T.O. Diener

Potato spindle tuber "virus". IV. A replicating, low molecular weight RNA.

Abstract Earlier results had indicated that the spindle tuber disease of potato is incited by free RNA, and that neither conventional virions nor proteins that could be construed as viral coat proteins are synthesized in infected plants. By a combination of density-gradient centrifugation and polyacrylamide gel electrophoresis, using internal marker RNAs, it is now shown that the infectious RNA occurs in the form of several species with molecular weights ranging from 2.5 × 10 4 to 1.1 × 10 5 daltons. No evidence for the presence in uninoculated plants of a latent helper virus was found. Thus, potato spindle tuber “virus” RNA, which is too small to contain the genetic information necessary for self-replication, must rely for its replication mainly on biosynthetic systems already operative in the uninoculated plant. Several possible mechanisms are discussed. The term “viroid” is proposed to designate potato spindle tuber “virus” RNA and other RNAs with similar properties.

Sensitive and Rapid Diagnosis of Potato Spindle Tuber Viroid Disease by Nucleic Acid Hybridization

A sensitive and reliable new method for the detection of potato spindle tuber viroid in potato tubers has been developed. The method is based on hybridization of highly radioactive recombinant DNA to viroid RNA that has been attached to a solid support. The method can be automated and permits the rapid testing of large numbers of tubers.

Potato spindle tuber viroid. X. Visualization and size determination by electron microscopy.

Purified potato spindle tuber viroid (PSTV) has been visualized by electron microscopy, using the protein monolayer spreading technique. When PSTV solutions in 4 M sodium acetate were spread onto distilled water, short structures, mostly in compact aggregates, were seen. No such structures could be seen with PSTV preparations that had been incubated with ribonuclease or in control preparations devoid of PSTV. When PSTV solutions in 8 M urea were spread onto distilled water or onto 0.015 M ammonium acetate (pH 8), large numbers of individual short strands were seen. No such strands could be discerned in control preparations or in PSTV preparations treated with ribonuclease. Average length of PSTV molecules was about 500 A. Heating of PSTV samples in 8 M urea for 10 min at 63°, followed by quenching in ice water and spreading, had no discernible effect on the appearance or the mean length of PSTV molecules. When PSTV was heat denatured in the presence of 1.1 M formaldehyde, followed by quenching and spreading onto distilled water, the length of PSTV molecules varied; some molecules, however, were up to 700 A long. To determine whether PSTV is a single- or a double-stranded molecule, PSTV in 8 M urea was mixed with native coliphage T7 DNA, or with heat-denatured, formylated carnation mottle virus RNA (CarMV RNA). PSTV and double-stranded T7 DNA had similar widths, but PSTV was clearly thicker than single-stranded CarMV RNA. Comparison of the lengths of T7 DNA and PSTV, spread in urea, showed that T7 DNA is about 280 times longer than PSTV; the comparison of denatured CarMV RNA with denatured PSTV indicated that CarMV RNA is probably about 17 times longer than PSTV. From these data, we conclude that PSTV is probably a single-stranded RNA with some kind of hairpin-like structure and with a molecular weight in the range of 8.0–9.0 × 104 daltons.

Chrysanthemum stunt: A viroid disease

Abstract Infectious material in crude extracts from chrysanthemum stunt-affected cineraria or chrysanthemum leaves sediments heterogeneously, some pelleting during low-speed centrifugation, some during high-speed centrifugation. High-speed supernatants, however, always contain appreciable amounts of infectious material. Treatment of crude extracts with chloroform-butanol, phenol, or ethanol does not reduce infectivity. The infectious agent is sensitive to treatment with ribonuclease, but not to treatment with deoxyribonuclease. The agent can be concentrated by ethanol precipitation and resuspension in a smaller buffer volume. Partial purification of the infectious agent by methods developed for the purification of potato spindle tuber viroid (PSTV), followed by analysis in linear-log sucrose density gradients and by electrophoresis in 20% polyacrylamide gels demonstrated that the stunt agent is a low molecular weight RNA. Its electrophoretic mobility is somewhat greater than that of PSTV. Thus, the causative agent of the chrysanthemum stunt disease is a viroid similar to, but distinct from, PSTV.

Potato spindle tuber viroid. XII. An investigation of viroid RNA as a messenger for protein synthesis

Abstract The ability of potato spindle tuber viroid (PSTV) RNA to act as a messenger RNA for protein synthesis has been investigated in several cell-free protein synthesizing systems. Extracts from wheat embryo, commercial wheat germ, Escherichia coli and Bacillus stearothermophilus , which are known to translate RNAs from viruses that do not infect these organisms, are unable to synthesize any specific proteins directed by PSTV. Amino acid incorporation is not stimulated much above the control without PSTV. This stimulation is less than 1% of the activity due to Qβ and brome mosaic virus RNAs, which can be translated into specific products under the same conditions. We tentatively conclude that this form of PSTV RNA is not a messenger for protein synthesis.

