Viroid RNA Research Articles

A Cholesterol and Actinide Dependent Shadow Biosphere of Archaea and Viroids in Neurodevelopmental Disorders

AimEndogenous digoxin has been related to the pathogenesis of autism, cerebral palsy and trisomy 21. The possibility of endogenous digoxin synthesis by endosymbiotic bacteria with a mevalonate pathway and cholesterol catabolism was considered and explored in the study. MethodsCholesterol substrate was added to the plasma of the patients and the generation of cytochrome F420, free RNA, free DNA, polycyclic aromatic hydrocarbon, hydrogen peroxide, serotonin, pyruvate, ammonia, glutamate, cytochrome C, hexokinase, ATP synthase, HMG CoA redutase, digoxin and bile acids were studied. The changes with the addition of antibiotics and rutile to the patient’s plasma were also studied.ResultsThe study showed rutile dependent increase in archaea and RNA viroids in the sera of these patients. The generation of cholesterol catabolites- polycyclic aromatic hydrocarbon, hydrogen peroxide, serotonin, pyruvate, ammonia, glutamate, digoxin and bile acids was increased in the presence of rutile suggesting a rutile dependent alternate biochemistry. The mevalonate pathway, ATP synthase and glycolysis in this archaea were also rutile dependent. The addition of antibiotics to the patient’s serum decreased all these activities suggesting their archaeal origin. ConclusionThe study demonstrates the existence of a shadow biosphere of rutile dependent archaea and viroids in these disease states. The archaeal cholesterol catabolism and viroidal RNA interference is crucial to the pathogenesis of these diseases. Key words: Autism; Cerebral palsy; trisomy 21; actinides; archaea; viroids; cholesterol oxidase; HMG CoA reductase

The Actinidic Archaea Related Lemurian Syndrome- Endomyocardial Fibrosis, Chronic Calcific Pancreatitis and Multinodular Goitre- Evolutionary Significance and Evidence for the Lemurian Supercontinent

Background: Endogenous digoxin has been related to the pathogenesis of chronic calcific pancreatitis, endomyocardial fibrosis, multinodular goitre and mucoid angiopathy which are seen in South India, South Africa, Australia and South America. Phytoplasmas, viroids and rutile in beach sands have been related to root wilt disease of coconut seen in the same endemic area. The possibility of endogenous digoxin synthesis by actinide based primitive organism like archaea with a mevalonate pathway and cholesterol catabolism as well as a RNA viroid role in pathogenesis of these endemic diseases was considered. The study aims to link actinidic nanoarchaea to etiology of these diseases and postulates their common geographic epidemiology to the existence of a pre-historic Lemurian supercontinent linking these geographic areas. It also postulates a role for actinidic surfaces in the evolution of life, eukaryote, primates and humans.Methods: 10 cases each of chronic calcific pancreatitis, endomyocardial fibrosis, multinodular goitre and mucoid angiopathy before starting treatment and 10 age and sex matched healthy controls from general population were chosen for the study. Cholesterol substrate was added to the plasma of the patients and the generation of cytochrome F420, free RNA, free DNA, polycyclic aromatic hydrocarbon, hydrogen peroxide, serotonin, pyruvate, ammonia, glutamate, cytochrome C, hexokinase, ATP synthase, HMG CoA reductase, digoxin and urease were studied. The changes with the addition of antibiotics and rutile to the patient’s plasma were also studied. The statistical analysis was done by ANOVA.Results: The parameters mentioned above were increased the patient’s plasma with addition of cholesterol substrate. The addition of antibiotics to the patient’s plasma caused a decrease in all the parameters while addition of rutile increased their levels. Conclusions: An actinide dependent shadow biosphere of archaea and viroids is described in endomyocardial fibrosis, chronic calcific pancreatitis, multinodular goitre and mucoid angiopathy contributing to their pathogenesis. The coexistence of EMF, CCP and MNG in South India, South Africa, Australia and South America is thus an indirect evidence for the existence of the Lemurian supercontinent containing these land masses and rich in actinidic minerals. It also is evidence of abiogenesis on radioactive actinidic beach sands through the process of surface metabolism in the course of evolution. This gives credence to the role of actinidic archaea as the third element that controls life and its role in the evolution of the multicellular eukaryote, primates and humans. Key words: Archaea; Viroids; Endomyocardial fibrosis; Chronic calcific pancreatitis; Multinodular goitre; Mucoid angiopathy; Lemuria

