Citrus viroid V: occurrence, host range, diagnosis, and identification of new variants.

The recently described Citrus viroid V (CVd-V) has been proposed as a new species of the genus Apscaviroid within the family Pospiviroidae. Analysis of 64 samples from different citrus-growing areas has shown that CVd-V is present in the United States, Spain, Nepal, and the Sultanate of Oman. CVd-V found in six sweet orange sources from the Sultanate of Oman was identical to the reference CVd-V variant, whereas three new variants with sequence identities of 98.6% (CVd-VCA), 97.3% (CVd-VST), and 94.9% (CVd-VNE) were identified in sources from California, Spain, and Nepal, respectively. These results suggest that this viroid has not emerged recently and that it is relatively widespread. Transmission assays to sweet orange, mandarin, and mandarin hybrids, clementine, satsuma, lemon, sour orange, Tahiti lime, Palestine sweet lime, calamondin, bergamot, and kumquat have shown that all these citrus species and citrus relatives are hosts for CVd-V. Several indexing approaches, including slot blot, northern blot hybridization, and reverse transcription-polymerase chain reaction, have been evaluated for detecting CVd-V, either using Etrog citron as an amplification host or directly from commercial species and cultivars.

A set of novel RNAs transcribed from the chloroplast genome accumulates in date palm leaflets affected by brittle leaf disease.

Brittle leaf disease or maladie des feuilles cassantes (MFC) is a lethal disorder of date palms that has assumed epidemic proportions in the oases of southern Tunisia. After a prolonged period during which palms are declining, the disease ends with the death of the palms. Whereas no pathogen could ever be associated with the disease, leaflets of affected palms have been previously shown to be deficient in manganese. Analysis of RNA preparations from leaflets of MFC-affected palms revealed the presence of a set of novel RNAs (MFC-RNAs) of sense and antisense polarities, which are homologous to various regions of the date palm chloroplast genome, such as the regions containing genes rrn5S-trnR(ACG) and trnM(CAU)-atpE. In the RNA preparations obtained from leaflets of affected palms, some of these RNAs are present as double-stranded species (MFC-dsRNAs), as witnessed by results from cellulose chromatography, end labeling, RNase digestion, and northern hybridization with strand specific probes. These MFC-RNAs represent a novel type of host-derived RNAs, and their presence in MFC-affected date palms is of diagnostic value.

Citrus viroid V: molecular characterization and synergistic interactions with other members of the genus Apscaviroid.

Studies on Atalantia citroides, a citrus relative, revealed the existence of a viroid not described previously. The new viroid has a GC-rich genome of 293-294 nucleotides and contains the central conserved region characteristic of members of the genus Apscaviroid, and the terminal conserved region present in this and other genera of the family Pospiviroidae. The secondary structure of minimum free energy predicted for the new viroid is a rod-like conformation with 68.7% paired nucleotides and showing sequence identities with other viroids always lower than 90%, the conventional limit that separates different species within a given genus. Infectivity assays showed that the new viroid induces mild but characteristic symptoms on the indicator Etrog citron. Co-inoculation of CVd-V with either Citrus bent leaf viroid or Citrus viroid III, two other members of the genus Apscaviroid infecting citrus, disclosed synergistic interactions manifested in enhanced leaf symptoms and very pronounced dwarfing. Viroid titers, however, remained unaltered in co-infected plants. Possible mechanisms underlying the observed synergistic effects are discussed. According to its molecular and biological properties and its unusual ability to replicate in A. citroides, the new viroid, tentatively named Citrus viroid V (CVd-V), should be considered a new species of the genus Apscaviroid.

Existence in vivo of the loop E motif in potato spindle tuber viroid RNA.

In vitro experiments have previously identified in potato spindle tuber viroid (PSTVd), the type member of the nuclear viroids, an element of local tertiary structure termed loop E. Here, by direct UV irradiation of PSTVd-infected tomato tissue and subsequent RNA analysis by denaturing polyacrylamide gel electrophoresis, northern blot hybridization and primer extension, we report that PSTVd (+) RNA also forms the loop E in vivo. These results provide strong support for the physiological relevance of this structural motif, which is involved in a wide range of functions including replication, host specificity and pathogenesis.

A real-time RT-PCR assay for quantifying the fitness of tobacco etch virus in competition experiments.

