist and replicate in two divergent hosts, plants and insects. There are fi ve groups of persistent-propagative plant viruses: rhabdoviruses, reoviruses, bunyaviruses, marafi viruses, and tenuiviruses. Throughout the transmission cycle of a persistent-propagative virus, there is a close association between virus and vector that is dependent on specifi c interactions. The –omic technologies that are now widely used for simultaneous examination of thousands of genes (genomics), mRNAs (transcriptomics), and proteins (proteomics) combined with highthroughput bioinformatic tools to extract a vast amount of information have become a popular approach to better understand virus-vector interactions. The integration of the available datasets that result from these –omic studies is contributing to the identifi cation of host factors that are required for the viral replication cycle. Current knowledge of the vector components that function in viral infection is still limited for the majority of persistent-propagative viruses. However, the emerging information on genomes, transcriptomes, and proteomes for insect vectors of plant viruses provides unique opportunities for studying the function of genes involved in virus attachment, acquisition, and transmission in different vector species. In this chapter we discuss the major groups of plant viruses transmitted in a persistent-propagative manner, the biology of these viruses, the interactions with their vectors, and the – omic technologies applied to study these virus-vector pathosystems.
... The viral core particle is composed of the inner core protein P3, which encloses P1, a putative RNA polymerase, P5, a guanylyltransferase, and P7, a protien with RNA-binding activity [8][9][10][11]. The outer capsid shell is composed of the major outer capsid protein P8, and the minor outer capsid proteins P2 and P9 [12][13][14][15][16]. The functions of nonstructural proteins in the insect vector have been determined using continuous cell cultures derived from N. cincticeps. ...
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.
Methods
RNA interference (RNAi) system targeting Pns4 gene of RDV was conducted. Double-stranded RNA (dsRNA) specific for Pns4 gene was synthesized in vitro, and introduced into cultured leafhopper cells by transfection or into insect body by microinjection. The effects of the knockdown of Pns4 expression due to RNAi induced by synthesized dsRNA from Pns4 gene on viral replication and spread in cultured cells and insect vector were analyzed using immunofluorescence, western blotting or RT-PCR assays.
Results
In cultured leafhopper cells, the knockdown of Pns4 expression due to RNAi induced by synthesized dsRNA from Pns4 gene strongly inhibited the formation of minitubules, preventing the accumulation of viroplasms and efficient viral infection in insect vector cells. RNAi induced by microinjection of dsRNA from Pns4 gene significantly reduced the viruliferous rate of N. cincticeps. Furthermore, it also strongly inhibited the formation of minitubules and viroplasms, preventing efficient viral spread from the initially infected site in the filter chamber of intact insect vector.
Conclusions
Pns4 of RDV is essential for viral infection and replication in insect vector. It may directly participate in the functional role of viroplasm for viral replication and assembly of progeny virions during viral infection in leafhopper vector.
... P3 and P8 are major components of the inner and outer protein shells that encapsidate the core, respectively [20,21]. P2 and P9 are minor components of the outer capsid [22,23]. The structural features and the process of assembly of RDV virions have been well studied [24,25]. ...
Background
Rice dwarf virus (RDV) is the causal agent of rice dwarf disease, which limits rice production in many areas of south East Asia. Transcriptional changes of rice in response to RDV infection have been characterized by Shimizu et al. and Satoh et al.. Both studies found induction of defense related genes and correlations between transcriptional changes and symptom development in RDV-infected rice. However, the same rice cultivar, namely Nipponbare belonging to the Japonic subspecies of rice was used in both studies.
Methods
Gene expression changes of the indica subspecies of rice, namely Oryza sativa L. ssp. indica cv Yixiang2292 that show moderate resistance to RDV, in response to RDV infection were characterized using an Affymetrix Rice Genome Array. Differentially expressed genes (DEGs) were classified according to their Gene Ontology (GO) annotation. The effects of transient expression of Pns11 in Nicotiana benthaminana on the expression of nucleolar genes were studied using real-time PCR (RT-PCR).
Results
856 genes involved in defense or other physiological processes were identified to be DEGs, most of which showed up-regulation. Ribosome- and nucleolus related genes were significantly enriched in the DEGs. Representative genes related to nucleolar function exhibited altered expression in N. benthaminana plants transiently expressing Pns11 of RDV.