Some physical and chemical properties of cucumber mosaic virus (strain Y) and of its isolated ribonucleic acid

Abstract Strain Y of cucumber mosaic virus (CMV-Y) contains 1.8% phosphorus and 16.4% nitrogen. Its base ratio is: adenine 24.3%, guanine 23.4%, cytosine 23.2%, and uracil 29.0%. CMV-Y has a ribonucleic acid (RNA) content of 18.5%. The sedimentation constant is 92 Svedberg units (S). In 0.02 M phosphate buffer of pH 7, CMV-RNA preparations (obtained by phenol extraction of several different virus batches) sedimented as paucidisperse mixtures of three components (A, B, and C) with sedimentation rates of 13.3 S, 19.6 S, and 22.9 S in which the latter was usually the major component. The molecular weight of the 22.9 S component of CMV-RNA was estimated to be in the range of 0.9–1.2 million. On the assumption that this represented a full complement of CMV-RNA, the molecular weight of CMV-Y was calculated to fall in the range of 4.9–5.8 million. Highly infectious CMV-RNA with the same physical properties was also prepared by degrading CMV-Y in 1.5 M KCl. The two fastest-sedimenting species in CMV-RNA, B and C, had the same sedimentation characteristics under the influence of either high (0.1 M ) or low (6 × 10 −4 M ) phosphate buffer concentration and under the influence of traces of Mg ++ ions or spermine. These “mergers” could be reversed in all but the low phosphate concentration treatment to the original three-component system. After heating of CMV-RNA up to 10 minutes at 80°C or 20 minutes at 60°C or after reacting with formaldehyde, a three-component system was retained although the slowest sedimenting component A had become somewhat polydisperse and its amount had increased at the expense of the fastest sedimenting species C. No significant loss of infectivity occurred upon heating CMV-RNA for 20 minutes at 60°C. Hypochromicity studies on CMV-Y and on CMV-RNA suggested that the RNA inside the virus possessed a large amount of secondary structure, which was retained in the isolated RNA. This was confirmed in optical density-temperature profiles of CMV-RNA. The above findings are discussed in relation to two alternative models for CMV-RNA: (1) CMV-RNA is a mixture of three molecular species with different molecular weights. (2) CMV-RNA is also a mixture of three molecular species, but the fastest-sedimenting ones (B and C) are of equal molecular weight, component C being ring-structured.

Potato spindle tuber virus: A plant virus with properties of a free nucleic acid: III. Subcellular location of PSTV-RNA and the question of whether virions exist in extracts or in situ

Abstract Bioassays of subcellular components isolated from potato spindle tuber virus-infected leaves showed that infectivity was mainly associated with the tissue debris and with purified nuclei. Fractions that contained soluble constituents, ribosomes, mitochondria, or chloroplasts were virtually noninfectious. Infectivity could be extracted with phosphate buffer from nuclei and from purified chromatin. Density-gradient centrifugation of nuclear or chromatin extracts, or of crude phosphate extracts of infected tissue, disclosed very slowly sedimenting infectious material ( ca. 10 S), as well as smaller amounts of faster-sedimenting infectious material. The latter material was heterogeneous and sedimented at rates of from 10 S to well beyond 200 S. Upon storage at 4°, the faster-sedimenting material lost infectivity as fast as, if not faster than, the 10 S material. Treatment of clarified crude extracts with deoxyribonuclease, or shaking with chloroform- n -butanol, or with phenol (alone or in conjunction with sodium dodecyl sulfate) had no appreciable effect on the sedimentation properties of the infectious material. Fast-sedimenting infectious material was also present in highly purified nucleic acid preparations from PSTV-infected tissue. Bioassays of extracts from PSTV-infected leaves that had been vacuum-infiltrated with solutions of nucleases indicated that in situ the infectious moiety of PSTV is sensitive to attack by ribonuclease. These results suggest that no virions exist in PSTV-infected tissue. If such entities do exist, they must be extremely labile and cannot be significant in the natural transmission of the disease agent. The polydisperse sedimentation behavior of the infectious material appears to be a property of PSTV-RNA; and it may be due to its peculiar structure or its unusual tendency to aggregate.

Virus infection and other factors affecting ribonuclease activity of plant leaves.

