Biomedical subjects

D L Nuss

Publications and source records attributed to D L Nuss.

At least 37 records · Page 2Linked to original sources

PubMed1994

Attenuation of fungal virulence by synthetic infectious hypovirus transcripts.

Noninfectious, cytoplasmically transmissible viral double-stranded RNAs of the genus Hypovirus cause reduced virulence (hypovirulence) in the chestnut blight fungus Cryphonectria parasitica, providing the basis for virus-mediated biological control of a fungal disease. Synthetic transcripts corresponding to a full-length hypovirus RNA coding strand are infectious when introduced into fungal spheroplasts by electroporation. Hypovirus infections were readily established in Cryphonectria parasitica and in related fungal species not previously reported to harbor viruses. These results demonstrate the use of a synthetic mycovirus transcript to expand fungal host range, thereby broadening the potential application of virus-mediated hypovirulence to control fungal pathogenesis.

Ascomycota↗

PubMed1994

cDNA-derived hypovirus RNA in transformed chestnut blight fungus is spliced and trimmed of vector nucleotides.

Unencapsidated double-stranded viral RNAs belonging to the genus Hypovirus attenuate virulence of the chestnut blight fungus, Cryphonectria parasitica. A full-length cDNA clone of hypovirus CHV1-713 double-stranded RNA was recently shown to be infectious when introduced into the C. parasitica genome by DNA-mediated transformation. In this study, we show that the viral RNA derived from the chromosomally integrated cDNA copy is trimmed of extraneous vector nucleotide sequences. The cDNA-derived viral RNA was also found to contain a 73-bp deletion located within the 5'-noncoding leader sequence as a result of a pre-mRNA splicing event. Implications of these results are discussed in terms of hypovirus RNA replication and anticipated field studies involving engineered hypovirulent C. parasitica strains.

Base Sequence↗

PubMed1993

Molecular analysis and overexpression of the gene encoding endothiapepsin, an aspartic protease from Cryphonectria parasitica.

The gene, epn-1, encoding endothiapepsin (Epn), an aspartic protease (AspP) synthesized and secreted by the ascomycete fungus responsible for chestnut blight, Cryphonectria (Endothia) parasitica, was identified and characterized. Inspection of the nucleotide and deduced amino acid (aa) sequences revealed perfect agreement with the experimentally derived 330-aa sequence of mature Epn [Barkholt, Eur. J. Biochem. 167 (1987) 327-338] and an additional 89 aa of putative preprosequence. Of the nine fungal AspP characterized to date, Epn was found to be most closely related to aspergillopepsin and penicillopepsin (52% and 55% identity, respectively), proteases produced by the ascomycetes Aspergillus awamori and Penicillium janthinellum, and least related to proteases produced by the yeasts Candida albicans and Saccharomyces cerevisiae (27% and 26% identity, respectively). Epn production was found to be the same in isogenic virus-free and virus-containing strains, indicating that this AspP is not down-regulated by the presence of a hypovirulence-associated viral double-stranded RNA, as has been reported for several other secreted C. parasitica gene products. Strains containing multiple copies of epn-1 were obtained by transformation with a plasmid vector containing the cloned epn-1. One of these strains was shown to produce seven to ten times more Epn than the parental wild-type strain.

Amino Acid Sequence↗

PubMed1993

Cyclophilin-dependent stimulation of transcription by cyclosporin A.

Exposure to cyclosporin A (CspA) increased laccase (lac-1) transcript accumulation in the chestnut blight fungus Cryphonectria parasitica. This response was suppressed by compounds that interfere with calcium-dependent signal transduction and by the presence of a virulence-attenuating mycovirus. CspA stimulated the accumulation of mRNA from a nonhomologous reporter fused to the lac-1 promoter, indicating that the increased transcript levels resulted from an increase in promoter activity. Based on the current model for the regulation of lac-1 transcription, these results suggest that CspA interferes with a negative regulatory pathway that normally constrains lac-1 promoter activity. Significantly, CspA did not stimulate lac-1 transcription in mutant strains deficient in CspA binding activity, directly demonstrating a requirement for the interaction of CspA and cyclophilin in the modulation of lac-1 transcription. Our results establish that CspA treatment can stimulate gene transcription and that cyclophilin is the cellular receptor that mediates this activity.