Conclusions
Induction of defense related genes is common for rice infected with RDV. There is a co-relation between symptom severity and transcriptional alteration in RDV infected rice. Besides ribosome, RDV may also target nucleolus to manipulate the translation machinery of rice. Given the tight links between nucleolus and ribosome, it is intriguing to speculate that RDV may enhance expression of ribosomal genes by targeting nucleolus through Pns11.
... Subsequently, RDV is transmitted to plants by the viruliferous vectors, most likely via a wound caused by feeding of the insects on the plants. The minor outer capsid protein P2 is essential for RDV infection to the vector insects (Yan et al., 1996; Tomaru et al., 1997; Omura et al., 1998). Viral particles containing P2 on the outer capsid layers can infect insect vectors through direct injection and by feeding insects through a membrane, whereas viral particles without P2 can infect insect vectors only through direct injection. ...
Rice dwarf virus and Rice gall dwarf virus, members of the genus Phytoreovirus in the family Reoviridae,are known as agents of rice disease, because their spread results in substantial economic damage in many Asian countries. These viruses are transmitted via insect vectors, and they multiply both in the plants and in the insect vectors. Structural information about the viruses and their interactions with cellular components in the life cycle are essential for understanding viral infection and replication mechanisms. The life cycle of the viruses involves various cellular events such as cell entry, synthesis of viral genome and proteins, assembly of viral components, viral egress from infected cells, and intra- and intercellular transports. This review focuses on the major events underlying the life cycle of phytoreoviruses, which has been visualized by various electron microscopy (EM) imaging techniques, including cryo-electron microscopy and tomography, and demonstrates the advantage of the advanced EM imaging techniques to investigate the viral infection and replication mechanisms.
... The core capsid is composed of P3, the major protein, which encloses P1, P5 and P6 (Ichimi et al., 2002; Omura et al., 1985). The outer capsid of the virus is composed of the P2 protein (Yan et al., 1996) and the major outer-capsid protein P8 (Noda et al., 1991 ). The other segments of dsRNA encode putative nonstructural (Pns) proteins, namely Pns4, Pns7, Pns9, Pns10, Pns11 and Pns12 (Koganezawa et al., 1990; Moriyasu et al., 2000 Moriyasu et al., , 2007 Noda et al., 1991). ...
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.
... The outer protein layer consists of P8 and P2. P8 is organized in T=13l lattice (Lu et al., 1998) (Nakagawa et al., 2003), and P2 is a minor protein that is needed for virus infection (Yan et al., 1996). ...
Symmetry is a key principle in viral structures, especially the protein capsid shells. However, symmetry mismatches are very common, and often correlate with dynamic functionality of biological significance. The three-dimensional structures of two isometric viruses, bacteriophage phi8 and the archaeal virus SH1 were reconstructed using electron cryo-microscopy. Two image reconstruction methods were used: the classical icosahedral method yielded high resolution models for the symmetrical parts of the structures, and a novel asymmetric in-situ reconstruction method allowed us to resolve the symmetry mismatches at the vertices of the viruses. Evidence was found that the hexameric packaging enzyme at the vertices of phi8 does not rotate relative to the capsid. The large two-fold symmetric spikes of SH1 were found not to be responsible for infectivity. Both virus structures provided insight into the evolution of viruses. Comparison of the phi8 polymerase complex capsid with those of phi6 and other dsRNA viruses suggests that the quaternary structure in dsRNA bacteriophages differs from other dsRNA viruses. SH1 is unusual