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.     

Separation of the plus and minus strands of cytoplasmic polyhedrosis virus and human reovirus double-stranded genome RNAs by gel electrophoresis.

The complementary strands of most of the genome double-stranded RNA segments of insect cytoplasmic polyhedrosis virus (CPV) and human reovirus are separated for the first time by agarose gel electrophoresis in in the presence of 7 M urea. CPV (+) strands and most reovirus (-) strands migrate faster than the corresponding strands of opposite polarity. Glyoxal treatment, which modifies guanine residues and prevents G-C basepairing, results in a loss of strand resolution and concomitantly a significant decrease in electrophoretic mobilities. Reovirus mRNAs synthesized in vitro with ITP substituted for GTP show similar decreased electrophoretic mobilities as the glyoxalated mRNAs. These results clearly indicate that the basis for (+) and (-) strand resolution is the presence of secondary structure formed mainly by G-C(U) base-pairs that are maintained during gel electrophoresis in the presence of 7 M urea. When the plus and minus strands of CPV genomes were separated and compared for protein synthesizing activity, it was found that only the plus strands were able to form stable 80S ribosome-RNA initiation complexes in wheat germ cell-free extracts.     

Mapping studies of the Peach latent mosaic viroid reveal novel structural features

Knowledge of the structure of a viroid is critically important to elucidate the roles played by the various RNA motifs in the steps of the viroid's life cycle. A new technique, RNA-selective 2'-hydroxyl acylation analysed by primer extension (SHAPE), has recently been shown to be fast, reliable and applicable to the study of various RNA molecules. Consequently, this method was used to probe sequence variants of Peach latent mosaic viroid (PLMVd). Initially, probing data from RNA strands of both polarities of the Siberian C variant confirmed the secondary structures previously determined using both conventional and fastidious approaches. Subsequently, analysis of an Alberta variant showed an identical structure for the strand of (-) polarity, but the (+) polarity strand exhibited two differences from the Siberian C variant: the P11-L11 stem-loop domain formed a cruciform structure, and nucleotides from loops L1 and L11 were involved in the formation of a pseudoknot. The existence of both of these motifs was confirmed by site-directed mutagenesis. The subsequent probing of 12 natural sequence variants led to the elucidation of the criteria governing the formation of this novel pseudoknot. Importantly, this study revealed that the heterogeneity of a viroid is not limited to its nucleotide sequence, but may also occur at the structural level.     

Elucidation of the structures of all members of the Avsunviroidae family

Viroids are small single‐stranded RNA pathogens which cause significant damage to plants. As their nucleic acids do not encode for any proteins, they are dependant solely on their structure for their propagation. The elucidation of the secondary structures of viroids has been limited because of the exhaustive and time‐consuming nature of classic approaches. Here, the method of high‐throughput selective 2′‐hydroxyl acylation analysed by primer extension (hSHAPE) has been adapted to probe the viroid structure. The data obtained using this method were then used as input for computer‐assisted structure prediction using RNAstructure software in order to determine the secondary structures of the RNA strands of both (+) and (–) polarities of all Avsunviroidae members, one of the two families of viroids. The resolution of the structures of all of the members of the family provides a global view of the complexity of these RNAs. The structural differences between the two polarities, and any plausible tertiary interactions, were also analysed. Interestingly, the structures of the (+) and (–) strands were found to be different for each viroid species. The structures of the recently isolated grapevine hammerhead viroid‐like RNA strands were also solved. This species shares several structural features with the Avsunviroidae family, although its infectious potential remains to be determined. To our knowledge, this article represents the first report of the structural elucidation of a complete family of viroids.     

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.     

Control of Replication in RNA Bacteriophages

The rates of viral RNA and protein syntheses for wild-type RNA bacteriophages and their nonpolar, coat protein amber mutants were determined in amber suppressor (S26R1E, Su-1 and H12R8a, Su-3) and nonsuppressor (AB259, S26, and Q13) strains of Escherichia coli in the presence of rifamycin. It was demonstrated that the rates of synthesis of phage-specific replicase and RNA minus strands drop off concurrently in both wild-type and coat protein mutant-infected Su(-) and Su(+) cells after 10 and 15 min postinfection, respectively. The rate of synthesis of RNA plus strands started to decline 5 to 10 min later in both cases. Excessive synthesis of replicase in the coat protein mutant-infected cells was accompanied by a similar overproduction of RNA minus strands, but not of plus strands. Partial suppression of protein synthesis in wild-type phage-infected cells abolishing coat protein control over replicase accumulation led to prolongation of replicase synthesis. Such an effect was observed also in coat protein mutant-infected cells, indicating that the excess of replicase itself may be capable of suppression of replicase synthesis in the absence of coat protein. The prolongation of replicase synthesis was followed by the prolonged synthesis of RNA minus strands in both cases. Moreover, replicase and minus strands were formed in nearly equal amounts when protein synthesis was partially inhibited. Assuming functional instability of phage RNAs, the observed coupling of replicase and minus-strand RNA synthesis offers a possibility for control of viral RNA replication by means of control of replicase synthesis on the translational level. A hypothesis is put forward to explain the molecular mechanism of such coupling between the syntheses of replicase and RNA minus strands.     

