Wound Tumor Virus Research Articles

Molecular analysis of six segments of Tobacco leaf enation virus, a novel phytoreovirus from tobacco

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

Molecular characterization of the largest and smallest genome segments, S1 and S12, of Rice gall dwarf virus

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.

Molecular characterization of the genome segments S4, S6 and S7 of rice gall dwarf virus

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

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

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

Sequence Analysis and GTP-Binding Ability of the Minor Core Protein P5 of <i>Rice Gall Dwarf Virus </i>

Analysis of the nucleotide sequence of the genome segment S5 of Rice gall dwarf virus (RGDV), which belongs to the genus Phytoreovirus, revealed that this segment encodes a putative protein of approximately 91 kDa. Antiserum raised against the protein reacted with a minor structural protein of similar size in RGDV, demonstrating that S5 encodes the structural protein of RGDV. The protein named P5 appeared to be a component of viral core particles. Although no overall homology was found to any other proteins, including those of animal-infecting reoviruses, P5 of RGDV exhibited a significant homology to P5 of Rice dwarf virus (50%), which might be a guanylyltransferase, and to P5 of Wound tumor virus (55%). These results, together with the observation that P5 bound guanosine triphosphate suggest that P5 of RGDV shows a guanylyltransferase activity and, moreover, that plant-infecting reoviruses have a similar functional and/or structural organization to that of animal-infecting reoviruses.

Evidence for a Phytoreovirus Associated with Tobacco Exhibiting Leaf Curl Symptoms in South Africa

ABSTRACT Three forms of tobacco leaf curl (termed classes I, II, and III, based on symptomatology) recently have been described in southern Africa. Numerous attempts to isolate virus particles responsible for a nongeminivirus-induced leaf curl disease (class I) of tobacco in South Africa have been unsuccessful. Recently, 12 dsRNA segments were isolated from tobacco exhibiting class I leaf curl symptoms, suggesting a possible reovirus genome. The objective of our study was to confirm whether the dsRNA segments are associated with a reovirus. Isolation of icosahedral particles with an outer core 60 to 65 nm in diameter and an inner core 40 to 45 nm in diameter was achieved. Twelve distinct nonpolyadenylated dsRNAs were isolated from purified virions, and the total molecular masses of the dsRNAs ranged from 17.86 to 18.40 x 10(6) Da in polyacrylamide and agarose gels, respectively. Using hybridization analysis, dsRNAs were identified as non-homologous distinct segments. Comparisons with other known reoviruses revealed a unique banding pattern that was most similar to the wound tumor virus (WTV), the type species of the genus Phytoreovirus. Hybridizations of WTV cloned DNA probes (segments S4 and S6 to S9) and dsRNAs from infected tobacco indicated no significant sequence similarity, whereas indirect enzyme-linked immunosorbent assay with a polyclonal antiserum to WTV showed strong positive cross-reactivity to tobacco virions. Our results indicate a virus with features consistent with those of phytoreoviruses. This is the first report of a plant reovirus in tobacco, the first record in Africa, and the second example of a field-isolated dicot phytoreovirus.

Polycistronic (tri- or bicistronic) phytoreoviral segments translatable in both plant and insect cells.

Genomic segment S12 of rice dwarf virus and segment S9 of wound tumor virus, both members of the genus Phytoreovirus, have small out-of-phase overlapping open reading frames (ORFs). Western blot (immunoblot) analysis revealed that rice dwarf virus S12 mRNA specified translation products from the large ORF and two overlapping small ORFs both in rice plant hosts and in Spodoptera frugiperda insect cells. These results provide the first example of a tricistronic mRNA for a segmented double-stranded RNA virus. Similarly, wound tumor virus S9 mRNA was found to direct the synthesis of protein products from both the large ORF and small out-of-frame ORF in S. frugiperda cells. Results of site-specific and deletion mutagenesis studies were consistent with a leaky scanning translation mechanism for the synthesis of the small ORFs.

Characterization of Genome Structure And Establishment of Vector Cell Lines for Plant Reoviruses

Publisher Summary Plant reoviruses are a unique group among plant pathogenic viruses. They are members of a family that includes viruses of both vertebrates and invertebrates, and all members share characteristic features: (1) they are leafhopper and planthopper borne in a persistent manner, (2) they contain spherical particles 60-70 nm in diameter, and (3) their genomes consist of 10-12 segmented double-stranded RNAs (dsRNAs). The classical study of plant reoviruses that attracted the attention of researchers focuses chiefly on the multiplication of the plant pathogenic viruses in their insect vectors, starting with Fukushi's finding in 1940, based on his spectacular experimental evidence that rice dwarf virus (RDV) multiplied in its insect vector; this finding later promoted “controversial discussions”. Subsequently, further evidence based on biological and electron microscopic studies fully supported Fukushi's discovery. Among the plant reoviruses, RDV and wound tumor virus (WTV) have been most extensively studied, with respect to their insect transmission, transovarial passage, virus particles, localization of the viruses in their plant and insect hosts, establishment of cell lines from embryonic tissue culture of the respective insect vectors, and genome characterization. This chapter reviews the recent findings concerning dsRNA genomes of plant reoviruses, especially the detailed characterization of all 12 genomic segments of RDV achieved in Japan. This is the first report of a successful genomic analysis of the 12 segmented dsRNAs of a virus belonging to the plant reoviruses.

