Viroids with hammerhead ribozymes: some unique structural and functional aspects with respect to other members of the group.

Viroids, subviral pathogens of plants, are composed of a single-stranded circular RNA of 246-399 nucleotides. Within the 27 viroids sequenced, avocado sunblotch, peach latent mosaic and chrysanthemum chlorotic mottle viroids (ASBVd, PLMVd and CChMVd, respectively) can form hammerhead structures in both of their polarity strands. These ribozymes mediate self-cleavage of the oligomeric RNAs generated in the replication through a rolling circle mechanism, whose two other steps are catalyzed by an RNA polymerase and an RNA ligase. ASBVd, and presumably PLMVd and CChMVd, replicate and accumulate in the chloroplast, whereas typical viroids replicate and accumulate in the nucleus. PLMVd and CChMVd do not adopt a rod-like or quasi rod-like secondary structure as typical viroids do but have a highly branched conformation. A pathogenicity determinant has been mapped in a defined region of the CChMVd molecule.     

Genomic Structure of Three Phenotypically Different Isolates of Peach Latent Mosaic Viroid: Implications of the Existence of Constraints Limiting the Heterogeneity of Viroid Quasispecies

The peach latent mosaic viroid (PLMVd) is used to study the interactions between a viroid containing hammerhead ribozymes and its natural host, peach. To gain insight into the molecular basis of the phenotypic effects observed upon viroid infection, sequence variants from three PLMVd isolates that differ in symptom expression on the peach indicator GF-305 have been characterized. Analysis of the primary structures of a total of 29 different sequence variants derived from a severe and two latent isolates has revealed a large number of polymorphic positions in the viroid molecule. The variability pattern indicates that preservation of the stability of both hammerhead structures and conservation of a branched secondary structure of the viroid molecule may be factors limiting sequence heterogeneity in PLMVd. Moreover, compensatory mutations in two hairpin loops of the proposed secondary structure, suggesting that a pseudoknot-like interaction may exist between them, have also been observed. Phylogenetic analysis has allowed the allocation of PLMVd molecules into three major groups. This clustering does not strictly correlate with the source isolate from which the variants were obtained, providing insights into the complex mixture of molecules which make up each isolate. Bioassays of individual PLMVd sequence variants on GF-305 peach seedlings have shown that the biological properties of the PLMVd isolates may be correlated with both the complexity of their viroid populations and the presence of specific sequence variants.     

Subcellular localization and rolling circle replication of peach latent mosaic viroid: hallmarks of group A viroids.

We characterized the peach latent mosaic viroid (PLMVd) replication intermediates that accumulate in infected peach leaves and determined the tissue and subcellular localization of the RNA species. Using in situ hybridization, we showed that PLMVd strands of both plus and minus polarities concentrate in the cells forming the palisade parenchyma. At the cellular level, PLMVd was found to accumulate predominantly in chloroplasts. Northern blot analyses demonstrated that PLMVd replicates via a symmetric mode involving the accumulation of both circular and linear monomeric strands of both polarities. No multimeric conformer was detected, indicating that both strands self-cleave efficiently via their hammerhead sequences. Dot blot hybridizations revealed that PLMVd strands of both polarities accumulate equally but that the relative concentrations vary by more than 50-fold between peach cultivars. Taken together these results establish two hallmarks for the classification of viroids. Group A viroids (e.g., PLMVd), which possess hammerhead structures, replicate in the chloroplasts via the symmetric mode. By contrast, group B viroids, which share a conserved central region, replicate in the nucleus via an asymmetric mechanism. This is an important difference between self-cleaving and non-self-cleaving viroids, and the implications for the evolutionary origin and replication are discussed.     

