Wound Tumor Virus Research Articles

Nucleotide sequence of rice dwarf virus genome segment 9.

The complete nucleotide sequence of the phytoreovirus rice dwarf virus (RDV) genome segment 9 is presented. It consisted of 1305 nucleotides and had an open reading frame that codes for a putative polypeptide of 351 amino acids. Mr of the protein was calculated to be 38,598. The terminal nucleotides 5' GGUAAA--GAU 3' were the same as those of RDV genome segment 10. The fourth nucleotide from the 3' end of an expected conserved sequence was a C rather than the U found in the previously sequenced genome segment 10. A structure similar to the segment-specific inverted repeat of wound tumour virus was also found in the terminal region of segment 9.

Assignment of wound tumor virus nonstructural polypeptides to cognate dsRNA genome segments by in vitro expression of tailored full-length cDNA clones

Presumptive full-length cDNA clones of 9 of the 12 wound tumor virus double-stranded RNA genome segments were tailored for efficient in vitro expression by a recently described strategy [ Z. Xu, J. V. Anzola, and D. L. Nuss (1987) DNA6, 505–513]. In vitro synthesized polypeptides specified by synthetic transcripts corresponding to the tailored cDNAs comigrated in polyacrylamide gels with in vivo synthesized viral-specific polypeptides. This analysis confirmed the functional integrity of the tailored cDNA clones and identified cognate genome segments which encode all five viral nonstructural polypeptides as well as four structural polypeptides; two which comprise the capsid, one located in the viral core and one associated with the outer protein coat.

Complete nucleotide sequence of wound tumor virus genomic segment S7.

Journal Article Complete nucleotide sequence of wound tumor virus genomic segment S7 Get access John V. Anzola, John V. Anzola Roche Institute of Molecular Biology, Roche Research CenterNutley, NJ 07110, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Zhengkai Xu, Zhengkai Xu Roche Institute of Molecular Biology, Roche Research CenterNutley, NJ 07110, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Donald L. Nuss Donald L. Nuss Roche Institute of Molecular Biology, Roche Research CenterNutley, NJ 07110, USA Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 17, Issue 8, 25 April 1989, Page 3300, https://doi.org/10.1093/nar/17.8.3300 Published: 25 April 1989 Article history Received: 15 February 1989 Published: 25 April 1989

Complete nucleotide sequence of wound tumor virus genomic segment S11.

Complete nucleotide sequence of wound tumor virus genomic segment S11.

Nucleotide sequence of segment S10 of the rice dwarf virus genome.

DNA complementary to the tenth largest (S10) of the 12 genome segments of rice dwarf virus (RDV) was cloned and its sequence was determined. This genome segment is 1319 nucleotides in length and has a single long open reading frame extending for 1059 nucleotides from the first AUG triplet (residues 27 to 29). The predicted translation product comprises 352 amino acids and has a mol. wt. of 39094. RDV transcripts synthesized in vitro have the same polarity as the plus strand of the genome. Terminal sequences were (+) 5' GGUA---UGAU 3' and (-) 3' CCAU---ACUA 5' which are similar to those of wound tumour virus RNA.

Probing the structure and function of viral RNA genomes.

AbstractThe majority of human, animal and plant viral pathogens possess genomes composed of RNA. The strategies evolved for expression and replication of viral RNA genomes can differ significantly from those utilized for expression and replication of host‐cell genetic material. Consequently, knowledge of the molecular details of these strategies can lead to a clearer understanding of the origin, evolution and control of viral pathogens. We describe recent progress in identifying important structural and functional domains of the RNA genomes and associated replicative enzymes for two very different viruses: vesicular stomatitis virus, which possesses a single‐stranded RNA genome of negative polarity, and wound tumor virus, which contains a genome composed of 12 discrete segments of double‐stranded RNA.

Segment-specific inverted repeats found adjacent to conserved terminal sequences in wound tumor virus genome and defective interfering RNAs.

