A naked plant-specific RNA ten-fold smaller than the smallest known viral RNA: the viroid

Viroids are subviral plant pathogens at the frontier of life. They are solely composed by a single-stranded circular RNA of 246-401 nt with a compact secondary structure. Viroids replicate autonomously when inoculated into their host plants and incite, in most of them, economically important diseases. In contrast to viruses, viroids do not code for any protein and depend on host enzymes for their replication, which in some viroids occurs in the nucleus and in others in the chloroplast, through a rolling-circle mechanism with three catalytic steps. Quite remarkably, however, one of the steps, cleavage of the oligomeric head-to-tail replicative intermediates to unit-length strands, is mediated in certain viroids by hammerhead ribozymes that can be formed by their strands of both polarities. Viroids induce disease by direct interaction with host factors, the nature of which is presently unknown. Some properties of viroids, particularly the presence of ribozymes, suggest that they might have appeared very early in evolution and could represent 'living fossils' of the precellular RNA world that presumably preceded our current world based on DNA and proteins.     

Viroids

Viroids are small, circular RNA pathogens, which infect several crop plants and can cause diseases of economic importance. They do not code for proteins but they contain a number of RNA structural elements, which interact with factors of the host. The resulting set of sophisticated and specific interactions enables them to use the host machinery for their replication and transport, circumvent its defence reactions and alter its gene expression. Although found in plants, viroids have a distant relative in the animal world: hepatitis delta virus (HDV), a satellite virus of hepatitis B virus, which has a similar rod-like structure and replicates in the nucleus of infected cells. Viroids have also a cellular relative: the retroviroids, found in some plants as independent (non-infectious) RNA replicons with a DNA copy. In this review, we summarize recent progress in understanding viroid biology. We discuss the possible role of recently identified viroid-binding host proteins as well as the recent data on the interaction of viroids with one part of the host's defence machinery, the RNA-mediated gene silencing and how this might be connected to viroid replication and pathogenicity.     

A long-distance translocatable phloem protein from cucumber forms a ribonucleoprotein complex in vivo with Hop stunt viroid RNA

Viroids are highly structured plant pathogenic RNAs that do not code for any protein, and thus, their long-distance movement within the plant must be mediated by direct interaction with cellular factors, the nature of which is presently unknown. In addition to this type of RNAs, recent evidence indicates that endogenous RNAs move through the phloem acting as macromolecular signals involved in plant defense and development. The form in which these RNA molecules are transported to distal parts of the plant is unclear. Viroids can be a good model system to try to identify translocatable proteins that could assist the vascular movement of RNA molecules. Here, we demonstrate by use of immunoprecipitation experiments, that the phloem protein 2 from cucumber (CsPP2) is able to interact in vivo with a viroid RNA. Intergeneric graft assays revealed that both the CsPP2 and the Hop stunt viroid RNA were translocated to the scion. The translocated viroid is symptomatic in the nonhost scion, indicating that the translocated RNA is functional. The CsPP2 gene was cloned and sequenced. The analysis of its primary structure revealed the existence of a potential double-spaced-RNA-binding motif, previously identified in a set of proteins that bind to highly structured RNAs, which could explain its RNA-binding properties. The possible involvement of this phloem protein in assisting the long-distance movement of the viroid RNA within the plant is discussed.     

Subviral pathogens of plants: viroids and viroidlike satellite RNAs.

Contrary to earlier beliefs, viruses are not the smallest causative agents of infectious diseases. Single-stranded RNAs as small as 246 nucleotides exist in certain higher plants and cause more than a dozen crop diseases. These RNAs have been termed viroids. Despite their extremely limited information content, viroids replicate autonomously in susceptible cells--that is, they do not require helper functions from simultaneously replicating conventional viruses. Viroids are covalently closed circular molecules with a characteristic rodlike secondary structure in which short helical regions are interrupted by internal and bulge loops. Viroids are not translated; they are replicated by a host enzyme (or enzymes) (probably RNA polymerase II) via oligomeric RNA intermediates by a rolling circle mechanism. Viroidlike satellite RNAs resemble viroids in size and molecular structure, but are found within the capsids of specific helper viruses on which they depend for their own replication. These RNAs are of great interest to molecular biology for at least two reasons: 1) they are the smallest and simplest replicating molecules known, and 2) they may represent living fossils of precellular evolution in a hypothetical RNA world.     