Chrysanthemum chlorotic mottle viroid RNA: dissection of the pathogenicity determinant and comparative fitness of symptomatic and non-symptomatic variants

Chrysanthemum chlorotic mottle viroid (CChMVd) is a small RNA (398-401nt) with hammerhead ribozymes in both polarity strands that mediate self-cleavage of the oligomeric RNA intermediates generated in a rolling-circle mechanism of replication. Within the in vivo branched RNA conformation of CChMVd, a tetraloop has been identified as a major determinant of pathogenicity. Here we present a detailed study of this tetraloop by site-directed mutagenesis, bioassay of the CChMV-cDNA clones and analysis of the resulting progenies. None of the changes introduced in the tetraloop, including its substitution by a triloop or a pentaloop, abolished infectivity. In contrast to observations for other RNAs, the thermodynamically stable GAAA tetraloop characteristic of non-symptomatic CChMVd-NS strains was not functionally interchangeable for other stable tetraloops of the UNCG family, suggesting that the sequence, rather than the structure, is the major factor governing conservation of this motif. In most cases, the changes introduced initially led to symptomless infections, which eventually evolved to be symptomatic concurrently with the prevalence in the progeny of the UUUC tetraloop characteristic of symptomatic CChMVd-S strains. Only in one case did the GAAA tetraloop emerge and eventually dominate the progeny in infected plants that were non-symptomatic. These results revealed two major fitness peaks in the tetraloop (UUUC and GAAA), whose adjacent stem was also under strong selection pressure. Co-inoculations with CChMVd-S and -NS variants showed that only when the latter was in a 100- or 1000-fold excess did the infected plants remain symptomless, confirming the higher biological fitness of the S variant and explaining the lack of symptom expression previously observed in cross-protection experiments.     

An extra nucleotide in the consensus catalytic core of a viroid hammerhead ribozyme: implications for the design of more efficient ribozymes.

Hammerhead ribozymes catalyze self-cleavage of oligomeric RNAs generated in replication of certain viroid and viroid-like RNAs. Previous studies have defined a catalytic core conserved in most natural hammerheads, but it is still unknown why some present deviations from the consensus. We have addressed this issue in chrysanthemum chlorotic mottle viroid (CChMVd), whose (+) hammerhead has an extra A (A10) between the conserved A9 and the quasi-conserved G10.1. Effects of insertions at this position on hammerhead kinetics have not hitherto been examined. A10 caused a moderate decrease of the trans-cleaving rate constant with respect to the CChMVd (+) hammerhead without this residue, whereas A10-->C and A10-->G substitutions had major detrimental effects, likely because they favor catalytically inactive foldings. By contrast, A10-->U substitution induced a 3-4-fold increase of the rate constant, providing an explanation for the extra U10 present in two natural hammerheads. Because A10 also occupies a singular and indispensable position in the global CChMVd conformation, as revealed by bioassays, these results show that some hammerheads deviate from the consensus due to the involvement of certain residues in critical function(s) other than self-cleavage. Incorporation of the extra U10 into a model hammerhead also caused a similar increase in the rate constant, providing data for a deeper understanding of the hammerhead structural requirements and for designing more efficient ribozymes.     

Additional roles of a peripheral loop-loop interaction in the Neurospora VS ribozyme

Many RNAs contain tertiary interactions that contribute to folding the RNA into its functional 3D structure. In the VS ribozyme, a tertiary loop-loop kissing interaction involving stem-loops I and V is also required to rearrange the secondary structure of stem-loop I such that nucleotides at the base of stem I, which contains the cleavage-ligation site, can adopt the conformation required for activity. In the current work, we have used mutants that constitutively adopt the catalytically permissive conformation to search for additional roles of the kissing interaction in vitro. Using mutations that disrupt or restore the kissing interaction, we find that the kissing interaction contributes ~1000-fold enhancement to the rates of cleavage and ligation. Large Mg(2+)-dependent effects on equilibrium were also observed: in the presence of the kissing interaction cleavage is favored >10-fold at micromolar concentrations of Mg(2+); whereas ligation is favored >10-fold at millimolar concentrations of Mg(2+). In the absence of the kissing interaction cleavage exceeds ligation at all concentrations of Mg(2+). These data provide evidence that the kissing interaction strongly affects the observed cleavage and ligation rate constants and the cleavage-ligation equilibrium of the ribozyme.     