Defective interfering (DI) RNAs are often associated with transmission-defective isolates of wound tumor virus (WTV), a plant virus member of the Reoviridae. We report here the cloning and characterization of WTV genome segment S5 [2613 base pairs (bp)] and three related DI RNAs (587-776 bp). Each DI RNA was generated by a simple internal deletion event that resulted in no sequence rearrangement at the deletion boundaries. Remarkably, although several DI RNAs have been in continuous passage for more than 20 years, their nucleotide sequences are identical to that of corresponding portions of segment S5 present in infrequently passaged, standard, transmission-competent virus. The positions of the deletion breakpoints indicate that the minimal sequence information required for replication and packaging of segment S5 resides within 319 bp from the 5' end of the (+)-strand and 205 bp from the 3' end of the (+)-strand. The terminal portions of segment S5 were found to contain a 9-bp inverted repeat immediately adjacent to the conserved terminal 5'-hexanucleotide and 3'-tetranucleotide sequences shared by all 12 WTV genome segments. The presence of a 6- to 9-nucleotide segment-specific inverted repeat immediately adjacent to the conserved terminal sequences was found to be a feature common to all WTV genome segments. These results reveal several basic principles that govern the replication and packaging of a segmented double-stranded RNA genome.

Regulation of expression of the wound tumor virus genome in persistently infected vector cells is related to change in translational activity of viral transcripts.

The interaction between a plant virus and its insect vector was studied at the molecular level by examining wound tumor virus (WTV) gene expression in cultured cells derived from its leafhopper vector. Infection of vector cells by WTV is noncytopathic and results in an acute phase (through day 5), followed by persistence beginning with the first cell passage. Viral-specific polypeptide synthesis and viral genome RNA accumulation increased to a maximum level during the first 5 days following inoculation and then decreased as infected cells were passaged (to 5 to 20% of the level observed during the acute phase by passages 10 to 15). In contrast, viral-specific mRNAs were present at approximately the same level in the acute phase and in the early stage (passage 10) of the persistent phase of infection. Although viral transcripts isolated at different times after inoculation exhibited identical electrophoretic migration patterns, they had different functional activities in cell-free translation systems. Transcripts isolated from persistently infected cells were inefficiently translated in vitro, reflecting the situation in infected cells. These results indicate that the decline in the level of viral polypeptide synthesis associated with the persistent phase of WTV infection is related to a change in the translational activity of viral transcripts.

Wound tumor virus polypeptide synthesis in productive noncytopathic infection of cultured insect vector cells

Inoculation of the leafhopper cell line AC-20 with wound tumor virus resulted in a productive noncytopathic infection with no detectable alteration of cellular protein synthesis. Virus-specific polypeptide synthesis, detectable by 8 h postinoculation, increased in a linear fashion, reaching a peak (approximately 10 to 15% of total protein synthesis) by 48 h postinoculation. The rate of viral protein synthesis continued at this level for several days but declined, relative to cellular protein synthesis, as infected cells were passaged. By passage 10, the synthesis of viral polypeptides was reduced to a level approximately 5% of that observed at 48 h postinoculation. Viral protein synthesis was not stimulated by superinfection. Viral antigens and infectious virus persisted in the majority (greater than 90%) of cells in an infected culture even after more than 100 passages. The synthesis of wound tumor virus polypeptides in infected insect vector cells appears to be regulated in a coordinated and selective manner.

Molecular cloning and characterization of the genome of wound tumor virus: A tumor-inducing plant reovirus

The double-stranded RNA genome of the tumor-inducing plant pathogen, wound tumor virus, was converted to double-stranded DNA and cloned into plasmid pBR322. Multiple apparent full-length copies of 9 of the 12 wound tumor virus genome segments were identified. The entire sequence of cloned genome segment S12, the smallest of the genome segments, was determined. This genome segment was found to be 851 nucleotides in length and to possess a single long open reading frame that extends 178 codons from the first AUG triplet (residues 35–37): information sufficient to encode a protein of the size estimated for the smallest of the previously identified wound tumor virus primary gene products, Pns 12. Sequence data obtained from analysis of cloned cDNA copies of several genome segments and from direct analysis of the 3′ termini of the double-stranded genome RNAs revealed that each wound tumor virus genome segment possesses the common terminal sequences: (+) 5′ GGUAUU … UGAU 3′ (-) 3′ CCAUAA … ACUA 5′.

Morphology and nucleic acid of rice gall dwarf virus.

