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 non-structural 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.

Tailored removal of flanking homopolymer sequences from cDNA clones.

Terminal homopolymer sequences introduced during the synthesis and cloning of cDNA molecules often interfere with subsequent expression of the cloned cDNA. We describe a general method for the removal of homopolymer flanking sequences from cDNA inserts and subsequent tailoring of the resulting termini. The cDNA insert containing homopolymer tails is first subcloned into the multiple cloning site of an appropriate transcription vector. cDNA copies are then generated from in vitro-synthesized transcripts using oligonucleotide primers complementary to the nucleotide sequences adjacent to the homopolymer tails. The resulting double-stranded cDNA contains the homopolymer flanking sequences as 3'-terminal extensions that are conveniently removed by the 3'----5' exonuclease activity of T4 DNA polymerase. If the oligonucleotide primers also contain 5'-terminal noncomplementary sequences that specify potential restriction endonuclease sites, those sites are subsequently generated by the 5'----3' polymerase activity of the T4 DNA polymerase. Thus, in the same reaction, flanking homopolymer sequences are removed and the resulting termini are tailored to specify desired sequences.

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'.

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.

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 sequence 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 (J. V. Anzol, 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.

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.

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 from 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 WTVNJ.