Abstract Rubbing of bean ( Phaseolus vulgaris L.) leaves, partial dehydration of cowpea [ Vigna sinensis (L.) Endl.] leaves, and inoculation of bean leaves with inactivated or viable pod mottle virus or of Datura stramonium L. leaves with tobacco mosaic virus resulted in increased ribonuclease activity of such leaves. The response after rubbing was more pronounced when the leaves were dusted with Carborundum prior to rubbing and when the medium contained Cu ++ or Zn ++ ions. The reaction was first noticeable approximately 2 hours after rubbing, reached its highest level 1 day after rubbing, and lasted for several days. Spraying of unwounded leaves with solutions of CuSO 4 also led to increased ribonuclease activity. Virus-infected leaves maintained higher levels of ribonuclease activity than healthy control leaves or leaves inoculated with inactivated virus. The effect of rubbing was not restricted to the upper epidermis, but extended into the mesophyll. Neither phosphatase nor phosphodiesterase activity significantly increased in bean or Datura leaves after rubbing of the leaves or after inoculation with virus. It is concluded that increased ribonuclease activity is not a specific response to virus infection, but occurs as a general shock reaction. In bean leaves with high ribonuclease activity, more lesions were produced and more pod mottle virus was synthesized than in leaves with lower ribonuclease activity. However, stimulation of ribonuclease activity by rubbing of bean leaves did not lead to increased pod mottle virus lesion formation or to stepped-up virus synthesis. The significance of these results is discussed with respect to a possible role of ribonuclease in the synthesis of viral nucleic acid.

Potato spindle tuber viroid: XIV. Replication in nuclei isolated from infected leaves

Abstract A method has been devised to isolated cell nuclei from uninfected or potato spindle tuber viroid (PSTV)-infected tomato leaves. Properties of the RNA-synthesizing system in nuclei purified from both sources were characterized and compared. Purified nuclei from uninfected and infected leaves have the ability to incorporate [ 3 H]UMP from [ 3 H]UTP into an acid-insoluble fraction. The overall characteristics of the reaction in the system with nuclei from infected leaves were similar to those in the system from uninfected leaves. With both sources of nuclei, [ 3 H]UMP was incorporated optimally at 30° and at or near pH 7.8. The reaction was inhibited by actinomycin D, deoxyribonuclease, and ribonuclease. It depended on the presence of all four nucleoside triphosphates, Mg 2+ ions, and an ATP-generating system. The reaction products were hydrolyzed by 1 N NaOH or ribonuclease, but not by 5% trichloroacetic acid at 70° or deoxyribonuclease. Isolation of low-molecular-weight RNAs from the in vitro reaction mixture and analysis by gel electrophoresis revealed that PSTV replication is sensitive to incubation with actinomycin D. These results, with in vivo evidence (Diener and Smith (1975) . Virology 63, 421–427.), indicate that DNA of the nuclei obtained from PSTV-infected tomato leaves may act as a template for the PSTV polymerization reaction.

The viroidlike structure and cellular location of anomalous RNA associated with the cadang-cadang disease.

Abstract The cadang-cadang associated RNA species, ccRNA-1 and ccRNA-2, were recovered from diseased coconut palm leaf using a range of procedures. These included varying the salt molarity, type of buffer, and reducing agent in the extraction medium, using either polyethylene glycol (PEG) or ammonium sulphate to precipitate the RNA-containing component from tissue extracts, and using three alternative procedures to extract nucleic acid from the PEG-precipitated material. The view that ccRNA-1 is uniquely associated with the disease was supported by showing that the similarly migrating RNA, which sometimes could be recovered from normal palms, differed structurally from ccRNA-1. Kinetics of digestion with ribonuclease A and the single-strand specific S1 endonuclease, and thermal denaturation studies, indicated that the ccRNA-1 molecule has both double- and single-stranded regions. Because disruption of base-pairing by melting or hot formamide treatment failed to affect the apparent molecular weight of ccRNA-1, it is concluded that ccRNA-1 has a looped or “hairpin” secondary structure, stabilized by base-pairing. Its buoyant density (1.62 g/cm3) falls between the expected values for a single-stranded and a double-stranded viral RNA. A comparative study with potato spindle tuber viroid (PSTV) showed that the S1 nuclease digestion kinetics of ccRNA-1 and PSTV were similar. In denaturing formamide polyacrylamide gels, the molecular weight of ccRNA-1 was below that of PSTV and was estimated to be 63,000–73,000. ccRNA-1 is soluble in 2 M LiCl, is able to penetrate 20% polyacrylamide gels, and its thermal denaturation profile responds to Mg2+ like that of citrus exocortis viroid RNA. The structure of ccRNA-1 therefore appears to resemble closely that of viroid RNA. Unlike the known viroids, ccRNA-1 is not sedimented from tissue extracts by low or high speed centrifugation, nor has its infectivity been demonstrated. Some variations in the ccRNA banding patterns in gels are described. The occasional appearance of duplex ccRNA-1 is attributed to the existence of two ccRNA-1 molecules which have the same number of base pairs, but which differ in having larger or smaller amounts of single-stranded RNA associated with them.