In negative stain preparations, the particles of rice gall dwarf virus (RGDV) had the form of angular icosahedra, approximately 65 nm in diameter, and resembled Phytoreovirus particles. The genomic double-stranded RNA of RGDV was fractionated by PAGE into 12 segments similar to those of the RNAs of the phytoreoviruses, wound tumor virus and rice dwarf virus, but of different mobilities. The RNAs were distinct from those of other plant reoviruses. RGDV therefore is confirmed as a new Phytoreovirus.

Variant dsRNAs associated with transmission-defective isolates of wound tumor virus represent terminally conserved remnants of genome segments

Variant double-stranded RNAs are often associated with the genome of transmission-defective isolates of wound tumor virus. These RNAs are replicated and packaged into virus particles in systemically infected plants and are transcribed in vitro by the virion-associated transcriptase. Direct physical evidence that the variant RNAs are remnants of particular WTV genome segments was provided by molecular hybridization studies. Subsequently, ribonuclease T1 digestion products of 3′-end-labeled genome and remnant RNAs were analyzed by one- and two-dimensional electrophoretic techniques. One-dimensional partial and complete digestion patterns were indistinguishable, indicating that the guanosine positions relative to the 3′ terminus of the corresponding strands of a particular genome segment and its remnant RNA are the same for at least 40 nucleotides from each end. Fingerprints of the 3′ terminal ribonuclease T1-resistant fragments were identical, showing that the nucleotide composition of the 3′ terminal ends of the corresponding strands of a particular genome segment and its remnant RNA are also identical. These results indicate that variant RNAs associated with transmission-defective WTV isolates are formed by deletion of an internal portion (as much as 85%) of genomic RNA segments yielding terminally conserved genomic remnants that are functional with respect to transcription, replication, and packaging.

Molecular Biology Of Wound Tumor Virus

Publisher Summary This chapter discusses the molecular details of wound tumor virus (WTV) transmission by insect vectors. The products of the WTV in vitro transcription reaction remain sensitive to ribonuclease digestion after incubation under conditions that would permit annealing of complimentary strands. This finding indicates that the transcripts are single-stranded and of the same polarity. A prerequisite to understanding the molecular biology of a virus is the identification of its primary gene products. If a virus is to be transmitted efficiently by an insect vector, it generally must not affect the vector adversely. For viruses that are transmitted in a propagative manner, mechanisms have evolved to allow efficient multiplication in the vector cells with no deleterious effects on cell functions. Attempts to understand the fine details of WTV transmission led to the establishment and utilization of a continuous cell culture system derived from the leafhopper vector. The complex process of biological transmission of WTV involves the acquisition of the virus by the leafhopper during feeding on an infected plant; sequential infection of the vector's filter chamber and ventriculus, hemolymph, and salivary glands; and passage of the virus to uninfected plants via the salivary fluid during subsequent feeding.

Resolution and genome assignment of mRNA transcripts synthesized in vitro by wound tumor virus

The 12 mRNA transcripts synthesized in vitro by purified wound tumor virus particles were resolved into 11 discrete species by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels containing 7 M urea. Transcripts were isolated and hybridized independently to a mixture of the 12 wound tumor virus double-stranded genome segments. Subsequent analysis of the hybrids revealed that each transcript hybridized selectively to one genome segment. In this manner, the assignment of 10 transcripts to their corresponding genome segments was established. The assignment of the remaining transcripts, which hybridized to an unresolved mixture of genome segments 4 and 5, was determined by analyzing the transcription products synthesized by an exvectorial isolate of WTV that lacks genome segment 5.

In Vitro Synthesis and Modification of mRNA by Exvectorial Isolates of Wound Tumor Virus

Nontransmissible (exvectorial) isolates of wound tumor virus retain the ability to catalyze in vitro synthesis of RNA. Furthermore, exvectorial virus particles exhibit mRNA-2'-O-methyltransferase activity even after long-term (30-year) passage in a host that lacks this enzyme activity.