Relative fitness determination has become a standard tool in experimental virus evolution studies. In this type of studies, the tested strain is mixed with a reference strain, which differs in an easy-to-score and genetically stable marker, and allowed to compete for a limited common pool of resources during a given number of generations. In this report, a TaqMan real-time PCR methodology is proposed for quantifying the relative fitness of tobacco etch potyvirus strains (TEV) in in planta mixed infections with a reference TEV strain. Two different forward primers along with a common reverse one are used into separated reactions mixes from the same RNA preparation. The reference strain, named TEV-PC1, was genetically engineered to carry a neutral marker in a highly conserved region of the RNA polymerase NIb gene. This marker allows tracking the frequency of both competitors during competition experiments by real-time quantitative PCR using specific primers. Both the reproducibility and sensitivity of the method have been explored. Reproducibility was assessed by running multiple competition experiments for the same genotype. Sensitivity was assessed by comparing the results of competition experiments against TEV-PC1 of 24 single-nucleotide substitutions mutants.

Diagnosis of "Maladie des feuilles cassantes" or Brittle leaf disease of date palms by detection of associated chloroplast encoded double stranded RNAs.

The "Maladie des feuilles cassantes" (MFC) or "Brittle leaf disease" of date palms is associated with the accumulation of two populations of small, chloroplast-encoded RNAs. A plasmid vector containing a cDNA with partial sequences of both of these RNA populations was used to synthesize a DIG-labeled bifunctional probe by PCR. The probe has been tested to detect, by molecular hybridization, MFC-associated RNAs from dsRNA-enriched palm leaflet preparations. Leaflet samples from MFC-affected date palm trees consistently gave a positive hybridization signal regardless of the date palm cultivar, severity of symptoms, or geographical location, whereas samples from date palm trees affected by other biotic and abiotic stresses tested negative. The assay is specific for MFC and can be used for early diagnostic purposes.

Close structural relationship between two hammerhead viroid-like RNAs associated with cherry chlorotic rusty spot disease.

Analysis of the population of cherry small circular RNAs (cscRNAs) from trees affected by cherry chlorotic rusty spot (CCRS) showed two groups of variants with similar sequence but differing in size (394-415 and 372-377 nt for cscRNA1 and cscRNA2, respectively) because of the presence or absence of a 27-nt fragment folding into a hairpin in their predicted quasi-rod-like secondary structures. These structures were preserved by co-variations and compensatory mutations, as well as by additional complex rearrangements. The variability also preserved the central conserved core and the stability of the helices of the plus and minus hammerhead ribozymes, supporting their role in replication of cscRNAs. The smaller variants most likely derive from the larger through recombination events. Possible functional relationships between cscRNAs and certain mycoviral-like double-stranded RNAs, also associated with CCRS, are discussed.

Identification in eggplant of a variant of citrus exocortis viroid (CEVd) with a 96 nucleotide duplication in the right terminal region of the rod-like secondary structure.

Analysis of eggplants, kept for years under greenhouse conditions after having been mechanically inoculated with nucleic acid preparations from field-grown eggplants containing two viroids, Eggplant latent viroid (ELVd) and Citrus exocortis viroid (CEVd), revealed the presence of an additional larger viroid RNA. Molecular characterization of this RNA showed that it is a stable 467-nt variant of CEVd with a 96-nt duplication of the right terminal region (CEVd-D96) that preserves the rod-like secondary structure. The coexistence in eggplant of CEVd (371nt) and CEVd-D96, and the fact that they have an almost identical sequence, strongly suggests the emergence of the latter from the former through an internal recombination mediated by a jumping RNA polymerase with low processivity. CEVd-D96 from eggplant is similar to the CEVd-D92 variant characterized previously in a hybrid tomato, suggesting that certain hosts may play a critical role in selecting and replicating this class of enlarged variants.

Eggplant latent viroid, the candidate type species for a new genus within the family Avsunviroidae (hammerhead viroids).

