George Bruening

Autolytic Processing of Dimeric Plant Virus Satellite RNA

Associated with some plant viruses are small satellite RNAs that depend on the plant virus to provide protective coat protein and presumably at least some of the proteins necessary for satellite RNA replication. Multimeric forms of the satellite RNA of tobacco ringspot virus are probable in vivo precursors of the monomeric satellite RNA. Evidence is presented for the in vitro autolytic processing of dimeric and trimeric forms of this satellite RNA. The reaction generates biologically active monomeric satellite RNA, apparently is reversible to form dimeric RNA from monomeric RNA, and does not require an enzyme for its catalysis.

Two proteins from cowpea mosaic virus

Abstract Cowpea mosaic virus protein was resolved into two polypeptide size classes by polyacrylamide gel electrophoresis in urea plus sodium dodecyl sulfate and by gel permeation chromatography in urea solution. Molecular weights of 22,000 and 42,000 were estimated for these size classes. Amino acid and peptide analyses indicated that each class consisted of a single kind of polypeptide chain. Evidence was obtained for the existence of some amino acid sequences common to the two proteins, but the possibility of a monomer-dimer relationship was excluded. Approximately equimolar amounts of the two proteins were found in each of the three centrifugal components of the virus: top, middle, and bottom.

Polyadenylate sequences in the ribonucleic acids of cowpea mosaic virus

Abstract Polyadenylate sequences at the 3′-termini of cowpea mosaic virus middle and bottom component RNAs were revealed both by the sequential removal of nucleotide residues and by the release of polyadenylate during nuclease digestion. The released polyadenylates from the two RNAs were similar in the distributions of their sedimentation rates. A weight average degree of polymerization of about 200 was estimated. No evidence was found for internal polyadenylate sequences or for terminal sequences of 16 or fewer residues.

Double-stranded DNA from cauliflower mosaic virus.

Abstract Extracted nucleic acid of cauliflower mosaic virus was confirmed as DNA on the basis of its susceptibility to nucleases and other criteria. The viral nucleic acid exhibits a sharp, cooperative-type melting profile with a transition midpoint at 87.2 ° in 0.15 M NaCl-0.015 M citrate and does not react with formaldehyde unless heated above 80 °. The DNA has a complementary base composition with the ratio of adenine: thymine and guanine:cytosine near unity and contains 43% guanine plus cytosine as shown by nucleotide analyses. The buoyant density of the DNA in cesium chloride is 1.702 g/ml. The separated viral DNA is infectious and exhibits the same buoyant densities after thermal denaturation and rapid cooling as before treatment, suggesting that the double-stranded DNA has the capacity for rapid renaturation.

Citrus exocortis viroid: nucleotide sequence and secondary structure of an Australian isolate

Exocortis or ‘scaly butt disease’ of citrus is distributed world-wide [I]. The causative agent is citrus exocortis viroid (CEV) [2,3] which is a member of that unique group of plant pathogens, the viroids, of which only 8 have been described [ 1,4,5]. Like other members of the group, CEV consists of a singlestranded covalently closed circular RNA molecule which is highly base-paired, rod-like, infectious and non-encapsidated [ 1,4]. The primary sequence and proposed secondary structure of only 3 viroids have been reported so far: potato spindle tuber viroid (PSTV) with 359 residues [6]; chrysanthemum stunt viroid (CSV) with 356 residues [7]; and avocado sunblotch viroid (ASBV) with 247 residues [8]. We report here the sequence and proposed secondary structure of the 371 residues of an Australian isolate of CEV and discuss the significance of the extensive sequence homology which exists between PSTV, CSV and CEV.

The inheritance of top component formation in cowpea mosaic virus

Abstract Two local lesion isolates of cowpea mosaic virus (CPMV) were fractionated first by velocity sucrose gradient centrifugation and then by equilibrium cesium chloride centrifugation. The isolate Sb24 yielded four principal components distinguishable by their buoyant densities: T, M, B U , and B L . The last two components have the same sedimentation coefficient and cannot be separated by sucrose gradient centrifugation. The isolate Sb2 yielded only M, B U , and B L . Some component combination of the form M + B U or M + B L appears to be necessary to initiate an infection. Inocula were prepared containing these pairs of components, either with both from one isolate or with one from isolate Sb2 and the other from Sb24. Inocula containing component M from isolate Sb24 caused T to be formed while progeny from inocula containing M from Sb2 tended to be free of T, regardless of the isolate which was the origin of the component B U or B L in the inoculum. Thus the inheritance of top component production seems to be through M, and the total genome of cowpea mosaic virus is apparently associated with two kinds of RNA molecules, one in the M particle and one in a B U or a B L particle.