because there are two major types of capsomers building up the capsid, both of which seem to be composed mainly of single beta-barrels perpendicular to the capsid surface. This indicates that the beta-barrel may be ancestral to the double beta-barrel fold. Virukset koostuvat yksinkertaisimmillaan perimäaineksesta (DNA tai RNA) ja sitä suojaavasta proteiinikuoresta. Proteiinikuoren rakenne on usein symmetrinen: monta kopiota samaa proteiinia nivoutuu yhteen säännölliseen muodostelmaan. Symmetria on yleensä joko helikaalinen (kierreportaat), jolloin virus on sauvamainen, tai ikosahedraalinen (5- ja 6-kulmioista ommeltu jalkapallo), jolloin syntyy pallomaisia viruksia. On kuitenkin tavallista, että jotkin viruksen toiminnan kannalta tärkeät rakenteet eivät noudata symmetriaa. Jalkapallossa esimerkiksi on vain yksi venttiilin paikka, eli yksi nahkapalasista poikkeaa muista. Viruksen tapauksessa taas vastaavalla tavalla muista poikkeavassa paikassa saattaa olla perimäaineksen pakkaamiseen tarvittava koneisto. Tässä työssä on tutkittu kahden pallomaisen viruksen, phi8:n ja SH1 kolmiulotteisia (3D) rakenteita. phi8 sairastuttaa erästä bakteeria, joka puolestaan sairastuttaa tiettyjä palkokasveja. SH1 sairastuttaa arkkieliöitä (bakteerien tapaisia yksisoluisia eliöitä), joita löytyy vaaleanpunaisista suolajärvistä Australiasta. Rakenteet määritettiin elektronimikroskooppikuvista laskennallisin keinoin. Perusajatus on, että kun kaksiulotteisissa mikroskooppikuvissa virus näkyy monesta eri suunnasta, nämä kuvat yhdistämällä saadaan selville viruksen 3D rakenne. Virukset erottuvat heikosti mikroskooppikuvissa, joten myös laskettu 3D rakenne on epäselvä. Sitä voidaan kuitenkin selkeyttää käyttäen hyväksi symmetriaa. Tällöin oletetaan, että virus on täysin symmetrinen, mistä seuraa, että laskettu 3D rakenne näyttää virheellisesti ne osat, jotka eivät seuraa symmetriaa. Esimerkiksi jalkapallon venttiilin paikka saattaisi ilmaantua 12 eri paikkaan tai kadota kokonaan näkyvistä. Tässä työssä jatkokehitettiin menetelmää, jonka avulla voidaan saada oikea kuva ventiilinpaikoista. Menetelmää sovellettiin molempiin tutkittuihin viruksiin. Virusten rakenteet kertovat niiden sukulaisuussuhteista, joten rakennetutkimus on myös sukututkimusta. Vain yhden phi8:n lähisukulaisen rakenne tunnettiin aiemmin, joten määritetty rakenne mahdollisti perheen sisäisen vertailun. SH1:n perheestä puolestaan ei ollut mitään tietoa, eikä sen rakennekaan nyt paljastanut varmuudella sen olevan sukua tunnetuille viruksille. On tosin mahdollista, että eräs yleinen viruskuoriproteiinityyppi on kehittynyt SH1:n tapaisen viruksen kuoresta.
... Both RDV and RGDV are unusual in their ability to multiply in both plant cells and insect-vector cells. Recent studies have revealed various functions for the viral proteins and their roles during viral replication: for example, in adsorption to and penetration of insect-vector cells (Yan et al., 1996; Wei et al., 2007), in propagation in the viroplasm (Wei et al., 2006c) and in intracellular movement of the virus within infected insect-vector cells (Wei et al., 2006bWei et al., , 2009b). The present study demonstrates that the viruses also appear to require a function that is associated with the P8 outer-capsid protein for the transport of infectious viral particles out of infected insect-vector cells. ...
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.
... The RDV outer shell is relatively unstable, and is lost during endocytosis. Intact RDV particles can enter and replicate in leafhopper (Nephotettix cincticeps) cells, but particles lacking a P2 protein due to chemical treatment or mutation cannot infect or attach to these cells and cannot be acquired by the leafhopper vector (134,171,194). The chemically treated virus that lacks P2 can replicate and is transmitted by the insect after injection into the hemocoel, but viruses carrying a mutant P2 do not replicate after hemocoel inoculation and are not transmitted. ...