Patterns of nuclease protection during strand exchange. recA protein forms heteroduplex DNA by binding to strands of the same polarity

recA protein, in the presence of ATP, polymerizes on single-stranded DNA (plus strand) to form a presynaptic nucleoprotein filament that pairs with linear duplex DNA and actively displaces the plus strand from the recipient molecule in a polarized fashion to form a new heteroduplex molecule. The interaction between recA protein and DNA during strand exchange was studied by labeling different strands and probing the intermediate with pancreatic deoxyribonuclease I (DNase I) or restriction endonuclease. The incoming single strand was resistant to DNase I in the original nucleoprotein filament and remained resistant even after extensive strand exchange had occurred. Both strands of the parental duplex molecule were sensitive to DNase I in the absence of joint molecule formation; but as strand exchange progressed following homologous pairing, increasing stretches of the parental plus strand became resistant, whereas the complementary parental minus strand remained sensitive to DNase I throughout the reaction. Except for a region of 50-100 base pairs at the end of the newly formed heteroduplex DNA where strand exchange was initiated, the rest of the heteroduplex region was resistant to cleavage by restriction endonucleases. The data suggest that recA protein promotes strand exchange by binding both the incoming and outgoing strands of the same polarity, whereas the complementary strand, which must switch pairing partners, is unhindered by direct contact with the protein.     

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:     

Studies on biosynthesis of tobacco mosaic virus. VII. Radioactivity of plus and minus strands in different forms of viral RNA after labelling of infected tobacco leaves

Tobacco leaves were labelled with tritiated undine for 30 or 120 minutes at different times after systemic infection with tobacco mosaic virus. RNA was extracted and separated into three fractions: one enriched in RF (replicative form), one enriched in RI (replicative intermediate), and one containing the bulk of single-stranded RNA. Radioactivity in plus strands (viral RNA) and minus strands (complementary RNA) was determined in each fraction by an isotope dilution assay. The amount of minus strands in the RP and RI fractions and the amount of plus strands in the single-stranded RNA fraction were also determined.Minus-strand synthesis was twice as high a few hours after the outbreak of visible symptoms as during the subsequent large accumulation of plus strands. At the early stage of virus production, the specific radioactivity of the minus strands was three- to fourfold that of the total RNA. Later it was about the same as that of the total RNA. As minus strands constitute a constant part of the total RNA at the later stages, this observation suggests that breakdown of minus strands is small.The specific radioactivity of minus strands was the same in corresponding RF and RI fractions. As the turn-over of minus strands appears to be small, a rapid interconversion of the two RNA types is indicated.In RF and RI the radioactivity in plus strands was between 6 and 50 times greater than that in minus strands. The specific radioactivity of plus strands was greater in RF and RI than in the single-stranded RNA, supporting the concept that both RF and RI have a precursor role for viral RNA.     

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.     

Secondary structure of Qβ RNA

The complete set of possible secondary structures of a variant Qβ RNA sequenced by Schaffner has been found using a computer program which allows G-U pairing as well as the usual Watson-Crick A-U and G-C pairing. Of special interest are those secondary structures with the highest double-strandedness. Omitting G-U pairing, we find the structure with the maximum double-strandedness has a pairing of 62% and exhibits a similarity to the clover leaf structure of tRNA. Including G-U pairing, the complementary strands of RNA are asymmetrical. We find maximum pairings of 71% for both the plus and minus strands. These structures also exhibit a cloverleaf structure. A similar analysis has been carried out for the secondary structure of a larger Qβ variant sequenced by Mills, Kramer and Spiegelman, but in this case there are a large number of secondary structures with the same maximum number of pairs and it is therefore not possible to select a unique structure with the maximum double-strandedness.     

Autonomous parvovirus LuIII encapsidates equal amounts of plus and minus DNA strands.

Autonomous parvoviruses are thought to uniquely encapsidate single-stranded DNA of minus polarity. In contrast, the defective adeno-associated viruses separately encapsidate equal amounts of plus and minus DNA strands. We reexamined the uniqueness of minus strand encapsidation for the autonomous parvoviruses. Although we found that Kilham rat virus and H-1 virus encapsidate varying but small amounts of complementary-strand DNA, it was unexpected to find that LuIII virus encapsidated equal amounts of plus and minus DNA. The extracted LuIII DNA possessed properties of double-stranded replicative-form DNA, including insensitivity to S1 endonuclease, cleavage by restriction enzymes, and conversion to unit-length, single-stranded DNA when electrophoresed under denaturing conditions. However, the inability of this DNA to form single-stranded DNA circles when denatured and then renatured in the presence of formamide and the lack of double-stranded DNA circle formation after treatment with exonuclease III and reannealing shows a lack of sequence homology of the 3' and 5' termini of LuIII DNA, in contrast to adeno-associated virus DNA. Digestion of LuIII double-stranded DNA with EcoRI and HincII and separation of plus and minus DNA strands on composite agarose-acrylamide gels identified a heterogeneity present only in the plus DNA strand. These results suggest that strand specificity of viral DNA encapsidation is not a useful property for differentiation between the autonomous and defective parvoviruses. Furthermore, encapsidation by LuIII of equal amounts of complementary DNA strands in contrast to encapsidation of minus strands by H-1 virus, when propagated in the same host cell type, suggests that selection of strands for encapsidation is a virus-coded rather than host-controlled event.