Citrus dwarfing viroid: effects on tree size and scion performance specific to Poncirus trifoliata rootstock for high-density planting

The term ‘transmissible small nuclear ribonucleic acids' (TsnRNAs) describes well-characterised viroid RNA species that do not induce any pronounced disease syndromes in specific citrus hosts, but rather act as regulatory genetic elements modifying tree performance. The canopy volume (CV) of 13-year-old navel orange trees (Citrus sinensis) on Poncirus trifoliata rootstock treated with TsnRNA-IIIb (syn. Citrus dwarfing viroid) was reduced by 45% and 53.5% in standard-density (6 m × 6.7 m) or high-density (3 m × 6.7 m) plantings, respectively. The total yield of eight consecutive harvests was not affected significantly by the TsnRNA-IIIb treatments or the two planting densities. However, the yield per land surface unit (Y/LSU) was almost doubled (increased by 97.5%) for the high-density plantings over the standard-density plantings of the untreated controls. The Y/LSU of the TsnRNA-IIIb treated navel orange trees in the standard-density planting was reduced by 32.7%. The TsnRNA-IIIb treatment in both planting densities concentrated significantly more fruit production (approximately 60%) in the economically advantageous middle canopy height zone (0.6–2.4 m) in comparison with the untreated controls (35%). Fruit grade, size, appearance, organoleptic characteristics or time of maturation of the TsnRNA-IIIb dwarfed navel trees were not significantly different between the two planting densities and the controls. Fruit with higher commercial value was produced in the TsnRNA-IIIb dwarfed navel trees in the high-density planting by 3.9% and 4.6% over the TsnRNA-IIIb or controls in standard-density planting respectively. The increase in Y/LSU and fruit value for the TsnRNA-IIIb treated navel trees in the high-density plantings in combination with the reduced management cost of dwarfed trees could result in substantial higher profits for a commercial grove despite the higher establishment cost of high-density plantings.

Ribosomal protein L5 and transcription factor IIIA from Arabidopsis thaliana bind in vitro specifically Potato spindle tuber viroid RNA

Potato spindle tuber viroid (PSTVd) contains an element of tertiary structure -loop E- also present in eukaryotic 5S rRNA. The ribosomal protein L5 and transcription factor IIIA (TFIIIA) from Arabidopsis thaliana bind 5S rRNA in vitro and in vivo, mediating different functions that include nucleocytoplasmic transport and transcription activation, respectively. We show that A. thaliana L5 and TFIIIA also bind PSTVd (+) RNA in vitro with the same affinity as they bind 5S rRNA, whereas the affinity for a chloroplastic viroid is significantly lower. These two proteins might participate in the synthesis and delivery of PSTVd RNA in vivo.

Trans -cleaving hammerhead ribozymes with tertiary stabilizing motifs: in vitro and in vivo activity against a structured viroid RNA

Trans-cleaving hammerheads with discontinuous or extended stem I and with tertiary stabilizing motifs (TSMs) have been tested previously against short RNA substrates in vitro at low Mg2+ concentration. However, the potential of these ribozymes for targeting longer and structured RNAs in vitro and in vivo has not been examined. Here, we report the in vitro cleavage of short RNAs and of a 464-nt highly structured RNA from potato spindle tuber viroid (PSTVd) by hammerheads with discontinuous and extended formats at submillimolar Mg2+. Under these conditions, hammerheads derived from eggplant latent viroid and peach latent mosaic viroid (PLMVd) with discontinuous and extended formats, respectively, where the most active. Furthermore, a PLMVd-derived hammerhead with natural TSMs showed activity in vivo against the same long substrate and interfered with systemic PSTVd infection, thus reinforcing the idea that this class of ribozymes has potential to control pathogenic RNA replicons.

