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Background: G-quadruplexes (G4s) are secondary structures in DNA and RNA that impact various cellular processes, such as transcription, splicing, and translation. Due to their numerous functions, G4s are involved in many diseases, making their study important. Yet, G4s evolution remains largely unknown, due to their low sequence similarity and the poor quality of their sequence alignments across several species. To address this, we designed a strategy that avoids direct G4s alignment to study G4s evolution in the 3 species kingdoms. We also explored the coevolution between RBPs and G4s. Methods: We retrieved one-to-one orthologous genes from the Ensembl Compara database and computed groups of one-to-one orthologous genes. For each group, we aligned gene sequences and identified G4 families as groups of overlapping G4s in the alignment. We analyzed these G4 families using Count, a tool to infer feature evolution into a gene or a species tree. Additionally, we utilized these G4 families to predict G4s by homology. To establish a control dataset, we performed mono-, di- and tri-nucleotide shuffling. Results: Only a few conserved G4s occur among all living kingdoms. In eukaryotes, G4s exhibit slight conservation among vertebrates, and few are conserved between plants. In archaea and bacteria, at most, only 2 G4s are common. The G4 homology-based prediction increases the number of conserved G4s in common ancestors. The coevolution between RNA-binding proteins and G4s was investigated and revealed a modest impact of RNA-binding proteins evolution on G4 evolution. However, the details of this relationship remain unclear. Conclusion: Even if G4 evolution still eludes us, the present study provides key information to compute groups of homologous G4 and to reveal the evolution history of G4 families.

Hop latent viroid (HLVd) is the biggest concern for cannabis and hop growers worldwide. Although most HLVd-infected plants remain asymptomatic, research on hops has demonstrated a decrease in both the α-bitter acid and terpene content of hop cones, which affects their economic value. The HLVd-associated “dudding” or “duds” disease of cannabis was first reported in 2019 in California. Since then, the disease has become widespread in cannabis-growing facilities across North America. Although severe yield loss associated with duds disease has been recorded, little scientific information is available to growers in order to contain HLVd. Consequently, this review aims to summarise all of the scientific information available on HLVd so as to be able to understand the effect of HLVd on yield loss, cannabinoid content, terpene profile, disease management and inform crop protection strategies.

Abstract Background Alternative splicing (AS) is an important mRNA maturation step that allows increased variability and diversity of proteins in eukaryotes. AS is dysregulated in numerous diseases, and its implication in the carcinogenic process is well known. However, progress in understanding how oncogenic viruses modulate splicing, and how this modulation is involved in viral oncogenicity has been limited. Epstein-Barr virus (EBV) is involved in various cancers, and its EBNA1 oncoprotein is the only viral protein expressed in all EBV malignancies. Methods In the present study, the ability of EBNA1 to modulate the AS of cellular genes was assessed using a high-throughput RT-PCR approach to examine AS in 1238 cancer-associated genes. RNA immunoprecipitation coupled to RNA sequencing (RIP-Seq) assays were also performed to identify cellular mRNAs bound by EBNA1. Results Upon EBNA1 expression, we detected modifications to the AS profiles of 89 genes involved in cancer. Moreover, we show that EBNA1 modulates the expression levels of various splicing factors such as hnRNPA1, FOX-2, and SF1. Finally, RNA immunoprecipitation coupled to RIP-Seq assays demonstrate that EBNA1 immunoprecipitates specific cellular mRNAs, but not the ones that are spliced differently in EBNA1-expressing cells. Conclusion The EBNA1 protein can modulate the AS profiles of numerous cellular genes. Interestingly, this modulation protein does not require the RNA binding activity of EBNA1. Overall, these findings underline the novel role of EBNA1 as a cellular splicing modulator.

