Plant Viral Satellite Research Articles

Betasatellite-encoded βC1 protein regulates helper virus accumulation by interfering with the ATP hydrolysis activity of geminivirus-encoded replication initiator protein.

Geminivirus-betasatellite disease complexes are an epidemic threat to the majority of economically important crops across the world. Plant virus satellites including betasatellites are maintained by their associated helper virus. Geminivirus-betasatellites influence viral pathogenesis by substantially increasing or decreasing their helper virus accumulation. In the present study, we attempted to understand the mechanistic details of the geminivirus-betasatellite interaction. Here, we used tomato leaf curl Gujarat virus (ToLCGV) and tomato leaf curl Patna betasatellite (ToLCPaB) as a model system. This study reveals that ToLCGV can efficiently trans-replicate ToLCPaB in Nicotiana benthamiana plants, but ToLCPaB greatly reduced the accumulation of its helper virus DNA. For the first time, we have identified that the ToLCPaB-encoded βC1 protein is able to interact with ToLCGV-encoded replication initiator protein (Rep). In addition, we demonstrate that the C-terminal region of βC1 interacts with the C-terminus of Rep (RepC) protein. Our previous study had established that βC1 proteins encoded by diverse betasatellites possess a novel ATP hydrolysis activity and the conserved lysine/arginine residues at positions 49 and 91 are necessary for this function. Here, we show that mutating lysine at positions 49 to alanine of βC1 (βC1K49A) protein did not affect its ability to interact with RepC protein. Biochemical studies performed with ATP hydrolysis activity-deficient K49A mutated βC1 (βC1K49A) and RepC proteins revealed that Rep-βC1 interaction interferes with the ATP hydrolysis activity of Rep protein. Further, we demonstrate that βC1 protein is able to interact with D227A and D289A mutated RepC proteins but not with D262A, K272A or D286A mutated RepC proteins, suggesting that the βC1-interacting region of Rep protein encompasses its Walker-B and B' motifs. The results of docking studies supported that the βC1-interacting region of Rep protein encompasses its motifs associated with ATP binding and ATP hydrolysis activities. Docking studies also provided evidence that the Rep-βC1 interaction interferes with the ATP binding activity of Rep protein. Together, our findings suggest that βC1 protein regulates helper virus accumulation by interfering with the ATP hydrolysis activity of helper virus Rep protein.

A plant virus satellite RNA directly accelerates wing formation in its insect vector for spread

Cucumber mosaic virus (CMV) often accompanies a short RNA molecule called a satellite RNA (satRNA). When infected with CMV in the presence of Y-satellite RNA (Y-sat), tobacco leaves develop a green mosaic, then turn yellow. Y-sat has been identified in the fields in Japan. Here, we show that the yellow leaf colour preferentially attracts aphids, and that the aphids fed on yellow plants, which harbour Y-sat-derived small RNAs (sRNAs), turn red and subsequently develop wings. In addition, we found that leaf yellowing did not necessarily reduce photosynthesis, and that viral transmission was not greatly affected despite the low viral titer in the Y-sat-infected plants. Y-sat-infected plants can therefore support a sufficient number of aphids to allow for efficient virus transmission. Our results demonstrate that Y-sat directly alters aphid physiology via Y-sat sRNAs to promote wing formation, an unprecedented survival strategy that enables outward spread via the winged insect vector.

Molecular interactions of plant viral satellites.

Plant viral satellites fall under the category of subviral agents. Their genomes are composed of small RNA or DNA molecules a few hundred nucleotides in length and contain an assortment of highly complex and overlapping functions. Each lacks the ability to either replicate or undergo encapsidation or both in the absence of a helper virus (HV). As the number of known satellites increases steadily, our knowledge regarding their sequence conservation strategies, means of replication and specific interactions with host and helper viruses is improving. This review demonstrates that the molecular interactions of these satellites are unique and highly complex, largely influenced by the highly specific host plants and helper viruses that they associate with. Circularized forms of single-stranded RNA are of particular interest, as they have recently been found to play a variety of novel cellular functions. Linear forms of satRNA are also of great significance as they may complement the helper virus genome in exacerbating symptoms, or in certain instances, actively compete against it, thus reducing symptom severity. This review serves to describe the current literature with respect to these molecular mechanisms in detail as well as to discuss recent insights into this emerging field in terms of evolution, classification and symptom development. The review concludes with a discussion of future steps in plant viral satellite research and development.

