A revised replication cycle for viroids: The role of longer than unit length RNA in viroid replication

Summary Longer than unit length plus and minus strand RNAs were detected in hop stunt viroid (HSV) infected cucumber leaf tissues by Northern blot hybridization analysis using strand-specific probes. To elucidate the role of these longer than unit length RNAs in the viroid replication cycle, we synthesized tandemly repeated plus and minus strand HSV RNAs in vitro from cloned HSV cDNA and assayed their infectivities. Two and four unit tandemly repeated plus strand RNAs were infectious, but one unit plus, and one, two and four unit minus strands were noninfectious. Taking these data into consideration, we propose a revised rolling circle model for viroid replication     

Tissue and intra-cellular distribution of coconut cadang cadang viroid and citrus exocortis viroid determined by in situ hybridization and confocal laser scanning and transmission electron microscopy

Confocal laser scanning microscopy and transmission electron microscopy (TEM) were used in conjunction with in situ hybridization techniques to compare and contrast the subnuclear (ultrastructural) and tissue (histological) localizations, respectively, of citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV). Both these viroids, which are members of the same taxonomic subgroup of viroids, were found in the vascular tissues as well as in the nuclei of mesophyll cells of infected host plants. At the subnuclear level, however, CEV was distributed across the entire nucleus, in contrast to CCCV which was mostly concentrated in the nucleolus with the remainder distributed throughout the nucleoplasm.     

Ribozymes: the hairpin and Varkud ribozymes are related.

The hairpin ribozyme of plant virusoids and the Varkud ribozyme from a retroplasmid of fungal mitochondria show notable similarities in sequence and secondary structure. Some more distant inter-relationships appear to exist between this pair, the viroid/virusoid hammerhead and the hepatitis delta ribozyme.     

Avocado sunblotch viroid: primary sequence and proposed secondary structure.

The sequence of the 247 nucleotide residues of the single strand circular RNA of avocado sunblotch viroid (ASBV) was determined using partial enzymic cleavage methods on overlapping viroid fragments obtained by partial ribonuclease digestion followed by 32p-labelling in vitro at their 5'-ends. ASBV is much smaller than potato spindle tuber viroid (PSTV; 359 residues) and chrysanthemum stunt viroid (CSV; 356 residues). A secondary structure model for ASBV is proposed and contains 67% of its residues base paired. In contrast to the extensive (69%) sequence homology of CSV with PSTV, only 18% of the ASBV sequence is homologous to PSTV and CSV. There are eight potential polypeptide translation products with chain lengths from 4 to 63 amino acid residues coded for by the plus (infectious) strand and four potential translation products (2 to 60 residues) coded for by the minus strand. An improved method is described for the synthesis of gamma-32p-ATP of high specific activity.     

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.     

In situ hybridization localizes avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang cadang viroid in the nucleus

Viroids, small single-stranded circular RNA molecules, are the smallest known infectious agents in Nature. The apparent inability of viroids to encode for proteins means that they must rely fully on host functions for their replication. The specific ultrastructural localization of viroids is fundamental to the determination of their replication strategies. In this paper the first in situ hybridization study to localize viroids within the cell at the electron microscope level is reported. Biotin-labelled RNA probes were used with subsequent detection by gold-labelled monoclonal anti-biotin antibodies to localize avocado sunblotch viroid and coconut cadang cadang viroid. Avocado sunblotch viroid was located in chloroplasts, mostly on the thylakoid membranes of cells from infected leaves of avocado (Persea americana). In contrast, coconut cadang cadang viroid was located in the nucleolus and nucleoplasm of cells of infected leaves of oil palm (Elaeis guineensis), with a higher concentration in the nucleolus. The results provide insight on the potential host RNA polymerases involved in the replication of these two viroids.     

Production and properties of monoclonal antibodies to Solanum nodiflorum mottle sobemovirus

Black raspberry necrosis virus (BRNV) reaches only very low concentrations in herbaceous plants and is difficult to maintain in culture. However, in a mixed culture with an unrelated virus, Solanum nodiflorum mottle (SNMV), in the genus Sobemovirus, the concentration of BRNV particles increases about 1000‐fold. In attempts to produce monoclonal antibodies (MAbs) to BRNV for diagnostic use, purified virus particles from the mixed virus culture were used as immunogen and the resultant antibodies screened against cultures of SNMV alone, BRNV+SNMV and healthy plant extracts. None of the virus‐specific MAbs obtained in this way was specific to BRNV but six were specific to SNMV. Although the original objective was not achieved, the SNMV MAbs were characterised and used to study serological properties of SNMV and other Sobemoviruses. Characterisation of the six SNMV MAbs showed that four were IgG3, one IgG1 and the other IgG2b. SNMV was detected by all six MAbs in ELISA, by five in Western blotting, by three in agarose gel double diffusion tests, but only one was suitable for trapping virus particles in immuno‐electron microscopy (IEM). In Western blotting using virus in sap extracts of Nicotiana clevelandii, each of the five MAbs detected a single major band of Mc. 31 000 in sap containing SNMV, and additional bands of lower mass attributed to degradation of coat protein. In various serological tests, no cross‐reactions were detected between SNMV and seven other viruses from the genus Sobemovirus. However, in IEM but not in Western blotting, significant cross‐reactions were observed between SNMV and Velvet tobacco mottle virus, another species from the genus Sobemovirus. The significance of these different findings is discussed.     

Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites

Virusoids are circular single-stranded RNAs dependent on plant viruses for replication and encapsidation. Virusoid replication appears to involve longer-than-unit-length plus and minus RNAs, indicating that unit-length plus RNA is generated by specific cleavage reactions. Here, we synthesize plus and minus partial-length RNAs of the 324-nucleotide virusoid from lucerne transient streak virus in vitro. Both RNAs self-cleave at a unique site in the presence of magnesium ions to give 5' hydroxyl and 2',3' cyclic phosphodiester termini. Conformations other than the native structures are necessary for cleavage. Similar secondary structures with considerable sequence homology are proposed for the active sites of these and other plant pathogenic RNAs. Our results are consistent with certain rolling-circle replication models.     

Studies on the transmission of velvet tobacco mottle virus by the mirid, Cyrtopeltis nicotianae

The minimum acquisition period of velvet tobacco mottle virus (VTMoV) by its mirid vector Cyrtopeltis nicotianae was about 1 min, with an increase in the rate of transmission (i.e. proportion of test plants infected) for acquisition periods up to 1000 min. Pre-acquisition starvation periods up to 18 h did not affect the rate of transmission. After an acquisition access period of 2 days, the minimum inoculation period was between 1 and 2 h and the rate of transmission increased with increasing inoculation time; when the acquisition access period was 1 h, or if vectors were fasted for 16 h after the 2 day acquisition, the rate of transmission was significantly lower. When mirids were transferred sequentially each day to a healthy plant after a 24 h acquisition feed, they transmitted intermittently for up to 10 days. Up to 50% of mirids transmitted after a moult and this was not due to the mirids probing the shed cuticles or exudates of infective insects. Mirids transmitted after a moult, following acquisition periods of 10, 100 or 1000 min. C. nicotianae transmitted solanum nodiflorum mottle virus (SNMV), sowbane mosaic virus (SoMV) and southern bean mosaic virus (SBMV), but not subterranean clover mottle virus (SCMoV), lucerne transient streak virus (LTSV), tobacco ringspot virus (TRSV), galinsoga mosaic virus (GMV), nor nicotiana velutina mosaic virus (NVMV). Tomato bushy stunt virus (TBSV) was transmitted to 1/58 test plants.     

2'' phosphomonoester, 3''-5'' phosphodiester bond at a unique site in a circular viral RNA.

Solanum nodiflorum mottle virus (SNMV) RNA2 is a single-stranded, covalently closed circular molecule. RNase T2 or nuclease P1 digests of this RNA contain a minor nucleotide of unusual chromatographic and electrophoretic mobility. This nucleotide is resistant to further digestion by T2 or P1 ribonucleases, or by alkali, but is sensitive to venom phosphodiesterase digestion. Alkaline phosphatase digestion yields a product which is RNase T2 and P1 sensitive. The products of these various digests show that the minor nucleotide is a ribonuclease-resistant dinucleotide carrying a 2' phosphomonoester group with the core structure C2'p3'p5'A. This dinucleotide is found in a unique RNase T1 product of SNMV RNA2, thus establishing a unique location in the sequence for the 2' phosphomonoester group at residue 49. Identical results have been obtained with a second related virus. The phosphomonoester group probably results from the RNA ligation event by which the molecules were circularised.     

Conformational Transitions in Viroids and Virusoids: Comparison of Results from Energy Minimization Algorithm and from Experimental Data

Abstract

Viroids are single-stranded circular RNA molecules of 240 to 400 nucleotides 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 replicate only as genomic part of a plant virus. Their structure and structural transitions have been investigated by thermodynamic, kinetic and hydrodynamic methods. The special features of the sequences of these RNAs, which are the basis for their secondary structures and structural flexibility, are investigated with theoretical methods.

A set of thermodynamic parameters for helix growth and loop formation is selected from the literature to calculate secondary structures and structural transitions of single-stranded RNAs. Appropriate modifications of the chosen parameter set are discussed.

For calculations we used either Tinoco-plots and the model of “cooperative helices” or the Zuker-program based on the exact algorithm of Nussinov et al, or both. Calculations were done for viroids and virusoids. As both are single-stranded, circular RNAs we had to modify the Zuker-program as described in the appendix.

