Donald L. Nuss

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.

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.

Structural and Functional Properties of Plant Reovirus Genomes

Publisher Summary This chapter focuses on recent advances in the characterization of plant reovirus genomes as they relate to the biological and molecular processes. The association of reoviruses with plant disease has been recognized for many years and a considerable literature has been assembled. The studies forming the basis of these reports have directly implicated the viruses as causal agents of disease. The chapter is restricted to those viruses known to multiply in plant hosts and the general characteristics of the plant reoviruses have been outlined, with the aim of providing a backdrop against which to judge the interest and importance of studies of their genomes. Over the past years, concentration has been on examination of the structural and functional properties of the WTV genome. In the process, gaining insight into the mechanisms involved in the recognition, sorting, and packaging of segmented RNA genome information that will have general application for understanding protein-RNA interactions in other viral and cellular systems.

Variation in the relative synthesis of immunoglobulin G and non-immunoglobulin G proteins in cultured MPC-11 cells with changes in the overall rate of polypeptide chain initiation and elongation

The synthesis of individual proteins in the mouse plasmacytoma cell MPC-11 is differentially inhibited when the rate of polypeptide chain initiation is reduced by exposure of cells to hypertonic medium. The synthesis of immunoglobulin G light and heavy chain polypeptides is 3.5 to 4-fold and 1.5 to 2-fold more resistant, respectively, than the synthesis of non-immunoglobulin G proteins when total protein synthesis is reduced by ~90%. In contrast, when polypeptide chain elongation is inhibited, the synthesis of the light and heavy chains is not more resistant than the synthesis of non-immunoglobulin G proteins. n nThe results with MPC-11 cells suggests that: (1) under standard growth conditions the relative synthesis of individual proteins is determined mainly, but not exclusively, by the relative amounts of the individual messenger RNA species present in the cell; (2) under conditions where the overall rate of polypeptide chain initiation is reduced the relative synthesis of individual proteins becomes more dependent upon the intrinsic ability of their corresponding mRNAs to form functional mRNA-ribosome initiation complexes.

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.

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.

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.

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.

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.

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.