Amino Acid Isomerases↗

PubMed1993

Mitotic stability and nuclear inheritance of integrated viral cDNA in engineered hypovirulent strains of the chestnut blight fungus.

Transmissible hypovirulence is a novel form of biological control in which virulence of a fungal pathogen is attenuated by an endogenous RNA virus. The feasibility of engineering hypovirulence was recently demonstrated by transformation of the chestnut blight fungus, Cryphonectria parasitica, with a full-length cDNA copy of a hypovirulence-associated viral RNA. Engineered hypovirulent transformants were found to contain both a chromsomally integrated cDNA copy of the viral genome and a resurrected cytoplasmically replicating double-stranded RNA form. We now report stable maintenance of integrated viral cDNA through repeated rounds of asexual sporulation and passages on host plant tissue. We also demonstrate stable nuclear inheritance of the integrated viral cDNA and resurrection of the cytoplasmic viral double-stranded RNA form in progeny resulting from the mating of an engineered hypovirulent C. parasitica strain and a vegetatively incompatible virulent strain. Mitotic stability of the viral cDNA ensures highly efficient transmission of the hypovirulence phenotype through conidia. Meiotic transmission, a mode not observed for natural hypovirulent strains, introduces virus into ascospore progeny representing a spectrum of vegetative compatibility groups, thereby circumventing barriers to anastomosis-mediated transmission imposed by the fungal vegetative incompatibility system. These transmission properties significantly enhance the potential of engineered hypovirulent C. parasitica strains as effective biocontrol agents.

Ascomycota↗

PubMed1993

Papain-like protease p29 as a symptom determinant encoded by a hypovirulence-associated virus of the chestnut blight fungus.

Viral double-stranded RNAs (dsRNAs) responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus, Cryphonectria parasitica, profoundly influence a range of host functions in addition to virulence. The 5'-proximal open reading frame, A, of the prototypical hypovirulence-associated viral dsRNA, L-dsRNA, present in hypovirulent strain EP713, was recently shown by DNA-mediated transformation analysis to suppress fungal sporulation, pigmentation, and accumulation of the enzyme laccase (G. H. Choi and D. L. Nuss, EMBO J. 11:473-477, 1992). We mapped this suppressive activity to the autocatalytic papain-like protease, p29, present within the amino-terminal portion of open reading frame A-encoded polyprotein p69. Mutational analysis revealed that the ability of p29 to alter fungal phenotype is dependent upon release from the polyprotein precursor but is independent of intrinsic proteolytic activity. Deletion of the p29-coding domain within the context of an infectious L-dsRNA cDNA clone resulted in a replication-competent viral dsRNA that exhibited intermediate suppressive activity while retaining the ability to confer hypovirulence. Thus, p29 is necessary but not sufficient for the level of virus-mediated suppression of fungal pigmentation, sporulation, and laccase accumulation observed for wild-type hypovirulent strain EP713 and is nonessential for viral RNA replication and virulence attenuation. These results also illustrate the feasibility of engineering infectious viral cDNA for construction of hypovirulent fungal strains with specific phenotypic traits.

Amino Acid Sequence↗

PubMed1992

Hypovirulence of chestnut blight fungus conferred by an infectious viral cDNA.

Strains of the chestnut blight fungus Cryphonectria parasitica that contain viral double-stranded RNAs often exhibit reduced virulence. Such hypovirulent strains act as biocontrol agents by virtue of their ability to convert virulent strains to hypovirulence after anastomosis. Transformation of virulent C. parasitica strains with a full-length complementary DNA copy of a hypovirulence-associated viral RNA conferred the complete hypovirulence phenotype. Cytoplasmic double-stranded RNA was resurrected from the chromosomally integrated complementary DNA copy and was able to convert compatible virulent strains to hypovirulence. These results establish viral double-stranded RNA as the casual agent of hypovirulence and demonstrate the feasibility of engineering hypovirulent fungal strains.

DNA, Viral↗

PubMed1992

A viral gene confers hypovirulence-associated traits to the chestnut blight fungus.