Structure and stability of RNA/RNA kissing complex: with application to HIV dimerization initiation signal

We develop a statistical mechanical model to predict the structure and folding stability of the RNA/RNA kissing-loop complex. One of the key ingredients of the theory is the conformational entropy for the RNA/RNA kissing complex. We employ the recently developed virtual bond-based RNA folding model (Vfold model) to evaluate the entropy parameters for the different types of kissing loops. A benchmark test against experiments suggests that the entropy calculation is reliable. As an application of the model, we apply the model to investigate the structure and folding thermodynamics for the kissing complex of the HIV-1 dimerization initiation signal. With the physics-based energetic parameters, we compute the free energy landscape for the HIV-1 dimer. From the energy landscape, we identify two minimal free energy structures, which correspond to the kissing-loop dimer and the extended-duplex dimer, respectively. The results support the two-step dimerization process for the HIV-1 replication cycle. Furthermore, based on the Vfold model and energy minimization, the theory can predict the native structure as well as the local minima in the free energy landscape. The root-mean-square deviations (RMSDs) for the predicted kissing-loop dimer and extended-duplex dimer are ∼3.0 Å. The method developed here provides a new method to study the RNA/RNA kissing complex.     

Common structural features of different viroids: serial arrangement of double helical sections and internal loops.

The thermodynamic parameters of five different highly purified viroid "species" were determined by applying UV-absorption melting analysis and temperature jump methods. Their thermal denaturation proved to be a highly cooperative process with midpoint-temperatures (Tm) between 48.5 and 51 degrees C in 0.01 M sodium cacodylate, 1 mM EDTA, pH 6.8. The values of the apparent reaction enthalpies of the different viroid species range between 3,140 and 3,770 kJ/mol. Although the cooperativity is as high as found in homogeneous RNA double helices the Tm-value of viroid melting is more than 30 degrees C lower than in the homogeneous RNA. In order to explain this deviation, melting curves were simulated for different models of the secondary structure of viroids using literature values of the thermodynamic parameters of nucleic acids. Our calculations show that the following refinement of our earlier model is in complete accordance with the experimental data: In their native conformation viroids exist as an extended rodlike structure characterized by a series of double helical sections and internal loops. In the different viroid species 250-300 nucleotides out of total 350 nucleotides are needed to interprete the thermodynamic behaviour.     

Nucleotide sequence and secondary structure of apple scar skin viroid.

The complete nucleotide sequence of apple scar skin viroid(ASSV) has been established, and a probable secondary structure is proposed. A single-stranded circular ASSV RNA consists of 330 nucleotides and can assume the rodlike conformation with extensive base-pairing characteristic of all the known viroids. ASSV shows low sequence homologies with other viroids and lacks the central conserved region. These indicate that ASSV should be allocated to a separate viroid group. However, homologous sequences with potato spindle tuber viroid(PSTV) in ASSV occur in limited and scattered regions of both viroids. These homologous regions fall within the particular domains in the viroid domain model which has been previously proposed by Keese and Symons(Proc. Natl. Acad. Sci. USA. 82, 4582-4586, 1985).     

The tolerance to exchanges of the Watson Crick base pair in the hammerhead ribozyme core is determined by surrounding elements

Tertiary interacting elements are important features of functional RNA molecules, for example, in all small nucleolytic ribozymes. The recent crystal structure of a tertiary stabilized type I hammerhead ribozyme revealed a conventional Watson-Crick base pair in the catalytic core, formed between nucleotides C3 and G8. We show that any Watson-Crick base pair between these positions retains cleavage competence in two type III ribozymes. In the Arabidopsis thaliana sequence, only moderate differences in cleavage rates are observed for the different base pairs, while the peach latent mosaic viroid (PLMVd) ribozyme exhibits a preference for a pyrimidine at position 3 and a purine at position 8. To understand these differences, we created a series of chimeric ribozymes in which we swapped sequence elements that surround the catalytic core. The kinetic characterization of the resulting ribozymes revealed that the tertiary interacting loop sequences of the PLMVd ribozyme are sufficient to induce the preference for Y3-R8 base pairs in the A. thaliana hammerhead ribozyme. In contrast to this, only when the entire stem-loops I and II of the A. thaliana sequences are grafted on the PLMVd ribozyme is any Watson-Crick base pair similarly tolerated. The data provide evidence for a complex interplay of secondary and tertiary structure elements that lead, mediated by long-range effects, to an individual modulation of the local structure in the catalytic core of different hammerhead ribozymes.     