Viroids are small, circular, highly structured pathogens that infect a broad range of plants, causing economic losses. Since their discovery in the 1970s, they have been considered as non-coding pathogens. In the last few years, the discovery of other RNA entities, similar in terms of size and structure, that were shown to be translated (e.g., cirRNAs, precursors of miRNA, RNA satellites) as well as studies showing that some viroids are located in ribosomes, have reignited the idea that viroids may be translated. In this study, we used advanced bioinformatic analysis, in vitro experiments and LC-MS/MS to search for small viroid peptides of the PSTVd. Our results suggest that in our experimental conditions, even though the circular form of PSTVd is found in ribosomes, no produced peptides were identified. This indicates that the presence of PSTVd in ribosomes is most probably not related to peptide production but rather to another unknown function that requires further study.

Viroids are non-coding RNA plant pathogens that are characterized by their possession of a high mutation level. Although the sequence heterogeneity in viroid infected plants is well understood, shifts in viroid population dynamics due to mutations over the course of infection remain poorly understood. In this study, the ten most abundant sequence variants of potato spindle tuber viroid RG1 (PSTVd) expressed at different time intervals in PSTVd infected tomato plants were identified by high-throughput sequencing. The sequence variants, forming a quasi-species, were subjected to both the identification of the regions favoring mutations and the effect of the mutations on viroid secondary structure and viroid derived small RNAs (vd-sRNA). At week 1 of PSTVd infection, 25% of the sequence variants were similar to the “master” sequence (i.e., the sequence used for inoculation). The frequency of the master sequence within the population increased to 70% at week 2 after PSTVd infection, and then stabilized for the rest of the disease cycle (i.e., weeks 3 and 4). While some sequence variants were abundant at week 1 after PSTVd infection, they tended to decrease in frequency over time. For example, the variants with insertions at positions 253 or 254, positions that could affect the Loop E as well as the metastable hairpin I structure that has been shown important during replication and viroid infectivity, resulted in decreased frequency. Data obtained by in silico analysis of the viroid derived small RNAs (vd-sRNA) was also analyzed. A few mutants had the potential of positively affecting the viroid’s accumulation by inducing the RNA silencing of the host’s defense related genes. Variants with mutations that could negatively affect viroid abundance were also identified because their derived vd-sRNA were no longer capable of targeting any host mRNA or of changing its target sequence from a host defense gene to some other non-important host gene. Together, these findings open avenues into understanding the biological role of sequence variants, this viroid’s interaction with host components, stable and metastable structures generated by mutants during the course of infection, and the influence of sequence variants on stabilizing viroid population dynamics.

Viroids are circular, highly structured, single-stranded, non-coding RNA pathogens known to infect and cause disease in several plant species. They are known to trigger the host plant’s RNA silencing machinery. The detection of viroid-derived small RNAs (vd-sRNA) in viroid-infected host plants opened a new avenue of study in host–viroid pathogenicity. Since then, several viroid research groups have studied the vd-sRNA retrieved from different host–viroid combinations. Such studies require the segregation of 21- to 24-nucleotide long small RNAs (sRNA) from a deep-sequencing databank, followed by separating the vd-sRNA from any sRNA within this group that showed sequence similarity with either the genomic or the antigenomic strands of the viroid. Such mapped vd-sRNAs are then profiled on both the viroid’s genomic and antigenomic strands for visualization. Although several commercial interfaces are currently available for this purpose, they are all programmed for linear RNA molecules. Hence, viroid researchers must develop a computer program that accommodates the sRNAs derived from the circular viroid genome. This is a laborious process, and consequently, it often creates a bottleneck for biologists. In order to overcome this constraint, and to help the research community in general, in this study, a python-based pattern matching interface was developed so as to be able to both profile and map sRNAs on a circular genome. A “matching tolerance” feature has been included in the program, thus permitting the mapping of the sRNAs derived from the quasi-species. Additionally, the “topology” feature allows the researcher to profile sRNA derived from both linear and circular RNA molecules. The efficiency of the program was tested using previously reported deep-sequencing data obtained from two independent studies. Clearly, this novel software should be a key tool with which to both evaluate the production of sRNA and to profile them on their target RNA species, irrespective of the topology of the target RNA molecule.