The majority of described plant viruses are transmitted by insects of the Hemipteroid assemblage that includes aphids, whiteflies, leafhoppers, planthoppers, and thrips. In this review we highlight progress made in research on vector interactions of the more than 200 plant viruses that are transmitted by hemipteroid insects beginning a few hours or days after acquisition and for up to the life of the insect, i.e., in a persistent-circulative or persistent-propagative mode. These plant viruses move through the insect vector, from the gut lumen into the hemolymph or other tissues and finally into the salivary glands, from which these viruses are introduced back into the plant host during insect feeding. The movement and/or replication of the viruses in the insect vectors require specific interactions between virus and vector components. Recent investigations have resulted in a better understanding of the replication sites and tissue tropism of several plant viruses that propagate in insect vectors. Furthermore, virus and insect proteins involved in overcoming transmission barriers in the vector have been identified for some virus-vector combinations.
... S1-encoded core protein P1 is considered the RNAdependent RNA polymerase (RdRp) of RDV (Suzuki et al., 1992). The S2-encoded 130-kDa protein (P2) is essential in virus attachment and/or penetration of virus into vector cells (Omura et al., 1994; Yan et al., 1996). S5-encoded P5 is assumed to be the mRNA guanyltransferase (Suzuki et al., 1996). ...
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 in Escherichia coli and 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.
... The lack of matched lattice symmetries suggests that the other minor proteins (e.g., P2 or P7) or the structural variations in the floor domains of P8 may be involved in accommodating the mismatched lattice symmetries of the outer and inner shells in assembly of the RDV particle (5; Molecular Plant Pathology On-Line). Indeed, it was found that the appearances of viral particles with or without P2 present were indistinguishable (19). Thus, the involvement of P2 or P7 in capsid formation is unclear. ...
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.
... The P2 protein was previously determined to be essential for RDV infection in its insect vectors and subsequent transmission to its host plants from these vectors (Yan et al., 1996;Tomaru et al., 1997;Omura et al., 1998). It was proposed that the P2 protein interacts with receptors encoded by the insect vector cells and this interaction was necessary for the recognition of virus particles by these insect cells (Omura and Yan, 1999). ...
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.
... The outer layer consists of three proteins, namely, P2, P8, and P9 (14,15,16,26,27). The minor outer capsid protein P2 is essential for the infection of insect vectors by RDV (18,22,25). It has been proposed that P2 interacts with receptors on insect vector cells and that this interaction is required for the recognition of viral particles by the insect cells (18). ...
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.
... In infected rice plants, RGDV is essentially restricted to phloem-related cells and induces tumors derived from the phloem while RDV is systemic in infected plants and does not cause hyperplasia [13]. RGDV particles retain p2 protein, a minor outer capsid protein, and infectivity irrespective of carbon tetrachloride (CCl 4 ) treatment while RDV particles lose P2 and infectivity by this treatment [10,14,15]; The optimum temperature for the activity of the RNA-dependent RNA polymerase is 25°C in RGDV but 35°C in RDV [16]. ...
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.
Viral plant diseases are a significant threat to global agriculture, causing substantial yield losses and economic damage. The transmission of plant viruses by insect vectors plays a crucial role in the spread and severity of these diseases. Understanding the molecular mechanisms underlying vector transmission and symptom expression is essential for developing effective management strategies. It provide an in-depth analysis of the current knowledge on the molecular mechanisms of vector transmission and symptom expression in viral plant diseases. We explore the intricate interactions between viruses, insect vectors, and host plants, highlighting the key factors involved in the transmission process. Viruses were said to be non-persistent if they were not retained by the vector for more than a few hours. Semi-persistent viruses are internalized in the insect by binding to chitin lining the gut, but do not appear to enter tissues. Persistent viruses, once acquired, were associated with the vector for the remainder of its life. These viruses required longer acquisition and inoculation times (hours to days) and latent periods of 1 day to several weeks. Furthermore, we discuss the various strategies employed by viruses to manipulate vector behaviour and enhance their spread. Additionally, we examine the molecular basis of symptom development in infected plants and how viral factors influence symptom expression. Through an extensive review of the literature, we aim to provide a comprehensive overview of the molecular mechanisms driving vector transmission and symptom expression in viral plant diseases.
Keywords: Non-persistent, semi-persistent, persistent viruses, acquisition time and retention time
Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.
Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a well-characterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 μm diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 Ångström were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.
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.
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.