Rice dwarf phytoreovirus segment S12 transcript is tricistronic in Vitro

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

First field isolation of wound tumor virus from a plant host: Minimal sequence divergence from the type strain isolated from an insect vector

A new strain of wound tumor virus (WTV) has been isolated form a periwinkle plant ( Catharanthus roseus)_that was among several used as bait plants in a blueberry field. The 12 segments of double-stranded RNA of the viral genome were isolated directly from infected tissue and found to have mobilities through agarose gels that were identical to those of the type strain WTV. Coupled complementary DNA (cDNA) and polymerase chain reactions (PCR) primed with oligonucleotides complementary to the termini of segments 4–12 of the type strain of WTV successfully amplified those segments. Amplification products of the 9 segments were of the size expected for the full-length segment, with no shorter than full-length products representing defective RNAs detected. PCR products representing segments 7, 11, and 12 were cloned and sequenced in their entirety. The sequence of each segment varied only slightly from the homologous segment of the type strain. Variation ranged from less than 1 % for segment 12 to approximately 3% for segment 7, but even these low levels of variation were much greater than the variation found in WTV isolates maintained in the laboratory. Most of the variation in each of the three segments was confined to the coding regions, and most of the differences were third position transitions. The new WTV strain has been designated WTV NJ.

Viruses in the Phytoreovirus Genus of the Reoviridae Family have the Same Conserved Terminal Sequences

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

Nucleotide sequences of genome segments S8, encoding a capsid protein, and S10, encoding a 36K protein, of rice gall dwarf virus

The nucleotide sequences of DNAs complementary to the eighth (S8) and the tenth (S10) largest of the 12 genome segments of rice gall dwarf virus (RGDV) were determined. The S8 and S10 segments consist of 1578 and 1198 nucleotides, each with a single open reading frame extending for 1278 nucleotides from nucleotide 21, and 960 nucleotides from nucleotide 22, respectively. S8 encodes a polypeptide of 426 amino acids with an Mr of 47419. The amino acid sequences of several peptide fragments of the major outer capsid protein reported as 45K were contained in the predicted polypeptide. This protein, renamed the 47K protein, showed high homology with the outer capsid proteins of rice dwarf virus (RDV) and wound tumour virus (WTV); there was 56, 52 and 48% amino acid sequence identity between RGDV and WTV, RGDV and RDV, and RDV and WTV, respectively. S10 had the coding potential for a polypeptide of 320 amino acids with an Mr of 36,095 (36K protein), which exhibits 32% and 35% amino acid sequence identity with the predicted translation product of RDV S9 and the P9 capsid protein encoded by WTV S11, respectively. The conserved terminal sequences 5' GG...GAU 3' which are present in all genome segments of WTV and RDV so far analysed, and in S9 of RGDV, were also found in RGDV S8 and S10. This conserved sequence together with the segment-specific inverted repeats found in the terminal sequence of RGDV S8 and S10 are thus characteristic structures common to all three phytoreoviruses. The nucleotide sequence of the region surrounding the inverted repeats was more similar between RGDV and WTV than between RGDV and RDV.

Structure-specific binding of wound tumor virus transcripts by a host factor: Involvement of both terminal nucleotide domains

A gel retardation assay was used to demonstrate binding of wound tumor virus transcripts by a protein component of leafhopper vector cell extracts. Comparative binding studies employing terminally modified and internally deleted transcripts established that the segment-specific inverted repeats present in the terminal domains of the viral transcripts were necessary but not sufficient for optimal binding. An additional involvement of internal sequences in either the formation or the stabilization of the binding complex was indicated. Results of competitive binding experiments confirmed the sequence- and structure-specificity of the protein-RNA interaction and revealed apparent differences in the ability of individual viral transcripts to form a stable binding complex. Possible implications of structure-specific interactions between wound tumor virus transcripts and a host component and the role of the terminal inverted repeats are discussed.

Segment 5 of the Rice Dwarf Virus Genome Encodes a Protein Highly Conserved within the Phytoreoviruses

The complete nucleotide sequence of segment 5 (S5) of the rice dwarf virus (RDV) genome was determined. RDV S5 is 2571 bp in length and has a single long open reading frame which encodes a polypeptide of 801 amino acids (Mr 90495). When compared to the wound tumor virus genome S5, there was 56.9% and 52.8% similarity in the nucleotide and amino acid sequences, respectively. This high similarity suggests that the S5 proteins of these viruses are functionally similar.