Viroids, small circular RNAs that replicate independently and in most cases incite diseases in plants, are classified into the families Pospiviroidae, composed of species with a central conserved region (CCR) and without hammerhead ribozymes, and Avsunviroidae, composed of three members lacking CCR but able to self-cleave in both polarity strands through hammerhead ribozymes. Here we report the biological and molecular properties of Eggplant latent viroid (ELVd). Purified circular ELVd induces symptomless infections when inoculated into eggplant seedlings. ELVd can be transmitted horizontally and through seed. Sequencing 10 complete cDNA clones showed that ELVd is a circular RNA of 332 to 335 nucleotides with high variability. This RNA can adopt a quasi-rod-like secondary structure of minimal free energy and alternative foldings that permit formation of stable hammerhead structures in plus and minus strands. The ribozymes are active in vitro and, most likely, in vivo. Considering the ELVd properties to be intermediate between those of the two genera of family Avsunviroidae, we propose ELVd as the type species of a third genus with the name ELAVIROID:

The DNA of a plant retroviroid-like element is fused to different sites in the genome of a plant pararetrovirus and shows multiple forms with sequence deletions.

Carnation small viroid-like RNA (CarSV RNA) and its homologous DNA are the two forms of a unique plant retroviroid-like system. CarSV RNA is a 275-nucleotide noninfectious viroid-like RNA, present in certain carnation plants, which can adopt hammerhead structures in both polarity strands and self-cleave accordingly. CarSV DNA is organized as a series of head-to-tail multimers forming part of extrachromosomal elements in which CarSV DNA sequences are fused to sequences of carnation etched ring virus (CERV), a plant pararetrovirus. Analysis of more than 30 CarSV-CERV DNA junctions showed that distinct regions of the viral genome seem able to interact with CarSV DNA. All these junctions were short nucleotide stretches common to both CarSV and CERV DNAs. This suggests a polymerase-driven mechanism for their origin involving an enzyme with low processivity, most likely the viral reverse transcriptase. This view was further supported by the observation that most of CarSV sequences forming part of the junctions correspond either to strong secondary structure motifs in the conformation proposed for CarSV RNA or to its self-cleavage sites, which may have facilitated polymerase jumping. Accompanying the most-abundant CarSV RNA, a series of CarSV RNAs with sequence deletions were previously characterized. Here we have identified some of their corresponding DNA forms, together with other CarSV DNA forms with deletions not found in any CarSV RNA species identified so far. Some of these CarSV DNA forms have also been detected fused to CERV sequences. The existence of these shortened CarSV DNA versions may provide a continuous input of their corresponding transcripts and explain the persistence of CarSV RNAs with defective hammerhead structures for which an RNA-RNA model of amplification seems unlikely.

Functional analysis of the interaction between VPg-proteinase (NIa) and RNA polymerase (NIb) of tobacco etch potyvirus, using conditional and suppressor mutants.

The tobacco etch potyvirus (TEV) RNA-dependent RNA polymerase (NIb) has been shown to interact with the proteinase domain of the VPg-proteinase (NIa). To investigate the significance of this interaction, a Saccharomyces cerevisiae two-hybrid assay was used to isolate conditional NIa mutant proteins with temperature-sensitive (ts) defects in interacting with NIb. Thirty-six unique tsNIa mutants with substitutions affecting the proteinase domain were recovered. Most of the mutants coded for proteins with little or no proteolytic activity at permissive and nonpermissive temperatures. However, three mutant proteins retained proteolytic activity at both temperatures and, in two cases (tsNIa-Q384P and tsNIa-N393D), the mutations responsible for the ts interaction phenotype could be mapped to single positions. One of the mutations (N393D) conferred a ts-genome-amplification phenotype when it was placed in a recombinant TEV strain. Suppressor NIb mutants that restored interaction with the tsNIa-N393D protein at the restrictive temperature were recovered by a two-hybrid selection system. Although most of the suppressor mutants failed to stimulate amplification of genomes encoding the tsNIa-N393D protein, two suppressors (NIb-I94T and NIb-C380R) stimulated amplification of virus containing the N393D substitution by approximately sevenfold. These results support the hypothesis that interaction between NIa and NIb is important during TEV genome replication.

Complexes containing both polarity strands of avocado sunblotch viroid: identification in chloroplasts and characterization.

