Cellular Microbiology (2008) doi:10.1111/j.1462-5822.2008.01231.x Microreview Viroids Efthimia Mina Tsagris,1,2* Introduction Ángel Emilio Martínez de Alba,1† Studies on viroids have led to the discovery of some of Mariyana Gozmanova1,2‡ and Kriton Kalantidis1,2 the most interesting principles of the biology of RNA: the 1Institute of Molecular Biology and Biotechnology, fact that a non-coding, non-translatable RNA can cause a Foundation for Research and Technology, disease (Diener, 1971), the extraordinary small size of their PO Box 1385, 71110 Heraklion, Greece. genome (Gross et al., 1978) and their circularity (Sänger 2Department of Biology, University of Crete, et al., 1976), which enables them to circumvent the pro- PO Box 2208, 71409 Heraklion, Greece. blems of linear genome replication such as the accurate replication of linear ends (Diener, 1989). One of the first Summary self-cleaving structures, the hammerhead ribozyme, was found in a satellite RNAvirus and a viroid RNA(Prody et al., Viroids are small, circular RNA pathogens, which 1986; Forster and Symons, 1987; Forster et al., 1987). infect several crop plants and can cause diseases Prior to the molecular characterization of the hepatitis delta of economic importance. They do not code for pro- virus (HDV), a circular (‘viroid-like’) RNA infecting human teins but they contain a number of RNA structural liver cells and associated with hepatitis B virus (Lai, 2005; elements, which interact with factors of the host. Taylor, 2006) viroids remained as an interesting, but The resulting set of sophisticated and specific inter- exotic example of a plant pathogen, investigated by some actions enables them to use the host machinery researchers in the field of plant pathology and molecular for their replication and transport, circumvent its plant virology, together with a group of biophysicists defence reactions and alter its gene expression. studying RNA structure. They recognized in this relatively Although found in plants, viroids have a distant abundant, natural RNA a perfect object to study RNA relative in the animal world: hepatitis delta virus structure transitions. Viroids have been the basis on which (HDV), a satellite virus of hepatitis B virus, which new experimental and computational methods have been has a similar rod-like structure and replicates in developed (Riesner, 1991; Steger and Riesner, 2003). the nucleus of infected cells. Viroids have also a A recent landmark of scientific breakthrough originating cellular relative: the retroviroids, found in some from viroid research is the discovery of RNA-mediated de plants as independent (non-infectious) RNA repli- novo DNA methylation, which was first described in trans- cons with a DNA copy. In this review, we summarize genic plants carrying copies of the viroid cDNA(Wasseneg- recent progress in understanding viroid biology. ger et al., 1994). Although this was an ‘artificial’ transgenic We discuss the possible role of recently identified system, it was quickly recognized that RNA-mediated DNA viroid-binding host proteins as well as the recent methylation is a mechanism that is a part of a whole battery data on the interaction of viroids with one part of the of responses that plants have towards environmental host’s defence machinery, the RNA-mediated gene changes and developmental programmes (Wassenegger, silencing and how this might be connected to viroid 2005; Henderson and Jacobsen, 2007). replication and pathogenicity. Several reviews have been published recently on viroids, showing the increasing scientific interest in these molecules as a model system and plant pathogen. In this review, we will discuss some of the most recent articles on Received 2 April, 2008; revised 20 August, 2008; accepted 25 August, viroids, and present some possible models concerning 2008. *For correspondence. E-mail [email protected]; Tel. their replication, biogenesis and evolution. (+30) 2810 394367; Fax (+30) 2810 394404. Present addresses: †Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia, Consejo Superior de Investigaciones Cientí- Basic properties and mode of replication of viroids ficas, Valencia, Spain; ‡University of Plovdiv, Department of Plant Physiology and Molecular Biology, 24, Tsar Assen St., 4000 Plovdiv Viroids have a size of ca 250–400 nucleotides. They in- Bulgaria. fect several crop plants, causing symptoms of differential © 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd 2 E. M. Tsagris, Á. E. Martínez de Alba, M. Gozmanova and K. Kalantidis severity, which range from mild effects such as hardly plant organ, the phloem and its surrounding cells visible growth reduction, up to deformation, necrosis (Fig. 1B and C). For cell-to-cell and long-distance trans- or chlorosis and severe stunting (Singh et al., 2003). port, viroids use the supracellular structure of vascular Some viroid strains do not cause symptoms at all and plants, where groups of cells are connected via special seem to behave as simple RNA replicons rather than channels, called plasmodesmata. Therefore, like plant