SUMMARYA 39 kDa protein, known as the viral spike protein or one of the protein components forming the viral spike, encoded by genomic segment 9 (S9) of Rice Ragged Stunt Oryzavirus (RRSV) was obtained by enzymatic cleavage of a fusion protein expressed by S9 cDNA in bacteria with proteinase factor Xa. The feeding of an insect vector — the rice brown planthopper (Nilaparvata lugens) on purified expressed 39 kDa protein before the inoculation of the insects on diseased rice plants could completely inhibit the vector transmission ability of the insect. The presence of a 32 kDa insect cell membrane protein which could bind to 39 kDa viral spike protein indicated that the inhibition might be resulted from the competition in the interactions of 39 kDa protein and intact virus with the virus receptors on the insect cells. These results suggest that the spike proteins of the plant reoviruses are essential for the virus infection in the interactions of virus, insect vectors and host plants. These results are also useful in the practical applications.
Rice ragged stunt oryzavirus (RRSV) replicates in both its insect vector, Nilaparvata lugens, and its plant host, rice, and has a complex multi-component particle bearing spikes on its outer surface. Transgenic rice lines expressing the 39 kDa spike protein showed good resistance to infection by RRSV. Furthermore, N. lugens fed on these plants prior to feeding on RRSV-infected plants were significantly protected against RRSV infection. The viral titre in insects initially fed on transgenic plants and then on RRSV-infected plants was inversely proportional to the levels of the 39 kDa protein expressed in the transgenic plants. This suggests that the 39 kDa protein interferes with the interaction between the intact virus particles and insect cell receptors and that the spike protein of RRSV contributes to vector specificity. This approach would probably be a more environment-friendly and sustainable method of virus control than by actual eradication of insect vectors.
Double-stranded RNA (dsRNA) viruses are a diverse group of viruses infecting hosts from bacteria to higher eukaryotes. Among the hosts are humans, domestic animals, and economically important plant species. Fine details of high-resolution virion structures have revealed common structural characteristics unique to these viruses including an internal icosahedral capsid built from 60 asymmetric dimers (120 monomers!) of the major coat protein. Here we focus mainly on the structures and assembly principles of large icosahedral dsRNA viruses belonging to the families of Cystoviridae and Reoviridae. It is obvious that there are a variety of assembly pathways utilized by different viruses starting from similar building blocks and reaching in all cases a similar capsid architecture. This is true even with closely related viruses indicating that the assembly pathway per se is not an indicator of relatedness and is achieved with minor changes in the interacting components.
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.
A transmission-defective (TD) isolate of rice dwarf phytoreovirus lacked the ability to infect cells when derived from the virus-free insect vector Nephotettix cincticeps. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified virus showed that among six structural proteins, the P2 outer capsid protein (encoded by genome segment S2) was absent from the TD isolate, whereas all six proteins were present in the transmission-competent (TC) isolate. P2 was not detected on immunoblots of rice plants infected with the TD isolate. Genome segment S2 and its transcript were detected in both TD and TC isolates. Sequence analysis of the S2 segment of the TD isolate revealed the presence of a termination codon due to a point mutation in the open reading frame, which might explain the absence of P2 in the TD isolate. These results demonstrate that the P2 protein is one of the factors essential for infection by the virus of vector cells and, thus, influences transmissibility by vector insects.
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.
A majority of the plant-infecting viruses and many of the animal-infecting viruses are dependent upon arthropod vectors for transmission between hosts and/or as alternative hosts. The viruses have evolved specific associations with their vectors, and we are beginning to understand the underlying mechanisms that regulate the virus transmission process. A majority of plant viruses are carried on the cuticle lining of a vector's mouthparts or foregut. This initially appeared to be simple mechanical contamination, but it is now known to be a biologically complex interaction between specific virus proteins and as yet unidentified vector cuticle-associated compounds. Numerous other plant viruses and the majority of animal viruses are carried within the body of the vector. These viruses have evolved specific mechanisms to enable them to be transported through multiple tissues and to evade vector defenses. In response, vector species have evolved so that not all individuals within a species are susceptible to virus infection or can serve as a competent vector. Not only are the virus components of the transmission process being identified, but also the genetic and physiological components of the vectors which determine their ability to be used successfully by the virus are being elucidated. The mechanisms of arthropod-virus associations are many and complex, but common themes are beginning to emerge which may allow the development of novel strategies to ultimately control epidemics caused by arthropod-borne viruses.