Small RNAs containing the pathogenic determinant of a chloroplast-replicating viroid guide the degradation of a host mRNA as predicted by RNA silencing

How viroids, tiny non-protein-coding RNAs (~250-400 nt), incite disease is unclear. One hypothesis is that viroid-derived small RNAs (vd-sRNAs; 21-24 nt) resulting from the host defensive response, via RNA silencing, may target for cleavage cell mRNAs and trigger a signal cascade, eventually leading to symptoms. Peach latent mosaic viroid (PLMVd), a chloroplast-replicating viroid, is particularly appropriate to tackle this question because it induces an albinism (peach calico, PC) strictly associated with variants containing a specific 12-14-nt hairpin insertion. By dissecting albino and green leaf sectors of Prunus persica (peach) seedlings inoculated with PLMVd natural and artificial variants, and cloning their progeny, we have established that the hairpin insertion sequence is involved in PC. Furthermore, using deep sequencing, semi-quantitative RT-PCR and RNA ligase-mediated rapid amplification of cDNA ends (RACE), we have determined that two PLMVd-sRNAs containing the PC-associated insertion (PC-sRNA8a and PC-sRNA8b) target for cleavage the mRNA encoding the chloroplastic heat-shock protein 90 (cHSP90), thus implicating RNA silencing in the modulation of host gene expression by a viroid. Chloroplast malformations previously reported in PC-expressing tissues are consistent with the downregulation of cHSP90, which participates in chloroplast biogenesis and plastid-to-nucleus signal transduction in Arabidopsis. Besides PC-sRNA8a and PC-sRNA8b, both deriving from the less-abundant PLMVd (-) strand, we have identified other PLMVd-sRNAs potentially targeting peach mRNAs. These results also suggest that sRNAs derived from other PLMVd regions may downregulate additional peach genes, ultimately resulting in other symptoms or in a more favorable host environment for viroid infection.     

Rapid formation of a solvent-inaccessible core in the Neurospora Varkud satellite ribozyme

We have used hydroxyl radicals generated by decomposition of peroxynitrous acid to study Mg(2+)-dependent structure and folding of the Varkud satellite (VS) ribozyme. Protection from radical cleavage shows the existence of a solvent-inaccessible core, which includes nucleotides near two three-helix junctions, the kissing interaction between stem-loops I and V and other nucleotides, most of which have also been implicated as important for folding or activity. Kinetic folding experiments showed that the ribozyme folds very quickly, with the observed protections completely formed within 2 s of addition of MgCl(2). In mutants that disrupt the kissing interaction or entirely remove stem-loop I, which contains the cleavage site, nucleotides in the three-helix junctions and a subset of those elsewhere remain protected. Unlike smaller ribozymes, the VS ribozyme retains a significant amount of structure in the absence of its substrate. Protections that depend on proper interaction between the substrate and the rest ribozyme map to a region previously proposed as the active site of the ribozyme and along both sides of helix II, identifying candidate sites of docking for the substrate helix.     

Structure–function analysis of the ribozymes of chrysanthemum chlorotic mottle viroid: a loop–loop interaction motif conserved in most natural hammerheads

Loop–loop tertiary interactions play a key role in the folding and catalytic activity of natural hammerhead ribozymes. Using a combination of NMR spectroscopy, site-directed mutagenesis and kinetic and infectivity analyses, we have examined the structure and function of loops 1 and 2 of the (+) and (–) hammerheads of chrysanthemum chlorotic mottle viroid RNA. In both hammerheads, loop 1 is a heptanucleotide hairpin loop containing an exposed U at its 5′ side and an extrahelical U at its 3′-side critical for the catalytic activity of the ribozyme in vitro and for viroid infectivity in vivo, whereas loop 2 has a key opened A at its 3′-side. These structural features promote a specific loop–loop interaction motif across the major groove. The essential features of this tertiary structure element, base pairing between the 5′ U of loop 1 and the 3′ A of loop 2, and interaction of the extrahelical pyrimidine of loop 1 with loop 2, are likely shared by a significant fraction of natural hammerheads.