Abstract It is well established that viroid derived small RNA (vd-sRNA) induces RNA silencing of endogenous mRNA. However, it remains not clear how exactly viroid infections can lead to severe symptom induction given the fact that fewer vd-sRNAs binding the specific target mRNAs were recovered from the infected plants. To answer this question, the two least expressed (+) and (−) strand vd-sRNAs of potato spindle tuber viroid (PSTVd) binding to both the 3′ UTR and the coding region of tomato mRNAs were analyzed by infecting tomato plants with two variants of PSTVd. As products of these putative target mRNAs are involved in plant phenotype, the effect of this viroid on these genes were analyzed by infecting tomato plants with two variants of PSTVd. The direct interaction between the vd-sRNAs and putative mRNAs was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Parallel analysis of RNA ends of viroid infected plants revealed the widespread cleavage of the target mRNAs in locations other than the vd-sRNA binding site during the viroid infection implying the viroid-infection induced vd-sRNA independent degradation of endogenous mRNAs during viroid infection.

The early 1970s marked two breakthroughs in the field of biology: (i) The development of nucleotide sequencing technology; and, (ii) the discovery of the viroids. The first DNA sequences were obtained by two-dimensional chromatography which was later replaced by sequencing using electrophoresis technique. The subsequent development of fluorescence-based sequencing method which made DNA sequencing not only easier, but many orders of magnitude faster. The knowledge of DNA sequences has become an indispensable tool for both basic and applied research. It has shed light biology of viroids, the highly structured, circular, single-stranded non-coding RNA molecules that infect numerous economically important plants. Our understanding of viroid molecular biology and biochemistry has been intimately associated with the evolution of nucleic acid sequencing technologies. With the development of the next-generation sequence method, viroid research exponentially progressed, notably in the areas of the molecular mechanisms of viroids and viroid diseases, viroid pathogenesis, viroid quasi-species, viroid adaptability, and viroid–host interactions, to name a few examples. In this review, the progress in the understanding of viroid biology in conjunction with the improvements in nucleotide sequencing technology is summarized. The future of viroid research with respect to the use of third-generation sequencing technology is also briefly envisaged.

RNA G-quadruplexes (rG4) are stable non-canonical secondary structures composed of G-rich sequences. Many rG4 structures located in the 5'UTRs of mRNAs act as translation repressors due to their high stability which is thought to impede ribosomal scanning. That said, it is not known if these are mRNA-specific examples, or if they are indicative of a global expression regulation mechanism of the mRNAs involved in a common pathway based on structure folding recognition. Gene-ontology analysis of mRNAs bearing a predicted rG4 motif in their 5'UTRs revealed an enrichment for mRNAs associated with the colorectal cancer pathway. Bioinformatic tools for rG4 prediction, and experimental in vitro validations were used to confirm and compare the folding of the predicted rG4s of the mRNAs associated with dysregulated pathways in colorectal cancer. The rG4 folding was confirmed for the first time for 9 mRNAs. A repressive effect of 3 rG4 candidates on the expression of a reporter gene was also measured in colorectal cancer cell lines. This work highlights the fact that rG4 prediction is not yet accurate, and that experimental characterization is still essential in order to identify the precise rG4 folding sequences and the possible common features shared between the rG4 overrepresented in important biological pathways.

Multiple human diseases including cancer have been associated with a dysregulation in RNA splicing patterns. In the current study, modifications to the global RNA splicing landscape of cellular genes were investigated in the context of Epstein-Barr virus-associated gastric cancer. Global alterations to the RNA splicing landscape of cellular genes was examined in a large-scale screen from 295 primary gastric adenocarcinomas using high-throughput RNA sequencing data. RT-PCR analysis, mass spectrometry, and co-immunoprecipitation studies were also used to experimentally validate and investigate the differential alternative splicing (AS) events that were observed through RNA-seq studies. Our study identifies alterations in the AS patterns of approximately 900 genes such as tumor suppressor genes, transcription factors, splicing factors, and kinases. These findings allowed the identification of unique gene signatures for which AS is misregulated in both Epstein-Barr virus-associated gastric cancer and EBV-negative gastric cancer. Moreover, we show that the expression of Epstein-Barr nuclear antigen 1 (EBNA1) leads to modifications in the AS profile of cellular genes and that the EBNA1 protein interacts with cellular splicing factors. These findings provide insights into the molecular differences between various types of gastric cancer and suggest a role for the EBNA1 protein in the dysregulation of cellular AS.