A viral double-stranded (ds)RNA associated with reduced virulence (hypovirulence) and the accompanying biological control of the chestnut blight fungus, Cryphonectria parasitica, was shown recently to contain two contiguous coding domains designated ORF A and ORF B. We report here that transformation of an isogenic virulent, dsRNA-free C. parasitica strain with a cDNA copy of ORF A conferred traits similar to those exhibited by the dsRNA-containing hypovirulent strain: characteristics included reduced pigmentation, reduced laccase accumulation and suppressed conidiation. However virulence was not reduced, indicating an apparent uncoupling of associated traits from hypovirulence. These results establish a direct cause and effect relationship between a viral dsRNA genetic element present in a hypovirulent C. parasitica strain and specific phenotypic traits. They demonstrate further that these traits are not the result of a general reaction of the fungus to the presence of the replicating viral RNA, but are caused by a specific viral coding domain.

Genes, Viral↗

PubMed1992

Regulatory pathways governing modulation of fungal gene expression by a virulence-attenuating mycovirus.

A viral double-stranded RNA associated with virulence attenuation (hypovirulence) in the chestnut blight fungus (Cryphonectria parasitica) has been shown by DNA-mediated transformation to be responsible for transmissible hypovirulence. In addition to reduced virulence, the fungal strain harboring this virus exhibits a diverse array of characteristics, termed hypovirulence-associated traits, which distinguish it from an isogenic virus-free strain. We have investigated one of these traits, suppressed lac-1 (laccase) transcript accumulation. Two different and opposing regulatory pathways appear to govern lac-1 transcript levels in the virus-free strain: a stimulatory pathway was found to be dependent on the inositol trisphosphate (IP3) and calcium second messenger systems. A second pathway limiting transcript accumulation was shown to require ongoing protein synthesis. Additionally, changes in the level of lac-1 transcript accumulation were found to be related to modulation of promoter activity and this activity was shown to be suppressed in the virus-containing strain. We conclude that this hypovirulence-associated virus interferes with transduction of an IP3-calcium-dependent signal that is required for stimulation of lac-1 transcription. The perturbation of such signal transduction pathways by hypovirulence-associated viruses may account for the manifold symptoms associated with transmissible hypovirulence.

Ascomycota↗

PubMed1992

Biological control of chestnut blight: an example of virus-mediated attenuation of fungal pathogenesis.

Environmental concerns have focused attention on natural forms of disease control as potentially safe and effective alternatives to chemical pesticides. This has led to increased efforts to develop control strategies that rely on natural predators and parasites or that involve genetically engineered microbial pest control agents. This review deals with a natural form of biological control in which the virulence of a fungal pathogen is attenuated by an endogenous viral RNA genetic element: the phenomenon of transmissible hypovirulence in the chestnut blight fungus, Cryphonectria parasitica. Recent progress in the molecular characterization of a hypovirulence-associated viral RNA has provided an emerging view of the genetic organization and basic expression strategy of this class of genetic elements. Several lines of evidence now suggest that specific hypovirulence-associated virus-encoded gene products selectively modulate the expression of subsets of fungal genes and the activity of specific regulatory pathways. The construction of an infectious cDNA clone of a hypovirulence-associated viral RNA represents a major advancement that provides exciting new opportunities for examining the molecular basis of transmissible hypovirulence and for engineering hypovirulent strains for improved biocontrol. These developments have significantly improved the prospects of using this system to identify molecular determinants of virulence and elucidate signal transduction pathways involved in pathogenic responses. In addition, novel approaches are now available for extending the application of transmissible hypovirulence for management of chestnut blight and possibly other fungal diseases.

Genes, Fungal↗

PubMed1991

Evidence for common ancestry of a chestnut blight hypovirulence-associated double-stranded RNA and a group of positive-strand RNA plant viruses.

Computer-assisted analysis of the putative polypeptide products encoded by the two open reading frames present in a large virus-like double-stranded RNA, L-dsRNA, associated with hypovirulence of the chestnut blight fungus, Cryphonectria parasitica, revealed five distinct domains with significant sequence similarity to previously described conserved domains within plant potyvirus-encoded polyproteins. These included the putative RNA-dependent RNA polymerase, RNA helicase, two papain-like cysteine proteases related to the potyvirus helper-component protease, and a cysteine-rich domain of unknown function similar to the N-terminal portion of the potyvirus helper-component protein. Phylogenetic trees derived from the alignment of the polymerase domains of L-dsRNA, a subset of positive-stranded RNA viruses, and double-stranded RNA viruses, using three independent algorithms, suggested that the hypovirulence-associated dsRNA and potyvirus genomes share a common ancestry. However, comparison of the organization of the conserved domains within the encoded polyproteins of the respective viruses indicated that the proposed subsequent evolution involved extensive genome rearrangement.