Rice dwarf virus crystals belong to space group I222 with cell parameters a = 770 (2), b = 795 (5), c = 814 (5) A and alpha = beta = gamma = 90 degrees. The unit cell of the crystal contains two viruses at the origin and body-centred positions. Using data synthesized from a rice dwarf virus model crystal in the space group I222, the possibility of ab initio phasing was thoroughly examined. The centric nature of the initial phases was unexpectedly broken by extensive iteration of the non-crystallographic symmetry averaging. The structure of rice dwarf virus was then solved with ab initio phasing up to 20 A resolution. The triangulation number determined by the present study is T = 13, which is different from the triangulation number, T = 9, previously determined by electron microscopy [Uyeda & Shikata (1982). Ann. Phytopathol. Soc. Jpn, 48, 295-300].
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 Spodoptera frugiperda cells infected with AcRS12, a baculovirus recombinant carrying a full-length cDNA of RDV S12. Purified Pns12 was also demonstrated to be phosphorylated in vitro by a kinase activity present in host (rice, barley, wheat, leafhopper) cells and non-host (tobacco, spinach, white clover, S. frugiperda, mosquito, mammals) cells as well. Immunocytochemical studies showed Pns12 accumulated in the cytoplasm of infected cells, and frequently localized in a slightly electron-dense patch. These results demonstrated that RDV Pns12 was a cytoplasmic nonstructural phosphoprotein.
This chapter focuses on the role of outer capsid protein in the transmission of Phytoreovirus by insect vectors. Using current research systems combined with reliable traditional experimental systems, the chapter discusses the viral proteins responsible for infection by Phytoreovirus of vector cells, with the goal of defining the mechanism of the vector transmission of such viruses. Clarification of the molecular mechanisms of viral multiplication in distinct hosts and of interkingdom differences in the expression of specific proteins should provide new insights into the roles of proteins that function in the virus–host relationship. Such investigations of the viral molecules that are essential for virus transmission by vectors and of the ways in which these molecules function should help develop measures for controling plant viruses that are transmitted by vectors. It might even be possible to generate transgenic plants that express proteins that interfere with the normal interactions between a virus and its vector.
Partial amino acid sequence of a minor 30 kDa polypeptide in purified Rice dwarf virus (RDV) was identical to the deduced amino acid sequence encoded by the dsRNA segment S9 of the virus. This polypeptide was specifically detected by Western blotting analysis with antibodies raised against the product of S9 expressed in a baculovirus system. Treatment of purified RDV particles with a relatively higher concentration of MgCl2 removed the polypeptide from core particles together with other outer capsid proteins. These results demonstrate that the 30 kDa polypeptide is a minor outer capsid protein that is encoded by genome segment S9 of RDV. This protein was named P9 protein.
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, besides its function in the assembly and subsequent packaging of viral dsRNA into core particles.
The sequence of the alpha-transinducing factor (alpha-TIF) of canine herpesvirus (CHV-l) was determined. Alignment of the predicted CHV-1 alpha-TIF amino acid sequence with other alpha-TIF homologues reveals a core region of similarity with divergent amino and carboxyl termini. Analysis of the CHV-1 infected cell protein 4 promoter region identified a region containing nine copies of a 52 bp repeat that showed significant up-regulation of transcription by alpha-TIF. This region contained an imperfect 'TAATGARAT' motif, the binding site for herpes simplex virus 1 alpha-TIF, with an imperfect Oct-1 binding site immediately following. The infectious laryngotracheitis virus alpha-TIF was also shown to up-regulate transcription through this region of the promoter. Transfection of CHV-1 genomic DNA failed to yield infectious virus in canine kidney cell lines. Co-transfection of genomic DNA and an alpha-TIF expression plasmid resulted in virus plaques, indicating a potential essential role for alpha-TIF in CHV-1 infection.
