Detlev Riesner

Structure and structure formation of viroids.

Abstract The structure of viroids and the mechanism of structure formation were investigated by different methods. Results from gel analysis, partial degradation pattern, electron microscopy, dye binding, hydrodynamic studies, and temperature-jump kinetics were interpreted in a common structural and mechanistic scheme. Gel analysis, electron microscopy and kinetic investigations show that viroids may assume the native as well as metastable conformations under the same conditions. The native conformation is obtained by complete renaturation, i.e. slow cooling throughout the transition range (e.g. 52 to 48 ° C for potato spindle tuber viroid (PST viroid) in 0.01 m -sodium cacodylate, pH 6.8). In contrast, metastable conformations were trapped if viroids were redissolved in the cold from their ethanol precipitate or if they were denatured and cooled quickly. The native secondary structure of the recently sequenced PST viroid (Gross et al. , 1978) was optimized for the free energy of base-pairing. The scheme agrees with that proposed by Gross et al. (1978), which was derived from chemical arguments. The extended structure does not undergo tertiary structure folding under a wide range of conditions, as was concluded from electron microscopy, sedimentation measurements and binding studies of ethidium bromide and a new dye specific for A · U pairs (2-(4′-aminophenyl)-5-(4′-methylpiperazin-1″yl)-benzirnidazol). Intermediate structures during viroid denaturation were analysed on theoretical and experimental grounds. The experimental data, in combination with the model calculations, show that all of the native base-pairs of viroids are dissociated in one highly co-operative main transition, and that during the same process very stable hairpins are formed that are not present in the native structure. The formation of stable hairpins induces a new type of long range cooperativity, which is responsible in part for the high co-operativity observed experimentally. This interpretation is in good agreement with kinetic results presented elsewhere (Henco et al. , 1979). In order to understand the uniqueness of viroids, the structure and the conformational transitions of circular RNA molecules of the same base composition as PST viroids but with 359 nucleotides arranged randomly, were studied theoretically. Common viroid features, such as the number of base-pairs, the high co-operativity and the formation of very stable hairpins, are found to be improbable in such random sequences. It is concluded that various viroid species, although differing in nucleotide sequence, follow common principles of structure and structure formation.

Conversion of circular viroid molecules to linear strands

We have shown that viroids are single-stranded covalently-closed RNA molecules with mol. wt -120 000. In their native state they exist as largely double-stranded rod-like structures which are characterized by a serial arrangement of helical segments and internal single-stranded loops [l-3 1. In our viroid preparations we find usually <l% linear molecules which were assumed to arise through ‘nicking’ of viroid circles during the isolation procedure [I]. For certain studies the availability of large quantities of linear viroid molecules would be of great advantage. It has been long established that biand trivalent metal cations catalyze the cleavage of RNA even at very low concentrations [4-61. Thus at slightly alkaline pH Mg*’ cleave RNA molecules generating 5’-hydroxyl end groups and 2’and 3’-phosphates, a reaction which has successfully been used to specifically cleave individual tRNA species [7]. We have applied this Mg’+-catalyzed phosphodiester cleavage to different viroids and found that one single nick occurred under a wide range of

Formation of a thermodynamically metastable structure containing hairpin II is critical for infectivity of potato spindle tuber viroid RNA.

The functional relevance of a hairpin II‐containing structure of viroid RNA was studied by site‐directed mutagenesis, thermodynamic calculations, experimental denaturation curves and infectivity tests. Hairpin II is formed during thermal denaturation of circular viroids or as part of a metastable structure during synthesis of viroid replication intermediates. In potato spindle tuber viroid (PSTVd), eight single‐site mutations were generated in the segments which form hairpin II. From the mutated viroid cDNA clones, linear RNA transcripts of PSTVd unit length were synthesized. The relevance of hairpin II for the mechanism of denaturation was confirmed quantitatively by optical denaturation curves and temperature‐gradient gel electrophoresis. Infectivity tests showed that the mutations in the core region of hairpin II reverted to the wild type sequence whereas the mutations in the peripheral regions of hairpin II remained genetically stable. These data are in accordance with the natural variance of hairpin II in other viroids of the PSTVd class. Thus, the integrity of the core of hairpin II is critical for infectivity. Hairpin II exhibits a strong similarity in sequence as well as in three‐dimensional structure to certain DNA GC‐clusters found in the 5′‐upstream regions of some genes in man, animals, viruses and plants. A hypothesis about a function of hairpin II as a binding site for host cell transcription factors is proposed.