Biochemical properties of three plant nucleases with anticancer potential

Biochemical and structural properties of three recombinant (R), highly homologous, plant bifunctional nucleases from tomato (R-TBN1), hop (R-HBN1) and Arabis brassica (R-ABN1) were determined. These nucleases cleave single- and double-stranded substrates, as well as both RNA and DNA with nearly the same efficiency. In addition, they are able to cleave several artificial substrates and highly stable viroid RNA. They also possess 3′-nucleotidase activity; therefore, they can be classified as nuclease I family members. Interestingly, poly(G) is resistant to cleavage and moreover it inhibits dsDNase, ssDNase and RNase activity of the studied nucleases. All three nucleases exhibit zinc-dependence and a strong stimulatory effect of Zn 2+ for dsDNA cleavage. 3-D models, predicted on the basis of experimental structure of P1 nuclease, show nine amino acid residues responsible for interactions with zinc atoms, located in the same positions as in P1 nuclease. It was also shown that R-TBN1, R-HBN1, and R-ABN1 are all N-glycosylated. Oligosaccharidic chains constitute about 16% of their MW. In addition, an anticancer potential of the R-ABN1 is compared in this work with previously tested R-TBN1, and R-HBN1. R-ABN1 injected intravenously showed 70% inhibitory effect on growth of human prostate carcinoma in athymic mice.

Effects of mixtures of citrus viroids as transmissible small nuclear RNA on tree dwarfing and commercial scion performance on Carrizo citrange rootstock

The term ‘transmissible small nuclear ribonucleic acids' (TsnRNAs) describes well characterised viroid RNA species that do not induce any disease syndromes in specific citrus hosts but rather act as regulatory genetic elements modifying tree performance. Twelve‐year‐old navel orange and 10‐year‐old Clementine mandarin trees on Carrizo citrange (Citrus sinensis×Poncirus trifoliata) rootstock treated with a mixture of three TsnRNAs (−Ia, syn. Citrus bent leaf viroid, +IIa, syn. Hop stunt viroid and +IIIb, syn. Citrus dwarfing viroid) were reduced in size by 33% and 43%, respectively. Clementine trees treated with a mixture of TsnRNA−Ia+IIa or −Ia+IIIb also had reduced canopy volume (CV) (∼38 and 31%, respectively), whereas trees treated with TsnRNA−IIa+IIIb showed little effect. The effects of the double TsnRNA treatments −Ia+IIa and −Ia+IIIb on Clementine canopy size and commercial performance were comparable and in some cases superior to that of the triple TsnRNA mixture. The TsnRNA−Ia+IIa treatment had the most attractive commercial traits with increased production of Clementine fruit per CV (23.6%), more fruit with high commercial value (31.7%), and more fruit optimally distributed in the canopy (68% of fruit between 0.5 and 2.5 m). None of the TsnRNA treatments affected the growth of Carrizo rootstock seedlings after 8 years in the field. Navel orange and Clementine scions treated with the same triple TsnRNA mixture expressed different trunk and fruit production patterns although effects on CV were similar.

Viroids: self‐replicating, mobile, and fast‐evolving noncoding regulatory RNAs

Viroids are small, circular, and noncoding RNAs that infect plants. They replicate in the nucleus or chloroplast and then traffic from cell to cell and from organ to organ to establish systemic infection. Viroids achieve nearly all of the biological functions by directly interacting with host cellular factors. Viroid replication, together with replication of human hepatitis delta virus, demonstrates the biological novelty and significance of RNA-dependent RNA polymerase activities of DNA-dependent RNA polymerases. Viroid systemic infection uncovers a new biological principle--the role of three-dimensional RNA structural motifs mediating RNA trafficking between specific cells. Viroid diseases are virtually the consequences of host gene regulation by noncoding RNAs. A viroid RNA has the highest in vivo mutation rate among all known nucleic acid replicons. The host range of many viroids is expanding, essentially as a result of continuing and fast evolution of noncoding sequences/structures to gain new biological functions. Here, I discuss recent progress in these areas, emphasizing the broad significance of viroid research to the discovery of fundamental biological principles.