Viroids: petite RNA pathogens with distinguished talents

Viroids are small, circular, single-stranded RNA molecules that cause several infectious plant diseases. Viroids do not encode any pathogen-specific peptides but nonetheless, the subviral pathogens replicate autonomously and spread in the plant by recruiting host proteins via functional motifs encoded in their RNA genome. During the past couple of years, considerable progress has been made towards comprehending how viroids interact with their hosts. Here, we summarize recent findings on the structure-function relationships of viroids, their strategies and mechanisms of replication and trafficking, and the identification and characterization of interacting host proteins. We also describe the impact of the RNA silencing machinery of plants on viroid RNAs and how this has started to influence our models of viroid replication and pathogenicity.     

Viroids: the minimal non-coding RNAs with autonomous replication

Viroids are small (246-401 nucleotides), non-coding, circular RNAs able to replicate autonomously in certain plants. Viroids are classified into the families Pospiviroidae and Avsunviroidae, whose members replicate in the nucleus and chloroplast, respectively. Replication occurs by an RNA-based rolling-circle mechanism in three steps: (1). synthesis of longer-than-unit strands catalyzed by host DNA-dependent RNA polymerases forced to transcribe RNA templates, (2). processing to unit-length, which in family Avsunviroidae is mediated by hammerhead ribozymes, and (3). circularization either through an RNA ligase or autocatalytically. Disease induction might result from the accumulation of viroid-specific small interfering RNAs that, via RNA silencing, could interfere with normal developmental pathways.     

Search for Viroids and Other Small RNAs Associated with Forest Damage

We examined whether viroids may be involved as the causative agents of forest damages which have been observed in Germany during the last decade. A crude RNA extract was prepared from healthy and diseased copper beech, spruce, and pine trees and was analysed by two-dimensional gel electrophoresis which resolves RNA bands in the molecular weight range from 25,000 to 10⁶. Viroids are known to consist of single stranded circular RNA and are clearly differentiable in the electrophoresis used here from cellular RNA. Viroids could not be found in any of the samples investigated. In all samples from diseased trees, however, other additional RNA bands were discovered which were more similar to cellular RNA than to viroids in their electrophoretic properties. It is uncertain whether these diseased-associated RNAs are due to an infection or originate from a modified nucleic acid metabolism.     

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.     

The Subviral RNA Database: a toolbox for viroids, the hepatitis delta virus and satellite RNAs research

Background  

Viroids, satellite RNAs, satellites viruses and the human hepatitis delta virus form the 'brotherhood' of the smallest known infectious RNA agents, known as the subviral RNAs. For most of these species, it is generally accepted that characteristics such as cell movement, replication, host specificity and pathogenicity are encoded in their RNA sequences and their resulting RNA structures. Although many sequences are indexed in publicly available databases, these sequence annotation databases do not provide the advanced searches and data manipulation capability for identifying and characterizing subviral RNA motifs.     