Virus infections are the cause of numerous plant disease syndromes that are generally characterized by the induction of disease symptoms such as developmental abnormalities, chlorosis, and necrosis. How viruses induce these disease symptoms represents a long-standing question in plant pathology. Recent studies indicate that symptoms are derived from specific interactions between virus and host components. Many of these interactions have been found to contribute to the successful completion of the virus life-cycle, although the role of other interactions in the infection process is not yet known. However, all share the potential to disrupt host physiology. From this information we are beginning to decipher the progression of events that lead from specific virus-host interactions to the establishment of disease symptoms. This review highlights our progress in understanding the mechanisms through which virus-host interactions affect host physiology. The emerging picture is one of complexity involving the individual effects of multiple virus-host interactions.
The complete nucleotide sequence of segment 7 of the rice dwarf virus (RDV) genome was determined. The segment was 1696 bp long and its plus-strand terminal sequence, 5'GGCAAA---UGAU3', was in agreement with the consensus sequence previously found in other segments of RDV. A 10 bp inverted repeat was found adjacent to the termini. A single long open reading frame extended for 1518 bp from the first AUG triplet (positions 26 to 28), and encoded a polypeptide of 506 amino acids (Mr 55,339). This protein had 32% identity in the amino acid sequence to the 57K protein encoded by segment 7 of the wound tumour virus genome. The translation product of transcript RNA made from 'tailored' cDNA of RDV segment 7 comigrated with the 60K core protein of RDV in 10% polyacrylamide gel and reacted with antiserum against the 60K core protein of RDV. Segment 7 of the RDV genome therefore codes for the 60K core protein.
SUMMARY Repeated selection of plants with unusually severe symptoms after their inoculation by insect vectors which had been injected with dilute inoculum from crude extracts of a stock culture (O strain) of rice dwarf virus (RDV) resulted in the emergence of a severe isolate (S strain). Of the 12 segments of RDV RNA, the fourth largest RNA of the S strain had an apparent Mr about 20000 larger than that of the corresponding segment of the O strain. The Mr of the protein corresponding to the M r 43000 protein of the O strain, which is located on the outside of the outer capsid, was 44000 in the S strain. The implication of the differences in the RNA and protein components between the S and O strains is discussed.
SUMMARY Infectivity assays of rice dwarf virus (RDV) were done by the fluorescent antibody focus counting technique on vector cell monolayers of the green rice leafhopper, Nephotettix cincticeps. The focus count method was shown to be an accurate and quantitative method for determining RDV infectivities. The optimal pH value for inoculation was about 6.0 in a solution containing 0.1 M-histidine HC1 and 0.01 M- MgC12. Below pH 5.5 and above 6.5, the infectivity of RDV dropped rapidly. The optimal adsorption period at 28 °C was dependent upon the RDV concentration. Optimal periods for adsorption with relative RDV concentrations of 10 -3, 10 -4 and 10 -4.5 were about 60, 90 and 120 min, respectively. The period from virion adsorption to penetration into the cell was about 90 to 120 rain. Infective progeny virions were first detected 12 h after the initial inoculation. From 12 to 20 h, the growth rate of the virus in the cells was exponential with a doubling time of about 96 rain, and then from 20 to 28 h there was little or no further increase in infective virus. When the infectivities of the same inocula were compared by using the focus count and vector insect injection methods, the dilution endpoints were approx. 10 -6 and 10 -4, respectively. The focus count assay method was thus about 100 times more sensitive than vector injection.
We have determined the complete nucleotide sequence of the largest segment S1 of rice dwarf phytoreovirus (RDV), a member of the family Reoviridae. S1 is 4423 nucleotides long with a segment-specific inverted repeat located adjacent to the conserved termini (5'GGCAAA---UGAU3'). A major open reading frame (bases 36 to 4367) on the S1 plus strand, which is preceded by a minicistron (bases 6 to 29), encodes the polypeptide (P1) consisting of 1444 amino acids with a M(r) of 164, 142. The sense-strand transcript derived from the full-length S1 cDNA, the minicistron of which was abolished, directed the synthesis of a polypeptide of 170 kDa in addition to smaller polypeptides in wheat germ extracts, and the 170-kDa product comigrated with the minor core protein in SDS-polyacrylamide gel. Thus, P1 is assumed to be localized in the viral core particle. The consensus sequence element conserved in RNA-dependent RNA polymerase is observed in the P1 amino acid sequence predicted from the nucleotide sequence. Based on the dendrogram established from the sequence alignment around the polymerase module region, and sequence identity within the alignment, P1 of plant-infecting RDV was evolutionarily compared with VP1, lambda 3, and VP1 of three other animal-infecting members of the family, rota-, reo-, and bluetongue viruses. Consequently, RDV S1 was shown to be more closely related to the rotavirus gene segment 1, in terms of molecular evolution, than the animal-infecting members are to one another.