The simplest known plant pathogens are the viroids. Because of their non-coding single-stranded circular RNA genome, they depend on both their sequence and their structure for both a successful infection and their replication. In the recent years, important progress in the elucidation of their structures was achieved using an adaptation of the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) protocol in order to probe viroid structures in solution. Previously, SHAPE has been adapted to elucidate the structures of all of the members of the family Avsunviroidae, as well as those of a few members of the family Pospiviroidae. In this study, with the goal of providing an entire compendium of the secondary structures of the various viroid species, a total of thirteen new Pospiviroidae members were probed in solution using the SHAPE protocol. More specifically, the secondary structures of eleven species for which the genus was previously known were initially elucidated. At this point, considering all of the SHAPE elucidated secondary structures, a classification system for viroids in their respective genera was proposed. On the basis of the structural classification reported here, the probings of both the Grapevine latent viroid and the Dahlia latent viroid provide sound arguments for the determination of their respective genera, which appear to be Apscaviroid and Hostuviroid, respectively. More importantly, this study provides the complete repertoire of the secondary structures, mapped in solution, of all of the accepted viroid species reported thus far. In addition, a classification scheme based on structural hallmarks, an important tool for many biological studies, is proposed.

To date, two plant genes encoding RNA-dependent RNA polymerases (RdRs) that play major roles in the defense against RNA viruses have been identified: (i) RdR1, which is responsible for the viral small RNAs (vsRNAs) found in virus-infected plants, and, (ii) RdR6, which acts as a surrogate in the absence of RdR1. In this study, the role of RdR6 in the defense against viroid infection was examined by knock-down of RdR6 followed by potato spindle tuber viroid (PSTVd) infection. The suppression of RdR6 expression increased the plant’s growth, as was illustrated by the plant’s increased height. PSTVd infection of RdR6 compromised plants resulted in an approximately three-fold increase in the accumulation of viroid RNA as compared to that seen in control plants. Additionally, RNA gel blot assay revealed an increase in the number of viroids derived small RNAs in RdR6 suppressed plants as compared to control plants. These data provide a direct correlation between RdR6 and viroid accumulation and indicate the role of RDR6 in the plant’s susceptibility to viroid infection.

Alternative splicing (AS) is a central mechanism of genetic regulation which modifies the sequence of RNA transcripts in higher eukaryotes. AS has been shown to increase both the variability and diversity of the cellular proteome by changing the composition of resulting proteins through differential choice of exons to be included in mature mRNAs. In the present study, alterations to the global RNA splicing landscape of cellular genes upon viral infection were investigated using mammalian reovirus as a model. Our study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in eukaryotic cells following infection with a human virus. We identify 240 modified alternative splicing events upon infection which belong to transcripts frequently involved in the regulation of gene expression and RNA metabolism. Using mass spectrometry, we also confirm modifications to transcript-specific peptides resulting from AS in virus-infected cells. These findings provide additional insights into the complexity of virus-host interactions as these splice variants expand proteome diversity and function during viral infection.