Amino Acid Sequence↗

PubMed1991

Gene expression by a hypovirulence-associated virus of the chestnut blight fungus involves two papain-like protease activities. Essential residues and cleavage site requirements for p48 autoproteolysis.

Proteolytic processing plays a fundamental role in gene expression of a recently characterized viral-like double-stranded RNA associated with biological control of the chestnut blight fungus. Polypeptide p29, a papain-like protease, was shown to autocatalytically release itself from the NH2 terminus of the polyprotein specified by the first of two encoded open reading frames, ORF A (Choi, G. H., Shapira, R., and Nuss, D. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1167-1171; Choi, G. H., Pawlyk, D. M., and Nuss, D. L. (1991) Virology 183, 747-752). The characterization of a second autocatalytic protease, p48, which is encoded by ORF B, is the subject of this report. Deletion analysis revealed that the catalytic domain resides within the carboxyl-terminal region, while site-specific mutational analysis identified Cys-341 and His-388 as residues essential for autoproteolysis. Autoproteolytic processing by p48 was also demonstrated when expressed in Escherichia coli and microsequence analysis of the recovered COOH-terminal cleavage product indicated that cleavage occurred between Gly-418 and Ala-419. The requirements for a functional cleavage site, including confirmation of the cleavage dipeptide, were defined by amino acid substitution analysis. Similarities between p29 and p48 suggest that the respective coding domains could have arisen as a result of a gene duplication event.

Amino Acid Sequence↗

PubMed1991

Cotranslational autoproteolysis involved in gene expression from a double-stranded RNA genetic element associated with hypovirulence of the chestnut blight fungus.

The genetic information responsible for reduced virulence (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica is thought to reside on cytoplasmically replicating double-stranded RNA (dsRNA) molecules. Cell-free translation of synthetic transcripts corresponding to the 5'-terminal 2640 nucleotides of the sense strand of the large dsRNA present in C. parasitica hypovirulent strain EP713 yielded two polypeptides with apparent molecular masses of 29 and 40 kDa. Nucleotide sequence analysis indicated that p29 and p40 were encoded by a single large open reading frame. The coding regions for p29 and p40 were mapped to nonoverlapping portions of the 5'- and 3'-terminal domains of the open reading frame, respectively. Kinetic analysis and in vitro translation studies with chimeric transcripts indicated that p29 is autocatalytically released from a nascent polyprotein during translation. Microsequence analysis of the amino terminus of radiolabeled p40 indicated that cleavage occurred between Gly-248 and Gly-249, consistent with translational mapping analysis. Examination of the p29 amino acid sequence revealed similarity to the Potyvirus-encoded cysteine-type proteinase HC-Pro. These results indicate the types of mechanism that operate during gene expression by hypovirulence-associated dsRNA genetic elements.

Amino Acid Sequence↗

PubMed1991

Virus-like genetic organization and expression strategy for a double-stranded RNA genetic element associated with biological control of chestnut blight.

The complete nucleotide sequence of the largest double-stranded (ds) RNA present in hypovirulent strain EP713 of the chestnut blight pathogen, Cryphonectria parasitica, was determined and the predicted genetic organization was confirmed by translational mapping analysis. The deduced RNA sequence was 12 712 bp in length, excluding the terminal poly(A):poly(U) homopolymer domain. The strand terminating with 3'-poly(A) contained two contiguous large open reading frames (ORF A and ORF B) beginning at nucleotide residues 496-498 and extending to nucleotide positions 11 859-11 861. The junction between ORF A and ORF B consisted of the sequence 5'-UAAUG-3', where UAA served as the termination codon for ORF A and AUG was the 5'-proximal initiation codon within ORF B. ORF A (622 codons in length, excluding the termination codon) was recently shown to encode two polypeptides, p29 and p40, which were generated from a nascent polyprotein by an autocatalytic event mediated by p29 (Choi et al., 1991). A similar autocatalytic event was observed during in vitro translation of ORF B (3165 codons in length) resulting in the release of a 48 kd polypeptide from the amino-terminal portion of the ORF B-encoded polyprotein. These results are discussed in terms of the opportunities they provide for elucidating the molecular basis of transmissible hypovirulence and possible origins of hypovirulence-associated dsRNAs.