Expression of Wound Tumor Virus Gene Products In Vivo and In Vitro

Infection of Agallia constricta vector cell monolayers with wound tumor virus results in the synthesis of 12 virus-specific polypeptides. Confirmation that these polypeptides are virus encoded rather than virus induced was obtained by cell-free translation of in vitro synthesized viral mRNA. In addition, transcription by purified wound tumor virus particles was coupled with translation of the resulting transcripts in a wheat embryo cell-free extract. Six previously described structural polypeptides, one presumptive structural polypeptide, and five previously unidentified nonstructural polypeptides were synthesized in infected vector cell monolayers, in cell-free extracts directed by in vitro synthesized viral mRNA, and in the homologous plant cell-free system, in which viral transcription was coupled with translation. Pulse-chase experiments revealed no evidence of precursor-product relationships for the wound tumor virus-specific polypeptides.

Neutralization of Infectivity of Potato Yellow Dwarf Virus and Wound Tumor Virus Assayed on Vector-Cell Monolayers

Neutralization of Infectivity of Potato Yellow Dwarf Virus and Wound Tumor Virus Assayed on Vector-Cell Monolayers

Development of a new medium for the culture of agallian leafhopper cells.

The optimal composition of a medium for tissue culture of cells from the leafhopperAgallia constricta was estimated from experiments in which the rate of growth of the cells was measured at different concentrations of each component. Wound tumor virus and theconstricta variety of potato yellow dwarf virus multiply, inA. constricta when these viruses are transmitted from one plant to another. Differences weredetected in the suitablility of different batches of fetal bovine serum (after heating at 56°C for 30 min), and histidine as components in the tissue culture medium. Without heat treatment even thebest fetal bovine serum was not suitable. Estimated optimal concentrations of fetal bovine serum, histidine, salts, dextrose, lactalbumin hydrolysate and yeast autolysate were determined. Fungizone (amphotericin B) at 50 or 100 mg per 1 caused harmful effects; effective concentrations of penicillin, neomycin and steptomycin did not. Combinations of histidine-HCl and histidine (free-base) made it possible to prepare buffered medium at my pH between 6.0 and 7.0. Optimal growth ofA. constricta cells occurred at pH 6.43, and ofAceratagallia sanguinolenta at pH 6.30. Osmotic pressures of the new medium between 360 and 405 mOSM were better than lower osmotic pressures. The new medium was still suitable for growth of the cells after, storage for 6 months at 4°C.

The comparative susceptibilities of cultured vector and nonvector leafhopper cells to three plant viruses

The susceptibilities of established leafhopper cell lines from Agallia constricta (AC), Aceratagallia sanguinolenta (AS), and Agalliopsis novella (AN) were compared with one another when inoculated with wound tumor virus (WTV), the constricta variety of potato yellow dwarf virus (CYDV), and the sanguinolenta variety of potato yellow dwarf virus (SYDV). The comparative susceptibility of an established cell line from Dalbulus elimatus (DE) to these viruses was also determined. D. elimatus is not closely related to agallian leafhoppers nor has it been shown to be a vector of any of the above viruses. Vector AC cells and AN cells were about equally susceptible to WTV. The susceptibility of nonvector AS cells to WTV was about 0 · 064 times that of the vector AC and AN cells. Despite the insect-virus relationships, all cell lines derived from agallian leafhoppers were about equally susceptible to CYDV and SYDV. Concentrations of WTV and CYDV which gave about 25% infection on vector AC cells failed to infect DE cells. On the contrary, SYDV infected the nonvector DE cells although the incidence of infection was only about 0.0027 times that of vector AS cells.

In vitro transcription of wound tumor virus RNA by virion-associated RNA transcriptase

The results of this communication indicate that wound tumor virus (WTV) has a virion-bound polymerase which can synthesize all 12 single-stranded RNA copies of the genome segments in vitro . Chymotrypsin treatment of the purified virus did not affect its transcriptase activity. Previous observations have shown that virus treated with chymotrypsin lacks the 131,000- and 96,000-dalton polypeptides. Since we have shown in the present study that the enzyme activity was retained following chymotrypsin treatment, we conclude that neither of these polypeptides are the polymerase. In addition, all cations tested, with the exception of Mg 2+ and Mn 2+ , did not enhance the enzyme activity. Phosphoenolpyruvate (PEP) together with PEP-kinase substantially increased in vitro transcription, and the transcribed products, when annealed with denatured WTV genome RNA, gave an electrophoretic profile identical to that of the native viral genome.