The primary structure of rice dwarf phytoreovirus (RDV) genome segment S3 was determined. RDV S3 consists of 3195 nucleotides. A 14-bp segment-specific inverted repeat is located immediately adjacent to the conserved terminal sequence (5'GGCAAA---UGAU3'). A single long open reading frame encoding for 1019 amino acids with an Mr of 114,289 is also identified. In order to investigate the localization of the predicted polypeptide, we determined the amino acid sequence of the 26-kDa peptide fragment obtained from the structural core protein digested by Staphylococcus aureus V8 protease. The sequence of the fragment was found in the translational product presumed from the nucleotide sequence of RDV S3, indicating that RDV S3 encodes the major structural core protein of 114 kDa.
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Selected References
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Omura T, Ishikawa K, Hirano H, Ugaki M, Minobe Y, Tsuchizaki T, Kato H. The outer capsid protein of rice dwarf virus is encoded by genome segment S8. J Gen Virol. 1989 Oct;70(Pt 10):2759–2764. [PubMed]
The nucleotide sequence of DNA complementary to the eighth largest (S8) of the 12 genome segments of rice dwarf virus was determined. This genome segment is 1424 nucleotides in length and has a single long open reading frame extending 1260 nucleotides from the first AUG triplet (residues 24 to 26). The predicted translational product comprises 420 amino acids and has an Mr of 46,422. The amino acid sequences of several peptide fragments of the major outer capsid protein were found to be contained in the predicted translational product of the above nucleotide sequence. This protein, previously reported to be 43K, is encoded by genome segment S8 and therefore renamed the 46K protein.
The complete nucleotide sequence of rice dwarf phytoreovirus genome segment 2 (S2) was determined to be 3,512 nucleotides long with one open reading frame initiating at nucleotide 15 and terminating at nucleotide 3363. The encoded polypeptide was predicted to have 1,116 residues with a relative molecular weight of 123 kD. Comparison of S2 of two isolates showed they had identical lengths and 97 and 98.3% nucleotide and amino acid sequence identities, respectively. A search of the Swiss-Prot data base (R 22.0) failed to find any proteins with significant homology to the S2-encoded protein. Determination of the nucleotide sequence of the S2 has completed the sequence determination of the genome of rice dwarf virus. Homology searches made for proteins encoded by each of the genomic segments showed that the polypeptide encoded by S11 has similarity to histone H1 protein and VP6 of blue tongue virus, indicating it might possess nucleic acid binding properties.
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.
Rice dwarf phytoreovirus (RDV) mRNA synthesized from purified virion has a cap structure, m7GpppAm-, which suggests the presence of guanylyltransferase activity in the virion. We attempted to identify the enzyme involved in the cap formation by using a nucleoside triphosphate binding assay. Incubation of virion with [alpha-32P]GTP resulted in labeling of an 89-kDa protein that had not previously been identified in purified virus preparations. Interestingly this protein also covalently bound UTP and ATP, which is not a property of the known guanylytransferases. RDV particles catalyzed GTP-PPi, dGTP-PPi, ATP-PPi, and UTP-PPi exchange reactions. In SDS-polyacrylamide gel electrophoresis, the 89-kDa protein comigrated with the S5-coded protein, P5, which had been expressed by a baculovirus vector. Moreover, the labeled 89-kDa protein was precipitated by an antiserum against this recombinant RDV P5. Careful reinvestigation of purified virus particles by SDS-polyacrylamide gel electrophoresis and Western blotting analyses showed that they contained a small amount of P5 (<0.5% of the total protein) within the core. These results may suggest that the minor core protein of RDV, which is coded by S5, is a candidate guanylyltransferase, although the biological significance of its ATP and UTP binding activities remains largely unknown.