G-quadruplexes (G4) are extremely stable secondary structures forming stacks of guanine tetrads. DNA G4 structures have been extensively studied, however, less is known about G4 motifs in mRNAs, especially in their coding sequences. Herein, we show that Aven stimulates the mRNA translation of the mixed lineage leukemia (MLL) proto-oncogene in an arginine methylation-dependent manner. The Aven RGG/RG motif bound G4 structures within the coding regions of the MLL1 and MLL4 mRNAs increasing their polysomal association and translation, resulting in the induction of transcription of leukemic genes. The DHX36 RNA helicase associated with the Aven complex and was required for optimal translation of G4 mRNAs. Depletion of Aven led to a decrease in synthesis of MLL1 and MLL4 proteins resulting in reduced proliferation of leukemic cells. These findings identify an Aven-centered complex that stimulates the translation of G4 harboring mRNAs, thereby promoting survival of leukemic cells.

We report the nucleotide sequences of three citrus viroids belonging to three different genera: Citrus exocortis viroid (CEVd), Hop stunt viroid (HSVd) and Citrus viroid-III (CVd-III) isolated from a single natural infected Citrus reticulata var. Clementine tree growing in a tree nursery in Manouba (near Tunis Capital). We describe the sequence variability of these viroids from their natural host without using an alternative passage by an indicator host or an artificial inoculation. This work confirms that naturally occurring viroid infections contain a mixture of sequence variants. These are the first sequences of citrus viroids from Africa.

In order to analyze the production of small RNA (sRNA) by viroids upon infecting the plants, the tomato plants (Solanum lycopersicum cultivar Rutgers) were inoculated with the variants of Potato spindle tuber viroid (PSTVd). After 21-days of postinoculation, total RNA was extracted and subjected for deep-sequencing using Illumina HiSeq platform. The primers were trimmed and only 21- to 24-nt long sRNAs were filtered after quality check of the raw data. The filtered sRNA population was then mapped against both the genomic (+) and antigenomic (−) strands of the respective PSTVd variants using standard pattern-matching algorithm. The profiling of viroid derived sRNA (vd-sRNA) revealed that the viroids are susceptible to host RNA silencing mechanism. High-throughput sequence data linked to this project have been deposited in the Gene Expression Omnibus (GEO) database under accession number GSE69225.

Viroids are small, circular, single stranded RNA molecules that infect plants. Since they are non-coding, their structures play a critical role in their life cycles. To date, little effort has been spend on elucidating viroid structures in solution due to both the experimental difficulties and the time-consuming nature of the methodologies implicated. Recently, the technique of high-throughput selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) was adapted for the probing of the members of family Avsunviroidae, all of whom replicate in the chloroplast and demonstrate ribozyme activity. In the present work, twelve viroid species belonging to four different genera of the family Pospiviroidae, whose members are characterized by the presence of a central conserved region (CCR) and who replicate in nucleus of the host, were probed. Given that the structures of five distinct viroid species from the family Pospiviroidae have been previously reported, an overview of the different structural characteristics for all genera and the beginning of a manual classification of the different viroids based on their structural features are presented here.

Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA. Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies.

Viroids are small circular single-stranded infectious RNAs characterized by a relatively high mutation level. Knowledge of their sequence heterogeneity remains largely elusive and previous studies, using Sanger sequencing, were based on a limited number of sequences. In an attempt to address sequence heterogeneity from a population dynamics perspective, a GF305-indicator peach tree was infected with a single variant of the Avsunviroidae family member Peach latent mosaic viroid (PLMVd). Six months post-inoculation, full-length circular conformers of PLMVd were isolated and deep-sequenced. We devised an original approach to the bioinformatics refinement of our sequence libraries involving important phenotypic data, based on the systematic analysis of hammerhead self-cleavage activity. Two distinct libraries yielded a total of 3,939 different PLMVd variants. Sequence variants exhibiting up to ∼17% of mutations relative to the inoculated viroid were retrieved, clearly illustrating the high level of divergence dynamics within a unique population. While we initially assumed that most positions of the viroid sequence would mutate, we were surprised to discover that ∼50% of positions remained perfectly conserved, including several small stretches as well as a small motif reminiscent of a GNRA tetraloop which are the result of various selective pressures. Using a hierarchical clustering algorithm, the different variants harvested were subdivided into 7 clusters. We found that most sequences contained an average of 4.6 to 6.4 mutations compared to the variant used to initially inoculate the plant. Interestingly, it was possible to reconstitute and compare the sequence evolution of each of these clusters. In doing so, we identified several key mutations. This study provides a reliable pipeline for the treatment of viroid deep-sequencing. It also sheds new light on the extent of sequence variation that a viroid population can sustain, and which may give rise to a quasispecies.