Sequence analysis of rice dwarf phytoreovirus genome segments S4, S5, and S6: Comparison with the equivalent wound tumor virus segments

The complete nucleotide sequences of genome segments S4, S5, and S6 of rice dwarf phytoreovirus (RDV) were determined. S4 and S5 consist of 2468 and 2570 base pairs, respectively, S5 thus being larger in size than S4, contrary to the situation suggested by their relative migration in a polyacrylamide gel. S6 is 1699 nucleotides long. The individual segments have segment-specific inverted repeats adjacent to the conserved terminal sequences (5′GGUAAA—UGAU3′ for S4, 5′GGCAAA—UGAU3′ for S5 and S6). S4, S5, and S6 each have single long open reading frames encoding 727, 801, and 509 amino acids, respectively. A low level of amino acid sequence homology was observed between RDV S4 and wound tumor virus (WTV) S4 (22.4%), and between RDV S6 and WTV S6 (20.2%). On the other hand, RDV S5 and WTV S5 show 52.0% amino acid sequence similarity, indicating that S5 is much more conserved than any other segments of RDV and WTV reported so far. Further comparative analyses indicate that the RDV segment shows a greater frequency of usage of codons XYG and XYC, and much less frequent usage of codon XYA than the equivalent WTV segment, this codon preference bias being more conspicuous than expected from the base contents.

Nucleotide sequence of rice dwarf virus genome segment 4

The complete nucleotide sequence of rice dwarf virus (RDV) genome segment 4 was determined. Genome segment 4 was 2468 nucleotides long and had a long open reading frame initiating at nucleotides 64 to 66 and terminating at 2245 to 2247. The deduced polypeptide contained 727 amino acid residues with an Mr of 79.8K. Amino acid sequences similar to a 'zinc-finger' and purine NTP-binding motifs were present in the deduced polypeptide. Considerable amino acid sequence homology was detected between genome segment 4 of RDV and wound tumor virus (WTV). One of the sequences similar to the 'zinc-finger' motif was present in a conserved region of the polypeptide of both viruses. However, the sequence similar to the purine NTP-binding motif was not present in the polypeptide encoded by genome segment 4 of WTV.

Nucleotide Sequence of Segment S9 of the Genome of Rice Gall Dwarf Virus

DNA complementary to the ninth largest (S9) of the 12 genome segments of rice gall dwarf virus (RGDV) was cloned and its sequence was determined. It is 1202 nucleotides in length and contains one open reading frame which extends for 969 nucleotides from nucleotide 26. It encodes a polypeptide of 323 amino acids with an Mr of 35,560. The dinucleotide sequence at the 5' end and the trinucleotide sequence at the 3' end of the plus strand, 5' GG--GAU 3', which are present in the RNA of both wound tumour virus (WTV) and rice dwarf virus (RDV), were also found in RGDV genome segment S9. The nucleotide sequences in the noncoding region at the 5' terminus and in the 15 nucleotides at the 3' terminus, which form an imperfect inverted repeat of 10 bp together with the 5' terminus, are approximately 70% homologous with those of the WTV genome segment S9, but only 30% and 50% homologous with the respective termini of RDV S9.

Sequence analysis and product assignment of segment 7 of the rice dwarf virus genome

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.

Complete nucleotide sequence of wound tumor virus genomic segments encoding nonstructural polypeptides

Sequence analysis of the genomic segments which encode the five wound tumor virus nonstructural polypeptides has been completed. The complete nucleotide sequences of segments S4 (2565 bp), S6 (1700 bp), S9 (1182 bp), and S10 (1172 bp) are presented in this report while the sequence of segment S12 (851 bp) has been described previously (T. Asamizu, D. Summers, M. B. Motika, J. V. Anzola, and D. L. Nuss, 1985, Virology 144, 398–409) . Comparison of the only published sequence for another member of the genus Phytoreovirus, that of rice dwarf virus segment S10, with the combined available wound tumor virus sequence data revealed similarity with WTV segment S10: 54.9 and 30.6% at the nucleotide and amino acid level, respectively. Although wound tumor virus and rice dwarf virus differ in plant host range, tissue specificity, vector range, and disease symptom expression, the level of sequence similarity shared by the two segments suggests a common origin for these viruses. The potential use of a phytoreovirus sequence database for predicting functions of viral encoded gene products is considered.

The 3′-terminal sequence of a wound tumor virus transcript can influence conformational and functional properties associated with the 5′-terminus

We recently reported the presence of segment-specific inverted repeats within the terminal regions of wound tumor virus genomic segments (1. V. Anzola, Z. Xu, T. Asamizu, and D. L. Nuss, 1987, Proc. Natl. Acad. Sci. USA, 84, 8301–8305). This report describes a series of experiments designed to investigate potential intramolecular interactions involving the 5′- and 3′-terminal domains of wound tumor virus transcripts. A series of transcription vectors were constructed which allowed the synthesis of an exact copy of the transcript corresponding to genomic segment S8 and four analogs that differed from the authentic sequence only at the immediate 3′-terminus. Modifications designed to extend or alter the 3′-terminal inverted repeat altered the in vitro translational efficiency of the transcript and the sensitivity of phosphodiester bonds within the immediate 5′-terminal domain to digestion by nuclease T1. These results were consistent with computer-assisted secondary structure analyses of the complete nucleotide sequence of transcripts corresponding to six genomic segments which predicted intramolecular interactions involving the terminal inverted repeats. Potential roles of the terminal domains in expression, sorting and packaging of a segmented RNA genome are considered.