Amino Acid Sequence↗

PubMed1991

The contribution of defective RNAs to the complexity of viral-encoded double-stranded RNA populations present in hypovirulent strains of the chestnut blight fungus Cryphonectria parasitica.

Hypovirulent strain EP713 of the chestnut blight fungus Cryphonectria (Endothia) parasitica harbors a family of viral encoded double-stranded (ds) RNAs thought to be responsible for the hypovirulence phenotype. These include L-dsRNA, described in the accompanying paper (Shapira et al., 1991); several prominent species in the estimated size range of 8 to 10 kb, referred to here as M-dsRNAs; and several smaller species designated S-dsRNAs which range in size from approximately 0.6 to 1.7 kb. The characterization of the M- and S-dsRNA species is the subject of this report. Results from polymerase chain reaction mapping and molecular hybridization analysis indicate that the M- and S-dsRNA species are internally deleted forms of L-dsRNA. Three different S-dsRNA species were cloned and sequenced. Each species contained a single deletion breakpoint and retained either 149, 155 or 156 bp of the terminus corresponding to the 5'-end of the coding strand and 440, 447 or 449 bp of the other terminus. Two of the S-dsRNA species contained, within the boundaries of the breakpoint, additional sequence information consisting of 42 bp or 95 bp that appeared to be unrelated to the L-dsRNA sequence. These results demonstrate that defective RNAs contribute significantly to the complexity of dsRNA populations found in hypovirulent strains of C. parasitica and provide a first approximation of the location of cis-acting signals involved in their replication.

Base Sequence↗

PubMed1991

Cloning of the maize rough dwarf virus genome: molecular confirmation of the plant-reovirus classification scheme and identification of two large nonoverlapping coding domains within a single genomic segment.

The segmented double-stranded RNA genome of maize rough dwarf virus, a plant-infecting reovirus of the genus Fijivirus, was cloned and partially characterized. Nucleotide sequence analysis of full-length cDNA clones corresponding to genomic segments S6, S7, and S8 revealed each segment to contain the conserved terminal oligonucleotide sequences (+) 5' AAGUUUUUU------UGUC 3' and adjacent, segment-specific, regions of inverted complementarity (inverted repeats), a structural motif previously reported for members of the genus Phytoreovirus. Genomic segment S6 was completely sequenced and found to consist of 2193 base pairs. Computer analysis indicated that the coding strand contained two large nonoverlapping open reading frames consisting of 363 and 310 codons and located in the 5'- and 3'-terminal domains, respectively. This was confirmed by cell-free translation studies with synthetic transcripts and denatured genomic RNA. However, only the product of the 5'-proximal open reading frame, a 40-kDa polypeptide, was efficiently expressed in vitro from the full-length S6 coding strand. This represents the first case in which a reovirus genomic segment was found to contain two large open reading frames in a nonoverlapping configuration, suggesting possible alternative strategies for regulation of gene expression by members of this genus. The combined results provide a molecular confirmation of the current classification scheme for plant-infecting reoviruses. Furthermore, the fact that the same terminal structural motif is conserved across genera provides additional evidence that these elements serve an important functional role during genome transcription or replication.

Amino Acid Sequence↗

PubMed1991

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.

Amino Acid Sequence↗

PubMed1991

The autocatalytic protease p29 encoded by a hypovirulence-associated virus of the chestnut blight fungus resembles the potyvirus-encoded protease HC-Pro.

Gene expression by a viral-like double-stranded RNA genetic element associated with reduced virulence (hypovirulence) of the chestnut blight fungus was recently shown to involve an autoproteolytic event which resulted in the release of an encoded protease, designated p29, from a polyprotein during translation. Mutational analysis of p29, described in this report, revealed that residues Cys-162 and His-215 are essential for autocatalytic cleavage. The results were also consistent with previous predictions that cleavage occurs between Gly-248 and Gly-249. Interestingly, p29 bears a striking resemblance to the potyvirus-encoded protease HC-Pro. Both proteases autocatalytically cleave at glycine dipeptides. In addition, there is a significant degree of similarity in the amino acid sequences flanking the essential Cys and His residues of the two proteases and in the spacing of these residues from their respective cleavage sites.

Amino Acid Sequence↗