Identification of Potato Spindle Tuber Viroid Small RNA in Orobanche Ramosa by Microarray

ABSTRACTPost transcriptional gene silencing (PTGS) in plants is reported as a defence mechanism against pathogens, like invading viruses and viroids, transposons and transgenes. The processing of double stranded RNAs to 21–24 nt duplex RNAs (small interfering RNAs) by RNAse III-type nuclease homologs-Dicer-like (DCL) enzymes is a key step of this process.We were interested whether the viroid RNA can trigger silencing in parasitic plant O.ramosa. Therefore we infected O.ramosa attached to tomato with Potato Spindle Tuber Viroid (PSTVd). The presence of small interfering RNAs derived from PSTVd has been detected by miRNA microarray.

ウイロイド研究の新展開

Viroids are autonomously replicating, small single-stranded circular RNA pathogens that cause diseases in infected, susceptible plants. They are non-coding RNA replicon which replicate depending on host transcriptional machinery and develop disease symptoms through interactions with cellular components of the host. The small size and unique molecular structure of viroid RNA makes them an attractive system to analyze molecular features responsible for pathogenesis, RNA transport, or molecular evolution and adaptation to specific host species. Here we show the latest progress in viroid research on new disease epidemics, molecular evolution and host adaptation, and pathogenesis in relation to viroid-induced RNA silencing.

Epidemiology, pathogenesis and management of hepatitis D: update and challenges ahead

Hepatitis D is caused by infection with the hepatitis D virus (HDV) and is considered to be the most severe form of viral hepatitis in humans. Hepatitis D occurs only in individuals positive for the HBV surface antigen (HBsAg) as HDV is a defective RNA viroid that requires HBsAg for transmission. At least eight different HDV genotypes have been described and each has a characteristic geographic distribution and a distinct clinical course. HDV and HBV coinfection can be associated with complex and dynamic viral dominance patterns. Chronic HDV infection leads to more severe liver disease than HBV monoinfection and is associated with accelerated fibrosis progression, earlier hepatic decompensation and an increased risk for the development of hepatocellular carcinoma. So far, only IFN-alpha treatment has proven antiviral activity against HDV in humans and has been linked to improved long-term outcomes. Studies conducted in the past 2 years on the use of PEG-IFN-alpha show that a sustained virologic response to therapy, measured in terms of undetectable serum HDV RNA levels, can be achieved in about one quarter of patients with hepatitis D. Novel alternative treatment options including prenylation inhibitors are awaiting clinical development for use in hepatitis D.

Deep sequencing of viroid-derived small RNAs from grapevine provides new insights on the role of RNA silencing in plant-viroid interaction.

BackgroundViroids are circular, highly structured, non-protein-coding RNAs that, usurping cellular enzymes and escaping host defense mechanisms, are able to replicate and move through infected plants. Similarly to viruses, viroid infections are associated with the accumulation of viroid-derived 21–24 nt small RNAs (vd-sRNAs) with the typical features of the small interfering RNAs characteristic of RNA silencing, a sequence-specific mechanism involved in defense against invading nucleic acids and in regulation of gene expression in most eukaryotic organisms.Methodology/Principal FindingsTo gain further insights on the genesis and possible role of vd-sRNAs in plant-viroid interaction, sRNAs isolated from Vitis vinifera infected by Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1) were sequenced by the high-throughput platform Solexa-Illumina, and the vd-sRNAs were analyzed. The large majority of HSVd- and GYSVd1-sRNAs derived from a few specific regions (hotspots) of the genomic (+) and (−) viroid RNAs, with a prevalence of those from the (−) strands of both viroids. When grouped according to their sizes, vd-sRNAs always assumed a distribution with prominent 21-, 22- and 24-nt peaks, which, interestingly, mapped at the same hotspots.Conclusions/SignificanceThese findings show that different Dicer-like enzymes (DCLs) target viroid RNAs, preferentially accessing to the same viroid domains. Interestingly, our results also suggest that viroid RNAs may interact with host enzymes involved in the RNA-directed DNA methylation pathway, indicating more complex scenarios than previously thought for both vd-sRNAs genesis and possible interference with host gene expression.