Multiple pathways of reversion in viroids for conservation of structural elements.

From site‐directed mutagenesis of potato spindle tuber viroid (PSTVd) it had been concluded earlier that the formation of a thermodynamically metastable structure containing hairpin II (HP II) is critical for infectivity. In order to differentiate between structural and sequence effects, in the present work base pairs in HP II were exchanged by site‐directed double mutations without significant alterations in the native rod‐like structure of PSTVd. The mutants were viable and genetically stable in the first generation, but one of the two mutations reverted to the wild‐type nucleotide in the second generation. Single‐site mutations in the stem of HP II, which had been described as revertants to the wild‐type sequence earlier, were analysed with respect to the time course of reversion and the sequence variation during reversion. All replicating sequence variants were separated by gel electrophoretic techniques and the sequences and their relative frequencies were determined. From both types of studies it can be concluded (i) that HP II is a functional element in the (−)strand replication intermediate, generated due to sequential folding during synthesis, and that it is essential for template activity of (+)strand synthesis; (ii) that G:U pairs are tolerated transiently in (−)strand HP II; the lower stability of such a HP II is compensated by additional mutations outside HP II which suppress the competition of a rod‐like structure; and (iii) that the reversions are generated spontaneously during (−)strand synthesis. Furthermore, the double‐stranded structure of HP II is the essential element for short term replication of PSTVd but the exact sequence of the wild‐type proves to be superior with regard to fitness and replicability of PSTVd.

Subcellular localization of viroids in highly purified nuclei from tomato leaf tissue.

Approximately 95% of the viroid RNA which is present in potato spindle tuber viroid (PSTV)‐infected tomato plant leaf issue, is associated with the nucleolar fraction obtained from purified nuclei. Viroids were released from the nucleolar fraction by increasing the ionic strength of the medium to 0.66 suggesting that viroid RNA is present in these subnuclear components in a protein‐nucleic acid complex. A purification procedure for nuclei from leaf tissue had to be newly developed; it involves two Percoll density centrifugations as final steps. The nuclei were sonicated and the sonicate fractionated into fractions either highly enriched in nucleoli or in broken chromatin and ribonucleoprotein particles. The viroid content in the different samples was determined by gel electrophoresis. Depending upon the progress of the disease, viroid copy numbers between 200 and 10,000 per cell were observed in homogenized tissue, purified nuclei and in the nucleolar fraction. In chloroplasts, practically no viroids were detected. The results are discussed in the light of current hypotheses about the replication, pathogenicity and origin of viroids.

Structure and function of viroids

Viroids are an independent class of plant pathogens which are distinguished from viruses by the absence of a protein coat and by their unusually small size. They are single-stranded circular RNAs composed of about 360 nucleotide residues. Sequence analysis and physicochemical studies of the potato spindle tuber viroid (PSTV) have shown that, as a result of intra-molecular base pairing, viroids form a unique rod-like secondary structure which is characterized by a serial arrangement of double-helical sections and internal loops. There is no indication for an additional tertiary structure because all parts of the molecule are freely accessible to ligand interaction. During the denaturation all of the native base pairs of viroids are dissociated in one highly cooperative transition, and in the same process very stable hairpins which are not present in the native structure are newly formed. Most of the properties of the structure and structural transitions of PSTV have been found also in citrus exocortis viroid, chrysanthemum stunt viroid and four different viroid-like RNAs associated with the cadang-cadang disease. The close similarity between these viroids is more expressed in the overall structure and in thermodynamic and functional domains than in the primary sequence. The stiffness of all viroids can be described by an unique persistence length of 300 å. Characteristically, regions of premelting, regions of stable hairpins, and the sequence UACUACCCGGUGG which is opposite to one of the stable hairpins, are the most conservative sequences in the molecules. Current hypotheses about the function of viroids are discussed on the basis of their structural and thermodynamic features. The suggestion that viroid RNA has features similar to DNA has been supported by the finding that they are replicated in vitro by the DNA-dependent RNA polymerase II of the host plant. The highly conserved sequence in viroids mentioned above corresponds very closely to a segment at the 5′-end of the small nuclear RNA U1 of eukaryotes. Because this segment is discussed in recent models, to be involved in the splicing process, a hypothesis is proposed in which viroids interfere with the splicing process leading to a pathogenic misregulation of mRNA processing.