The Use of an Abrasive in the Isolation of Cowpea Leaf Protoplasts which Support the Multiplication of Cowpea Mosaic Virus

Abstract Protoplasts were isolated from primary leaves of cowpeas (Vigna sinensis) by a technique that makes stripping of the leaf epidermis unnecessary. Powdered abrasive was applied to the lower epidermis and spread about by brief brushing. Subsequent enzymatic digestion released more than five times as many protoplasts from treated, as compared to untreated, leaves and in less than one-third the digestion time. Only the upper epidermis and vascular tissue remained undigested. Protoplasts cultured in a modified White medium regenerated cell walls over a period of four days. Freshly isolated protoplasts were inoculated with cowpea mosaic virus. Infectivity, as detected by local lesion assay of protoplast crude extracts, increased over a period of 40 hr to a level consistent with >5 × 105 virions per protoplast.

A possible mechanism for the inhibition of plant viruses by a peptide from Phytolacca americana.

Abstract An inhibitor of virus transmission isolated from pokeweed was tested for its ability to inhibit in vitro polypeptide synthesis in systems employing ribosomes of wheat, cowpea, and pokeweed. Only the pokeweed ribosome system was resistant to inhibition. This suggests that the inhibitor acts in vivo by blocking the messenger function of a potentially infective virus RNA.

Oligomers of avocado sunblotch viroid are found in infected avocado leaves

The most abundant form of avocado sunblotch viroid (ASBV) in extracts of infected leaves of avocado (Persea americana) is a single-stranded covalent RNA circle [l] of 247 residues [2]. Highly purified preparations of the circular viroid infect avocado and induce the sunblotch disease [3]. ASBV has a limited sequence homology with 3 other viroids which have extensive sequence homology with each other [2,4]; potato spindle tuber viroid (PSTV), chrysanthemum stunt viroid (CSV) and citrus exocortis viroid (CEV). The level of ASBV in nucleic acid extracts from leaves of infected avocado leaves from different trees varied over a 10 OOO-fold range [5], while there was a lOOO-fold variation in the concentration of ASBV in extracts prepared from 6 branches of the same tree [6]. We report here that extracts of leaf tissue from strongly infected avocado seedlings contain an oligomeric series of RNAs which are integral multiples of the unit length ASBV and are of the same (+) polarity. Complementary (-) RNAs are also present but in much lower concentrations.

Satellite tobacco ringspot virus RNA: Biological activity of DNA clones and their in vitro transcripts

The satellite RNA of tobacco ringspot virus (STobRV RNA) replicates in association with tobacco ringspot virus (TobRV), apparently by means of intermediates that are multimeric, tandem repeats of STobRV RNA (M. C. Kiefer, S. D. Daubert, I. R. Schneider, and G. Bruening, 1982, Virology 121, 262-273) and which are capable of autolytic cleavage to produce active monomeric RNA (G. A. Prody, J. T. Bakos, J. M. Buzayan, I. R. Schneider, and G. Bruening, Science, in press). We have prepared plasmids that contain circularly permuted dimeric and trimeric cDNA forms of the 359 residue monomeric STobRV RNA sequence. The dimeric and trimeric DNA inserts contain contiguous, unpermuted monomeric and dimeric STobRV RNA sequences, respectively. Monomeric RNAs of both the encapsidated, (+), and the complementary, (-), polarities were prepared in vitro: transcripts of cloned sequences were initiated at the bacteriophage SP6 promoter, and these autolytically processed to generate RNA with the electrophoretic mobility of monomeric STobRV RNA. Monomeric (+)RNA transcripts and double-stranded DNA with a permuted trimeric sequence were biologically active, as judged by their ability to engender encapsidated STobRV RNA when inoculated to plants in the presence of TobRV. Biological activity was not detected with monometric RNA transcripts of the (-) polarity or with single-stranded DNAs that contained permuted dimeric sequences of either polarity.