Some mathematical refinements concerning error minimization in the genetic code

The genetic code is known to have a high level of error robustness and has been shown to be very error robust compared to randomly selected codes, but to be significantly less error robust than a certain code found by a heuristic algorithm. We formulate this optimization problem as a Quadratic Assignment Problem and use this to formally verify that the code found by the heuristic algorithm is the global optimum. We also argue that it is strongly misleading to compare the genetic code only with codes sampled from the fixed block model, because the real code space is orders of magnitude larger. We thus enlarge the space from which random codes can be sampled from approximately 2.433 × 10(18) codes to approximately 5.908 × 10(45) codes. We do this by leaving the fixed block model, and using the wobble rules to formulate the characteristics acceptable for a genetic code. By relaxing more constraints, three larger spaces are also constructed. Using a modified error function, the genetic code is found to be more error robust compared to a background of randomly generated codes with increasing space size. We point out that these results do not necessarily imply that the code was optimized during evolution for error minimization, but that other mechanisms could be the reason for this error robustness.     

Early fixation of an optimal genetic code

The evolutionary forces that produced the canonical genetic code before the last universal ancestor remain obscure. One hypothesis is that the arrangement of amino acid/codon assignments results from selection to minimize the effects of errors (e.g., mistranslation and mutation) on resulting proteins. If amino acid similarity is measured as polarity, the canonical code does indeed outperform most theoretical alternatives. However, this finding does not hold for other amino acid properties, ignores plausible restrictions on possible code structure, and does not address the naturally occurring nonstandard genetic codes. Finally, other analyses have shown that significantly better code structures are possible. Here, we show that if theoretically possible code structures are limited to reflect plausible biological constraints, and amino acid similarity is quantified using empirical data of substitution frequencies, the canonical code is at or very close to a global optimum for error minimization across plausible parameter space. This result is robust to variation in the methods and assumptions of the analysis. Although significantly better codes do exist under some assumptions, they are extremely rare and thus consistent with reports of an adaptive code: previous analyses which suggest otherwise derive from a misleading metric. However, all extant, naturally occurring, secondarily derived, nonstandard genetic codes do appear less adaptive. The arrangement of amino acid assignments to the codons of the standard genetic code appears to be a direct product of natural selection for a system that minimizes the phenotypic impact of genetic error. Potential criticisms of previous analyses appear to be without substance. That known variants of the standard genetic code appear less adaptive suggests that different evolutionary factors predominated before and after fixation of the canonical code. While the evidence for an adaptive code is clear, the process by which the code achieved this optimization requires further attention.     

Dynamics and interactions of viroids

Viroids are single stranded circular RNA molecules of 120,000 daltons which are pathogens of certain higher plants and replicate autonomously in the host cell. Virusoids are similar to viroids in respect to size and circularity but do replicate only as a part of a larger plant virus. The structure and structural transitions have been investigated by thermodynamic, kinetic and hydrodynamic methods and have been compared to results from calculations of the most favorable native structures and the denaturation process. The algorithm of Zuker et al. was modified for the application to circular nucleic acids. For viroids the calculations confirm our earlier theoretical and experimental results about the extended native structure and the highly cooperative transition into a branched structure. Virusoids, although described in the literature as viroid-like, show less base pairing, branching in the native secondary structure, and only low cooperativity during denaturation. They resemble more closely the properties of random sequences with length, G:C content, and circularity as in viroids but sequences generated by a computer. The comparison of viroids, virusoids and circular RNA of random sequences underlines the uniqueness of viroid structure. The interactions of viroids with dye and oligonucleotide-ligands and with RNA-polymerase II from wheat germ, which enzyme replicates viroids in vitro, has been studied in order to correlate viroid structure and its ability for specific interactions. Specificity of the interactions may be interpreted on the basis of the neighbourhood of double stranded and single stranded regions. In the host cell viroids are localized in the cell nucleus; they may be detected as free nucleic acids and in high molecular weight complexes together with other RNA and proteins.     

Discovering viroids--a personal perspective

During 1970 and 1971, I discovered that a devastating disease of potato plants is not caused by a virus, as had been assumed, but by a new type of subviral pathogen, the viroid. Viroids are so small--one fiftieth of the size of the smallest viruses--that many scientists initially doubted their existence. We now know that viroids cause many damaging diseases of crop plants. Fortunately, new methods that are based on the unique properties of viroids now promise effective control.