RNA analysis by nondenaturing polyacrylamide gel electrophoresis and Northern blot hybridization of avocado chloroplasts purified from protoplasts of leaves infected by avocado sunblotch viroid (ASBVd) revealed the main ASBVd-specific bands found previously in preparations of total leaf RNA: the monomeric, dimeric and subgenomic RNAs, and two bands, x and y. After RNase treatment in high ionic strength, bands x and y remained resistant, indicating a high content of double-stranded RNAs, whereas the other viroid-specific and cellular RNAs were degraded. Analysis by denaturing polyacrylamide gel electrophoresis and Northern blot hybridization showed that the major constituents of the purified y and x bands were the monomeric circular and linear ASBVd forms of both polarities, but band y contained additionally multimeric ASBVd RNAs, also of both polarities, that probably cause its slower migration in nondenaturing gels. After RNase treatment, the composition of band y was essentially unaffected, but only the monomeric linear ASBVd RNAs of both polarities was recovered from band x. However, in the presence of higher RNase concentrations, band y was converted into band x, indicating that they are closely related. The structure of complexes x and y, containing minus ASBVd strands and particularly the monomeric circular form, supports a role of replicative intermediates in the symmetric rolling circle mechanism proposed for ASBVd, whereas their localization in the chloroplast is strong evidence in favor of this organelle as the replication site of ASBVd.

Reverse transcription polymerase chain reaction protocols for cloning small circular RNAs.

A protocol is described for general application for cloning small circular RNAs which requires only minimal amounts of template (approximately 50 ng) of unknown sequence. Both cDNA strands are synthesized with a 26-mer primer whose six 3'-terminal positions are totally degenerate in two consecutive reactions catalyzed by reverse transcriptase and DNA polymerase, respectively. The cDNAs are then PCR-amplified, using a 20-mer primer with the non-degenerate sequence of the previous primer, cloned and sequenced. This information permits the synthesis of one or more pairs of specific and adjacent primers for obtaining full-length cDNA clones by a protocol which is also described.

Secondary structures in the capsid protein coding sequence and 3' nontranslated region involved in amplification of the tobacco etch virus genome.

The 3'-terminal 350 nucleotides of the tobacco etch potyvirus (TEV) genome span the end of the capsid protein (CP)-coding sequence and the 3' nontranslated region (NTR). The CP-coding sequence within this region contains a 105-nucleotide cis-active element required for genome replication (S. Mahajan, V. V. Dolja, and J. C. Carrington, J. Virol. 70:4370-4379, 1996). To investigate the sequence and secondary structure requirements within the CP cis-active region and the 3' NTR, a systematic linker-scanning mutagenesis analysis was done. Forty-six mutations, each with two to six nucleotide substitutions, were introduced at consecutive hexanucleotide positions in the genome of a recombinant TEV strain expressing a reporter protein (beta-glucuronidase). Genome amplification activity of each mutant in the protoplast cell culture system was measured. Mutations that severely debilitated genome amplification were identified throughout the CP-coding cis-active sequence and at several distinct locations within the 3' NTR. However, based on a computer model of RNA folding, mutations that had the most severe effects mapped to regions that were predicted to form base-paired secondary structures. Linker-scanning mutations predicted to affect either strand of a base-paired structure within the CP-coding cis-active sequence, a base-paired structure between two segments of the CP-coding cis-active sequence and a contiguous 14-nucleotide segment of the 3' NTR, and a base-paired structure near the 3' terminus of the 3' NTR inactivated genome amplification. Compensatory mutations that restored base pair interactions in each of these regions restored amplification activity, although to differing levels depending on the structure restored. These data reveal that the 3' terminus of the TEV genome consists of a series of functionally discrete sequences and secondary structures and that the CP-coding sequence and 3' NTR are coadapted for genome amplification function through a requirement for base pair interactions.

RNA binding activity of NIa proteinase of tobacco etch potyvirus.

The C-terminal domain of NIa protein (NIaPro) from tobacco etch potyvirus (TEV) is a sequence-specific proteinase required for processing of the viral polyprotein. This proteinase also interacts with NIb, the TEV RNA-dependent RNA polymerase. NIaPro and two NIaPro-containing polyproteins (NIa and 6/NIa) were analyzed from extracts of recombinant Escherichia coli. Using RNA-protein blot and UV-crosslinking assays, NIaPro and the NIaPro-containing polyproteins were shown to possess RNA-binding activity. NIaPro bound nonspecifically to several RNAs, including plus- and minus-strands of the TEV 5' and 3' noncoding regions. Saturation binding data obtained using the UV-crosslinking assay were consistent with a possible cooperative RNA-binding activity of NIaPro. In addition, the RNA-binding activities of NIaPro and full-length NIa protein were similar. Based on its RNA-binding activity and other known functions, NIaPro or a NIaPro-containing polyprotein is proposed to serve one or more direct roles during TEV RNA synthesis.