Calculations are done for different viroids, i.e. potato spindle tuber, citrus exocortis, chrysanthemum stunt, coconut cadang-cadang, and avocado sunblotch, and for two virusoids, i.e. the circular RNAs of Solanum nodiflorum mottle virus, and velvet tobacco mottle virus. 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 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 statistical sequences. The comparison of viroids, virusoids, and circular RNA or random sequences confirms the uniqueness of viroid structure.     

Complementary RNA Species Isolated from Vesicular Stomatitis (HR Strain) Defective Virions

The wild-type strain of vesicular stomatitis virus (VSV) contains in its complete virion (VSV-1, B particles) a minus strand RNA. The principle defective particle of the wild-type strain (VSV-111, T particles) contains a shorter minus strand, homologous to part of the VSV-1 genome. Neither virion contains any detectable complementary (plus) strand RNA. In contrast, a preparation of a heat-resistant (HR) strain of VSV containing defective virions was found to contain both plus (21%) and minus strand RNA, present in several distinct size classes. It was found that the RNA in the HR virion preparation was at least 94% single-stranded and principally (96%) in ribonucleoprotein complexes. On extraction the plus and minus strand RNA species partially annealed to give a population of double- and multistranded RNA species. A small amount of RNA polymerase activity was associated with the HR defective virus preparation.     

Stiffness of viroids and viroid-like RNA in solution.

The sedimentation coefficients of the potato spindle tuber viroid, four viroid-like RNAs from cadang-cadang-disease, circular RNA from velvet tobacco mottle virus, circular RNA from Solanum nodiflorum mottle virus and double stranded RNA5 from cucumber mosaic virus were measured in the analytical ultracentrifuge. The numbers of nucleotides of the RNA species varied between 246 and 670. The hydrodynamic models of rigid rods and flexible cylinders were applied for the interpretation of the sedimentation coefficients. Double-stranded RNA5 from cucumber mosaic virus with 335 basepairs fits the model of a rigid rod with an hydrated diameter of 29 A. Potato spindle tuber viroid and the four viroid-like RNA species of cadang-cadang-disease form a homologous series of flexible cylinders with a Kuhn's statistical length lambda-1 of 600 A. The circular RNA from the two viruses mentioned above are more flexibel than the viroids and viroid-like RNAs. The hydrodynamic interpretation is in accordance with thermodynamic data and secondary structure models. In two of the RNAs from cadang-cadang, cruciform structures would also be possible on the basis of the nucleotide sequence. The hydrodynamic data, however, favour clearly the extended structure over the cruciform.     

Prominent polypurine and polypyrimidine tracts in plant viroids and in RNA of the human hepatitis delta agent.

To seek patterns of nucleotide usage in the three types of circular subviral RNA pathogens, trimer frequencies and nearest-neighbor biases were studied in 12 plant viroid sequences; five sequences of circular plant viral satellite RNAs; and the sequence of RNA from the human hepatitis delta agent. The viroids and RNA of the delta agent contain tracts of polypurines and polypyrimidines which make up substantial portions of their genomes. Such tracts are not common in the virusoids or in the satellite RNA of tobacco ringspot virus. Viroids, the delta hepatitis agent, and the circular satellite RNAs of certain plant viruses have several features in common: all have circular genomic RNA and replicate through an RNA to RNA rolling circle replication cycle. However, virusoids and related satellite RNAs are directly or indirectly dependent on their helper viruses for replication, while the delta agent and viroids are not. The difference in the pattern of nucleotide usage between the plant viral satellite RNAs on the one hand, and viroids and delta RNA on the other, may relate to this difference in replication strategy.     

Replication of in vitro constructed viroid mutants: location of the pathogenicity-modulating domain of citrus exocortis viroid

Sequence variants from field isolates of citrus exocortis viroid (CEV) that cause either mild or severe symptoms on tomato plants have previously been classified into two groups, A and B. These groups differ primarily in two domains, P_L and P_R, of the proposed native structure. Infectivity studies with full-length cDNA clones of variants from each class have now directly confirmed the original correlation between Class A sequences and the severe phenotype and between Class B sequences and the mild phenotype. Direct evidence for this correlation could only be obtained by using individual sequence variants since field isolates of CEV have been shown to contain a mixture of RNA species. The construction and infectivity of chimaeric cDNA clones derived from mild and severe sequence variants of CEV has demonstrated that novel, infectious viroid molecules can be generated in vitro, and that P_L is the pathogenicity-modulating domain. The role of the P_R domain is not known but infectivity experiments with one chimaeric cDNA clone suggest that it may influence the efficiency of the infection or replication process of the viroid in the plant.