The Biology of Viroid-Host Interactions

Viroids are single-stranded, circular, and noncoding RNAs that infect plants. They replicate in the nucleus or chloroplast and then traffic cell-to-cell through plasmodesmata and long distance through the phloem to establish systemic infection. They also cause diseases in certain hosts. All functions are mediated directly by the viroid RNA genome or genome-derived RNAs. I summarize recent advances in the understanding of viroid structures and cellular factors enabling these functions, emphasizing conceptual developments, major knowledge gaps, and future directions. Newly emerging experimental systems and research tools are discussed that are expected to enable significant progress in a number of key areas. I highlight examples of groundbreaking contributions of viroid research to the development of new biological principles and offer perspectives on using viroid models to continue advancing some frontiers of life science.

RNAi‐mediated resistance to Potato spindle tuber viroid in transgenic tomato expressing a viroid hairpin RNA construct

Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistant to degradation by RNA interference (RNAi). In this article, we report that transgenic tomato plants expressing a hairpin RNA (hpRNA) construct derived from Potato spindle tuber viroid (PSTVd) sequences exhibit resistance to PSTVd infection. Resistance seems to be correlated with high-level accumulation of hpRNA-derived short interfering RNAs (siRNAs) in the plant. Thus, although small RNAs produced by infecting viroids [small RNAs of PSTVd (srPSTVds)] do not silence viroid RNAs efficiently to prevent their replication, hpRNA-derived siRNAs (hp-siRNAs) appear to effectively target the mature viroid RNA. Genomic mapping of the hp-siRNAs revealed an unequal distribution of 21- and 24-nucleotide siRNAs of both (+)- and (-)-strand polarities along the PSTVd genome. These data suggest that RNAi can be employed to engineer plants for viroid resistance, as has been well established for viruses.

An artificial chimeric derivative of Citrus viroid V involves the terminal left domain in pathogenicity

The recently described Citrus viroid V (CVd-V) induces, in Etrog citron, mild stunting and very small necrotic lesions and cracks, sometimes filled with gum. As Etrog citron plants co-infected with Citrus dwarfing viroid (CDVd) and CVd-V show synergistic interactions, these host-viroid combinations provide a convenient model to identify the pathogenicity determinant(s). The biological effects of replacing limited portions of the rod-like structure of CVd-V with the corresponding portions of CDVd are reported. Chimeric constructs were synthesized using a novel polymerase chain reaction-based approach, much more flexible than those based on restriction enzymes used in previous studies. Of the seven chimeras (Ch) tested, only one (Ch5) proved to be infectious. Plants infected with Ch5 showed no symptoms and, although this novel chimera was able to replicate to relatively high titres in singly infected plants, it was rapidly displaced by either CVd-V or CDVd in doubly infected plants. The results demonstrate that direct interaction(s) between structural elements in the viroid RNA (in this case, the terminal left domain) and as yet unidentified host factors play an important role in modulating viroid pathogenicity. This is the first pathogenic determinant mapped in species of the genus Apscaviroid.