Experimental Transmission of Pospiviroid Populations to Weed Species Characteristic of Potato and Hop Fields

ABSTRACT Weed plants characteristic for potato and hop fields have not been considered in the past as potential hosts that could transmit and lead to spreading of potato spindle tuber (PSTVd) and hop stunt (HSVd) viroids, respectively. To gain insight into this problem, we biolistically inoculated these weed plants with viroid populations either as RNA or as cDNA. New potential viroid host species, collected in central Europe, were discovered. From 12 weed species characteristic for potato fields, high viroid levels, detectable by molecular hybridization, were maintained after both RNA and DNA transfers in Chamomilla reculita and Anthemis arvensis. Low viroid levels, detectable by reverse transcription-PCR (RT-PCR) only, were maintained after plant inoculations with cDNA in Veronica argensis and Amaranthus retroflexus. In these two species PSTVd concentrations were 105 and 103 times, respectively, lower than in tomato as estimated by real-time PCR. From 14 weeds characteristic for hop fields, high HSVd levels were detected in Galinsoga ciliata after both RNA and DNA transfers. HSVd was found, however, not to be transmissible by seeds of this weed species. Traces of HSVd were detectable by RT-PCR in HSVd-cDNA-inoculated Amaranthus retroflexus. Characteristic monomeric (+)-circular and linear viroid RNAs were present in extracts from weed species propagating viroids to high levels, indicating regular replication, processing, and circularization of viroid RNA in these weed species. Sequence analyses of PSTVd progenies propagated in C. reculita and A. arvensis showed a wide spectrum of variants related to various strains, from mild to lethal variants; the sequence variants isolated from A. retroflexus and V. argensis exhibited similarity or identity to the superlethal AS1 viroid variant. All HSVd clones from G. ciliata corresponded to a HSVdg variant, which is strongly pathogenic for European hops.

Helix-coil transitions in double-stranded viral RNA. Fine resolution melting and ionic strength dependence

Helix-coil transitions of double-stranded RNA from reovirus and infectious bursal disease virus were measured optically in aqueous medium of different ionic strengths. In RNA from reovirus four transitions and in RNA from infectious bursal disease virus two transitions were resolved and evaluated quantitatively. The ionic strength dependence of their midpoint temperatures dTm/dlog[Na+] were 13.5 +/- 0.3 degrees C for reovirus RNA and 14.9 +/-0.7 degrees C for infectious bursal disease virus RNA. The midpoint temperatures extrapolated to 1 M ionic strength were 102.9, 104.3, 105.6, and 108.8 +/- 0.3 degrees C for reovirus RNA, and 108.8 +/- 1.8 and 109.6 +/- 1.0 degrees C for infectious bursal disease virus RNA. The G + C content of the regions in reovirus RNA melting in the different transitions were determined from the spectrum of the hypochromicity. The quantitative interpretation of the data is carried out on the basis of the ion condensation theory. It is estimated for double-stranded RNA of 100% G + C, that dTm/dlog[na+] = 8.4 degrees C. The two-dimensional dependence Tm = Tm (ionic strength, G + C content) is given. The ionic strength dependence in different double-stranded RNAs is correlated to the spacing of the phosphate backbone, secondary structure, and tertiary structure.

Melting curves on less than 1 μg of nucleic acid

Abstract Two types of microcuvettes were developed for the registration of melting curves in normal uv-spectrophotometers. Nucleic acid (0.5–1 μg) is required to determine changes in absorption with an accuracy of 0.1 mA. The quartz cuvette allows the registration of equilibrium melting curves up to 100°C. The other type of cuvette made from gilded brass allows to measure slow temperature jumps up to 80°C and equilibrium melting curves up to 70°C. The time range of ∼3 s to several hours is accessible complementing the Eigen/De Mayer temperature-jump technique (1963, in Techniques of Organic Chemistry, Vol. VIII/2, pp. 895–1054, Wiley, New York). Temperature jumps of high amplitude as well as jumps to lower temperatures are possible. Examples of measurements on viroids and double-stranded viral RNA are presented.