Alzheimer's disease (AD) etiological studies suggest that an elevation in amyloid-β peptides (Aβ) level contributes to aggregations of the peptide and subsequent development of the disease. The major constituent of these amyloid peptides is the 1 to 40–42 residue peptide (Aβ40−42) derived from amyloid protein precursor (APP). Most likely, reducing Aβ levels in the brain may block both its aggregation and neurotoxicity and would be beneficial for patients with AD. Among the several possible ways to lower Aβ accumulation in the cells, we have selectively chosen to target the primary step in the Aβ cascade, namely, to reduce APP gene expression. Toward this end, we engineered specific SOFA-HDV ribozymes, a new generation of catalytic RNA tools, to decrease APP mRNA levels. Additionally, we demonstrated that APP-ribozymes are effective at decreasing APP mRNA and protein levels as well as Aβ levels in neuronal cells. Our results could lay the groundwork for a new protective treatment for AD.

The HDV ribozyme's folding pathway is, by far, the most complex folding pathway elucidated to date for a small ribozyme. It includes 6 different steps that have been shown to occur before the chemical cleavage. It is likely that other steps remain to be discovered. One of the most critical of these unknown steps is the formation of the trans Watson-Crick GU base pair within loop III. The U(23) and G(28) nucleotides that form this base pair are perfectly conserved in all natural variants of the HDV ribozyme, and therefore are considered as being part of the signature of HDV-like ribozymes. Both the formation and the transformation of this base pair have been studied mainly by crystal structure and by molecular dynamic simulations. In order to obtain physical support for the formation of this base pair in solution, a set of experiments, including direct mutagenesis, the site-specific substitution of chemical groups, kinetic studies, chemical probing and magnesium-induced cleavage, were performed with the specific goal of characterizing this trans Watson-Crick GU base pair in an antigenomic HDV ribozyme. Both U(23) and G(28) can be substituted for nucleotides that likely preserve some of the H-bond interactions present before and after the cleavage step. The formation of the more stable trans Watson-Crick base pair is shown to be a post-cleavage event, while a possibly weaker trans Watson-Crick/Hoogsteen interaction seems to form before the cleavage step. The formation of this unusually stable post-cleavage base pair may act as a driving force on the chemical cleavage by favouring the formation of a more stable ground state of the product-ribozyme complex. To our knowledge, this represents the first demonstration of a potential stabilising role of a post-cleavage conformational switch event in a ribozyme-catalyzed reaction.

Influenza A virus seasonal outbreaks and occasional pandemics represent a global health threat. The high genetic instability of this virus permits rapid escape from the host immune system and emergence of resistance to antivirals. There is thus an urgent need to develop novel approaches for efficient treatment of newly emerging strains. Based on a sequence alignment of representatives from every subtype known to infect humans, we identified nucleic acid regions that are conserved amongst these influenza A populations. We then engineered SOFA-HDV-Ribozymes as therapeutic tools recognizing these conserved regions to catalytically cleave the corresponding viral mRNA targets. The most promising ribozymes were chosen based on an initial in silico screening, and their efficacy was assessed using in vitro cleavage assays. Further characterization of their antiviral effect in cell culture and in mice led to the gradual identification of prophylactic SOFA-HDV-Ribozyme combinations, providing proof-of-principle for the potential of this novel strategy to develop antivirals against genetically highly variable viruses.