Pepper chat fruit viroid: Biological and molecular properties of a proposed new species of the genus Pospiviroid

In autumn 2006, a new disease was observed in a glasshouse-grown crop of sweet pepper ( Capsicum annuum L.) in the Netherlands. Fruit size of the infected plants was reduced up to 50%, and plant growth was also slightly reduced. Here we show that the disease is caused by a previously non-described viroid. The pepper viroid is transmitted by both mechanical inoculation and pepper seeds and, when inoculated experimentally, it infects several solanaceous plant species inducing vein necrosis and reduced fruit and tuber size in tomato and potato, respectively. The viroid RNA genome consists of 348 nucleotides and, with minor modifications, it has the central conserved and the terminal conserved regions characteristic of members of the genus Pospiviroid. Classification of the pepper viroid within the genus Pospiviroid is further supported by the presence and structure of hairpins I and II, the presence of internal and external RY motifs, and phylogenetic analyses. The primary structure of the pepper viroid only showed a maximum of 66% nucleotide sequence identity with other viroids, which is far below the main species demarcation limit of 90%. According to its biological and molecular properties, we propose to assign the pepper viroid to a new species within the genus Pospiviroid, and to name this new species Pepper chat fruit viroid.

Interplay between viroid-induced pathogenesis and RNA silencing pathways

Of all known plant pathogens, viroids have the lowest biological complexity. Their genome consists of a naked RNA without protein-encoding capacity. However, viroids contain sufficient genetic information to establish infection in susceptible hosts. The process by which this tiny RNA subverts the plant cell machinery by coercing the host to express symptoms of viroid infection is the 'Holy Grail' that has been searched for since the first viroid-induced disease was described. Recently, a large body of evidence has led to the emergent view that RNA silencing has a crucial role in viroid pathogenesis and evolution. Here, we chronologically analyse the relevant findings supporting this idea and propose a model to explain the possible interrelation between the trans-acting small interfering RNA (ta-siRNA) biogenesis pathway and viroid replication and pathogenesis.

Viroids: Uniquely Simple and Tractable Models to Elucidate Regulation of Cell-to-Cell Trafficking of RNA

Cell-to-cell trafficking of RNA is an emerging biological principle that integrates systemic gene regulation, viral infection, cellular antiviral response, and cell-to-cell communication. The mechanisms regulating this trafficking remain to be understood. A testable hypothesis is that many RNAs have distinct structural motifs to direct trafficking across various cellular boundaries. The identification of structural motifs in a noncoding viroid RNA required for trafficking between specific cell types in a plant provides compelling genetic evidence to support this hypothesis. Here we discuss the unique features of viroids as well as experimental data to demonstrate these RNAs as simple and highly tractable models to elucidate the general mechanisms of RNA trafficking.

Structure–function analysis of the ribozymes of chrysanthemum chlorotic mottle viroid: a loop–loop interaction motif conserved in most natural hammerheads

Loop–loop tertiary interactions play a key role in the folding and catalytic activity of natural hammerhead ribozymes. Using a combination of NMR spectroscopy, site-directed mutagenesis and kinetic and infectivity analyses, we have examined the structure and function of loops 1 and 2 of the (+) and (–) hammerheads of chrysanthemum chlorotic mottle viroid RNA. In both hammerheads, loop 1 is a heptanucleotide hairpin loop containing an exposed U at its 5′ side and an extrahelical U at its 3′-side critical for the catalytic activity of the ribozyme in vitro and for viroid infectivity in vivo, whereas loop 2 has a key opened A at its 3′-side. These structural features promote a specific loop–loop interaction motif across the major groove. The essential features of this tertiary structure element, base pairing between the 5′ U of loop 1 and the 3′ A of loop 2, and interaction of the extrahelical pyrimidine of loop 1 with loop 2, are likely shared by a significant fraction of natural hammerheads.

Apricot is a new host ofApple scar skin viroid

Apple scar skin viroid (ASSVd), a pathogenic RNA viroid, infects apple and pear trees. To determine if ASSVd can also infect apricot, we performed reverse transcription-polymerase chain reaction (RT-PCR), spot hybridisation andin situ RT-PCR and detected ASSVd-specific sequences from apricot. We cloned and sequenced the apricot-derived ASSVd and registered it in GenBank.In situ RT-PCR further confirmed the existence of ASSVd in apricot leaf tissues. Our data indicate that apricot is a novel host of ASSVd. These findings provide the basis for the rapid identification of ASSVd in apricot.