A 451-nucleotide circular RNA from cherry with hammerhead ribozymes in its strands of both polarities.

The sequence of 451 nucleotides of a cherry small circular RNA (csc RNA1) associated with a cherry disease has been determined. Both csc RNA1 and its complementary strand can form hammerhead structures similar to those found previously in other plant and animal small RNAs. In the branched secondary structure of lowest free energy of csc RNA1, the sequences involved in the hammerhead structures, which comprise approximately one-fourth of this RNA, are found opposite each other, forming part of a rod-like segment. Plus- and minus-strand full-length transcripts of csc RNA1 self-cleaved during transcription and after purification, as predicted by the hammerhead structures, which are stable and very probably act as single hammerhead structures. The minus-strand hammerhead structure of csc RNA1 is exceptional in having a central loop with only 11 conserved nucleotides, a situation previously observed in only one other natural hammerhead structure. Both hammerhead structures of csc RNA1 are also peculiar in having an A instead of a C preceding the self-cleavage sites. The in vivo concentration of the plus strand of csc RNA1 is only slightly higher than that of its complementary strand, and significant fractions of both strands are extracted from the tissue in the form of a complex. csc RNA1 has sequence similarities to viroids and especially to some viroid-like satellite RNAs; they also share some characteristics of their corresponding hammerhead structures with these satellite RNAs. These observations, together with the association in symptomatic tissue of csc RNA1 with a set of presumably viral double-stranded RNAs, suggest that csc RNA1 is a new viroid-like satellite RNA.

A group I plant intron accumulates as circular RNA forms with extensive 5' deletions in vivo.

Analysis by a PAGE approach for detecting small circular RNAs showed the existence of one such molecular species (RNA 1) accumulating at high levels in cherimoya. Sequencing of cDNA clones of RNA 1 revealed a size of 281 nt and a sequence identical to the 3'-terminal region of the 494-nt tRNALeu(UAA) group I intron from cherimoya. Northern blot hybridizations with a probe complementary to RNA 1 showed that this RNA coexists in vivo with its corresponding linear form, with the presumed full-length intron, and with minor amounts of two additional small circular species (RNAs 2 and 3). RNAs 2 and 3 had sizes of 216 and 156 nt, respectively, and sequences identical to different moieties of the 3'-terminal region of the tRNALeu(UAA) intron. The three cyclization sites giving rise to RNAs 1, 2, and 3, located within loop 8, are preceded by CUU or UUU trinucleotides and followed by sequences capable of forming base pairing interactions with the internal guide sequence characteristic of group I introns. The good correlation observed between the stabilities of these interactions and the in vivo accumulation levels of the corresponding cherimoya circular RNAs support the hypothesis that they emerge through a common mechanism similar to that advanced previously for the generation of circular RNAs derived from other group I introns. The lack of interactions of similar stabilities in tobacco, in which no circular RNAs derived from the tRNALeu(UAA) intron were detected, is consistent with this proposal, although other factors are also probably important in the synthesis and accumulation of the small circular RNAs in cherimoya.

A general strategy for cloning viroids and other small circular RNAs that uses minimal amounts of template and does not require prior knowledge of its sequence.

Two PCR-based methods are described for obtaining clones of small circular RNAs of unknown sequence and for which only minute amounts are available. To avoid introducing any assumption about the RNA sequence, synthesis of the cDNAs is initiated with random primers. The cDNA population is then PCR-amplified using a primer whose sequence is present at both sides of the cDNAs, since they have been obtained with random hexamers and then a linker with the sequence of the PCR primer has been ligated to their termini, or because the cDNAs have been synthesized with an oligonucleotide that contains the sequence of the PCR primer at its 5' end and six randomized positions at its 3' end. The procedures need only approximately 50 ng of purified RNA template. The reasons for the emergence of cloning artifacts and precautions to avoid them are discussed.