For a long time nucleic acid-based approaches directed towards controlling the propagation of Hepatitis C Virus (HCV) have been considered to possess high potential. Towards this end, ribozymes (i.e. RNA enzymes) that specifically recognize and subsequently catalyze the cleavage of their RNA substrate present an attractive molecular tool. Here, the unique properties of a new generation of ribozymes are taken advantage of in order to develop an efficient and durable ribozyme-based technology with which to target HCV (+) RNA strands. These ribozymes resulted from the coupling of a specific on/off adaptor (SOFA) to the ribozyme domain derived from the Hepatitis Delta Virus (HDV). The former switches cleavage activity "on" solely in the presence of the desired RNA substrate, while the latter was the first catalytic RNA reported to function naturally in human cells, specifically in hepatocytes. In order to maximize the chances for success, a step-by-step approach was used for both the design and the selection of the ribozymes. This approach included the use of both bioinformatics and biochemical methods for the identification of the sites possessing the greatest potential for targeting, and the subsequent in vitro testing of the cleavage activities of the corresponding SOFA-HDV ribozymes. These efforts led to a significant improvement in the ribozymes' designs. The ability of the resulting SOFA-HDV ribozymes to inhibit HCV replication was further examined using a luciferase-based replicon. Although some of the ribozymes exhibited high levels of cleavage activity in vitro, none appears to be a potential long term inhibitor in cellulo. Analysis of recent discoveries in the cellular biology of HCV might explain this failure, as well as provide some ideas on the potential limits of using nucleic acid-based drugs to control the propagation of HCV. Finally, the above conclusions received support from experiments performed using a collection of SOFA-HDV ribozymes directed against HCV (-) strands.

Through evolution, enzymes have developed subtle modes of activation in order to ensure the sufficiently high substrate specificity required by modern cellular metabolism. One of these modes is the use of a target-dependent module (i.e. a docking domain) such as those found in signalling kinases. Upon the binding of the target to a docking domain, the substrate is positioned within the catalytic site. The prodomain acts as a target-dependent module switching the kinase from an off state to an on state. As compared to the allosteric mode of activation, there is no need for the presence of a third partner. None of the ribozymes discovered to date have such a mode of activation, nor does any other known RNA. Starting from a specific on/off adaptor for the hepatitis delta virus ribozyme, that differs but has a mechanism reminiscent of this signalling kinase, we have adapted this mode of activation, using the techniques of molecular engineering, to both catalytic RNAs and DNAs exhibiting various activities. Specifically, we adapted three cleaving ribozymes (hepatitis delta virus, hammerhead and hairpin ribozymes), a cleaving 10-23 deoxyribozyme, a ligating hairpin ribozyme and an artificially selected capping ribozyme. In each case, there was a significant gain in terms of substrate specificity. Even if this mode of control is unreported for natural catalytic nucleic acids, its use needs not be limited to proteinous enzymes. We suggest that the complexity of the modern cellular metabolism might have been an important selective pressure in this evolutionary process.

HepatitisD(delta) virus (HDV) is an infectious agent that propagates in hepatocytes only in the presence of hepatitis B virus, causing fulminant or chronic hepatitis with liver cirrhosis. HDV is a 36 nm particle that includes a circular RNA genome of 1.7 kilobases with an extensive internal complementary that allows it to fold into a rod-like structure. The relationships among genotypes, sequence variability, geographical distribution and disease severity of HDV remain unknown. Consequently, in the present study, the complete nucleotide sequence of anHDVisolated from a Canadian patient was determined. The viral RNA from serum was amplified using reverse transcription coupled to polymerase chain reaction amplification. The resulting complementary DNA was cloned and sequenced. Sequence analysis revealed that this new isolate contained 1672 nucleotides corresponding to genotype 1, which has a worldwide distribution. Sequencing of four independent clones revealed 17 substitutions, corresponding to an overall sequence variability of 1%. Surprisingly, seven mutations were found in the 48-nucleotide region located between the two highly conserved self-catalytic motifs. This is the first demonstration that many substitutions are identified in this region of HDV, and prompts the present authors to define it as a hypervariable region.