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D L Nuss

Publications and source records attributed to D L Nuss.

At least 55 records · Page 3Linked to original sources
PubMed1990

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.

Animals↗
PubMed1989

Characterization of double-stranded RNA genetic elements associated with biological control of chestnut blight: organization of terminal domains and identification of gene products.

We have determined the organization within the terminal domains of the major large double-stranded RNA genetic elements associated with the hypovirulent strain EP713 of the chestnut blight pathogen Cryphonectria (Endothia) parasitica. Only the polyadenylated strand contained long open reading frames. Furthermore, only RNA of the same polarity as the polyadenylated strand was detectable in a single-stranded form, indicating that the polyadenylated strand is the coding or plus strand. The organization of the 5'-proximal portion of the plus strand consisted of a 495 nucleotide non-coding leader sequence followed by two overlapping open reading frames. The first, ORF1, extended 957 nucleotides while the second, ORF2, began 68 nucleotides upstream of the ORF1 termination codon and extended at least 1412 nucleotides. No open reading frames of significant size were detected within 0.8 kb of the poly(A) tail. In vitro translation of synthetic transcripts containing ORF1 yielded a polypeptide of Mr 29 kd. The ORF1 product was also detected in lysates of the hypovirulent strain but was absent in lysates of the isogenic virulent strain. It represents the first protein to be identified as a gene product encoded by a hypovirulence-associated double-stranded RNA genetic element.

Amino Acid Sequence↗
PubMed1989

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.

Base Sequence↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1987

cDNA probes of individual genes of human rotavirus distinguish viral subgroups and serotypes.

The use of cDNA probes for detection of rotaviruses has been investigated using plasmids containing inserts specific for each of the eleven genes of human rotavirus strain Wa. In a dot-blot detection system in which radioactive DNA probes were hybridized to viral RNA extracted from cultivatable rotavirus strains, cDNAs of genes 7, 8, 10 and 11, were found to be the most reliable probes for detecting a range of rotavirus strains. Unexpectedly, rotaviruses could be distinguished with respect to subgroup and subtype specificities when cDNAs of genes 6 and 9, which encode the immunologically relevant proteins VP6 (group-specific antigen) and VP7 (type-specific antigen), were used as probe, even though the nucleic acid sequences of these genes are known to have a high degree of sequence homology.

Child, Preschool↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1987

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.

Cloning, Molecular↗
PubMed1986

Structural properties of double-stranded RNAs associated with biological control of chestnut blight fungus.

Double-stranded RNAs (ds RNAs) are thought to be the cytoplasmic determinants responsible for the phenomenon of transmissible hypovirulence in the chestnut blight fungus Endothia parasitica [Murr.] Anderson. The three major ds RNA components associated with the North American hypovirulent strain, Grand Haven 2, were characterized with respect to molecular-hybridization specificity and RNase T1-digestion patterns. The large (L-RNA; approximately 9 kilobase pairs) and middle-sized (M-RNA; approximately 3.5 kilobase pairs) ds RNA components cross-hybridized under stringent conditions and exhibited indistinguishable partial and complete RNase T1 digestion patterns relative to their 5' and 3' termini. These results suggest that M-RNA was derived from L-RNA by an internal deletion event. The small (S-RNA; approximately 1 kilobase pair) RNA was unrelated to L- and M-RNA by these criteria. However, all three ds RNA components contained RNase T1-resistant oligonucleotides at one 5' terminus and at the corresponding 3' terminus of the complementary strand. These RNase T1-resistant species exhibited properties consistent with stretches of poly(uridylic acid) and poly(adenylic acid), respectively. The combined results are discussed in terms of the structural organization of hypovirulence-associated ds RNA molecules and their similarities to "double-stranded" RNA molecules observed in plant and animal cells infected with single-stranded RNA viruses.

Journal Article↗
PubMed1986

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.

Cells, Cultured↗
PubMed1985

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'.

Base Sequence↗
PubMed1985

Diagnosis of rotavirus infection with cloned cDNA copies of viral genome segments.

The diagnostic potential of cloned cDNA copies of human rotavirus (strain WA) genome segments for the detection of rotavirus in clinical specimens has been determined. A hybridization assay in which a mixture of 32P-labeled cDNAs representing the 11 rotavirus segments was used as a probe compared favorably with three frequently used diagnostic tests for rotavirus in terms of both specificity and sensitivity. Significantly, clinical isolates could be readily distinguished when cloned cDNA copies of individual genome segments were used independently as a probe. In assays in which genome RNA from rotaviruses of known subgroups and serotypes were tested, cloned probes that encode nonstructural viral proteins hybridized efficiently to genome RNAs of all strains, whereas cloned probes corresponding to genome segments 6 and 9 exhibited the potential for differentiating strains of different subgroups and serotypes. Cloned cDNA copies of rotavirus genome segments therefore offer considerable potential for improved general diagnosis of rotavirus in clinical specimens, as well as for epidemiological studies in which virus isolates can be distinguished on the basis of nucleotide sequence homology of individual genome segments.

Cloning, Molecular↗
PubMed1985

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.

Animals↗