pathogens. However, symptoms depend very much viruses but unlike animal viruses, viroid RNA does not on environmental conditions and may change during have to cross the plasma membrane for long-distance infection as has also been found with plant viruses transport. It does, however, cross the borders of the (Semancik, 2003). nucleus via the nuclear pore or the chloroplast mem- Viroids do not have coding capacity and so far no branes. The precise mechanism by which this transfer viroid-coded protein has been detected in infected plants. is executed is not known. Viroids are not encapsidated. They replicate in the plant The entire replication and spread of viroids depends hosts by RNA–RNA transcription, in a ‘rolling-circle’ mode on host factors. DNA-dependent RNA polymerase II (Branch and Robertson, 1984). During replication, oligo- has been shown to replicate nuclear localized viroids meric linear RNAs of plus polarity are formed, which are (reviewed in Tabler and Tsagris, 2004), while it has been cleaved to monomers and ligated into circles (Fig. 1A). suggested that the nuclear-encoded, but chloroplast- Oligomeric forms of (-) polarity are also present as repli- localized DNA-dependent RNA polymerase (NEP) repli- cative intermediates. Circular molecules of minus polarity cates the chloroplastic viroids (Flores et al., 2004). have been detected in plants only in some members Viroids and HDV seem to be able to delude specific of the family Avsunviroidae, which bear self-cleaving DNA-dependent RNA polymerases of their host cells, ribozyme structures in their RNA genomes. Viroid local- offering genomic and antigenomic RNAs as a compe- ization is either nuclear (family Pospiviroidae) or chloro- titive template to the normal DNA templates of these plastic (family Avsunviroidae), where they replicate with enzymes. Or, alternatively, they just ‘remind’ to these the aid of host-encoded DNA-dependent RNA poly- enzymes their ‘RNA World’ attributes. In order to be able merases. Viroids can therefore be considered as para- to compete with the normal DNA templates, these RNA sites of the transcriptional machinery of the organelles replicons must have a competitive advantage: either a (nucleus or chloroplast), in contrast to most plant RNA high-affinity binding site, or the independence of acces- viruses, which replicate in the cytoplasm and can be sory trans-acting factors for recognition of the primitive therefore regarded as parasites of the translational ‘promoter’ by the DNA-dependent RNA polymerase, or machinery of the cell. both. In some plants, non-infectious circular RNA transcripts The viroids Potato spindle tuber viroid (PSTVd) and of similar size to viroids have been detected, which Citrus exocortis viroid (CEVd) are both replicated in contain hammerhead ribozyme structures (Darós and the nucleus, most probably by the same enzyme. PSTVd Flores, 1995; Hegedus et al., 2001). These transcripts has been shown to be transcribed in vitro and in vivo exist in the genome of their hosts as extra chromosomal by DNA-dependent RNA polymerase II (Mühlbach and or integrated concatameric DNA copies; if they replicate, Sänger, 1979; Rackwitz et al., 1981) and CEVd exists in they do so via oligomeric forms of plus and minus polarity. a complex with at least the large subunit of the DNA- As they are usually found in plants associated with a dependent RNA polymerase II (Warrilow and Symons, pararetrovirus they may use the machinery of the virus to 1999). Putative initiation sites for PSTVd have been copy and integrate their cDNA in the host’s genome. They determined, using different methods with varying results. have been named ‘retroviroids’ (Darós and Flores, 1995). Earlier work has identified nucleotide G168, localized One can speculate on whether viroids evolved from ret- at the right terminal part (Tabler and Tsagris, 1990), as roviroids, or vice versa, and what the intermediate steps transcription initiation site but nucleotides U359, C1 and have been. others (Kolonko et al., 2006) have also been identified Viroids infect the epidermis of their hosts after as potential transcription initiation sites. The methods mechanical damage of the plant cell wall. Unlike most used did not, however, allow the identification of a other plant viruses, they do not have important natural genuine 5′-triphosphate transcript. vectors from the animal kingdom (Singh et al., 2003). For Avocado sunblotch viroid (ASBVd), a viroid RNA After entry to the first cell layers of the leaves and initial pathogen replicating in the chloroplast, it has been pro- replication, they are transported to neighbouring cells, posed that a nuclear-encoded DNA-dependent RNA poly- reaching the vascular tissues, from which they travel to merase (NEP) is involved in its replication (Navarro et al., newly emerging young leaves or other sink tissues like 2000). NEP is a nuclear-encoded enzyme, which is roots, together with photoassimilates (Ding et al., 2005). transported to the chloroplast and functions there during Infected plants contain viroid RNA in the conductive chloroplast development.