How can plant virus satellite RNAs alter the effects of plant virus infection? A study of the changes in the Nicotiana benthamiana proteome after infection by Peanut stunt virus in the presence or absence of its satellite RNA

Peanut stunt virus (PSV), which belongs to the Cucumovirus genus, is a pathogen of legumes. Certain PSV strains associated with a satellite RNA (satRNA) modify the symptoms of infected plants and interfere with plant metabolism. We used PSV-P genomic transcripts (GTs) with and without PSV-P satRNA and a comparative proteomic 2D-DIGE/MS study to assess their effects on Nicotiana benthamiana infection. When the proteomes of the PSV-P genomic transcripts-infected (no satRNA present) and mock-inoculated plants were compared 29 differentially regulated proteins were found. When comparisons were made for plants infected with PSV-P-GT in the presence or absence of satRNA, and for mock-infected plants and those infected with the satRNA-associated PSV-P-GT, 40 and 60 such proteins, respectively, were found. The presence of satRNA mostly decreased the amounts of the affected host proteins. Proteins involved in photosynthesis and carbohydrate metabolism, for example ferredoxin-NADP-reductase and malate dehydrogenase, are among the identified affected proteins in all comparisons. Proteins involved in protein synthesis and degradation were also affected. Such proteins include chaperonin 60β--whose abundance of the proteins changed for all comparisons--and aminopeptidase that is a satRNA- or PSV-P-GT/satRNA-responsive protein. Additionally, the levels of the stress-related proteins superoxide dismutase and acidic endochitinase Q increased in the PSV-P-GT- and PSV-P-GT/satRNA-infected plants. This study appears to be the first report on plant proteome changes in response to a satRNA presence during viral infection and, as such, may provide a reference for future studies concerning the influence of satRNAs during viral infections.

Gene from a novel plant virus satellite from grapevine identifies a viral satellite lineage

We have identified the genome of a novel viral satellite in deep sequence analysis of double-stranded RNA from grapevine. The genome was 1,060 bases in length, and encoded two open reading frames. Neither frame was related to any known plant virus gene. But translation of the longer frame showed a protein sequence similar to those of other plant virus satellites. Other than in commonalities they shared in this gene sequence, members of that group were extensively divergent. The reading frame in this gene from the novel satellite could be translationally coupled to an adjacent reading frame in the -1 register, through overlapping start/stop codons. These overlapping AUGA start/stop codons were adjacent to a sequence that could be folded into a pseudoknot structure. Field surveys with PCR probes specific for the novel satellite revealed its presence in 3% of the grapevines (n = 346) sampled.

Exploring the diversity of plant DNA viruses and their satellites using vector-enabled metagenomics on whiteflies.

Current knowledge of plant virus diversity is biased towards agents of visible and economically important diseases. Less is known about viruses that have not caused major diseases in crops, or viruses from native vegetation, which are a reservoir of biodiversity that can contribute to viral emergence. Discovery of these plant viruses is hindered by the traditional approach of sampling individual symptomatic plants. Since many damaging plant viruses are transmitted by insect vectors, we have developed “vector-enabled metagenomics” (VEM) to investigate the diversity of plant viruses. VEM involves sampling of insect vectors (in this case, whiteflies) from plants, followed by purification of viral particles and metagenomic sequencing. The VEM approach exploits the natural ability of highly mobile adult whiteflies to integrate viruses from many plants over time and space, and leverages the capability of metagenomics for discovering novel viruses. This study utilized VEM to describe the DNA viral community from whiteflies (Bemisia tabaci) collected from two important agricultural regions in Florida, USA. VEM successfully characterized the active and abundant viruses that produce disease symptoms in crops, as well as the less abundant viruses infecting adjacent native vegetation. PCR assays designed from the metagenomic sequences enabled the complete sequencing of four novel begomovirus genome components, as well as the first discovery of plant virus satellites in North America. One of the novel begomoviruses was subsequently identified in symptomatic Chenopodium ambrosiodes from the same field site, validating VEM as an effective method for proactive monitoring of plant viruses without a priori knowledge of the pathogens. This study demonstrates the power of VEM for describing the circulating viral community in a given region, which will enhance our understanding of plant viral diversity, and facilitate emerging plant virus surveillance and management of viral diseases.

Complex formation of quercetin with lanthanum enhances binding to plant viral satellite double stranded RNA

Due to the broad spectrum of biological activities of flavonoids, their target molecules in the cell are intensively studied. We examined the interactions of the flavonoid quercetin (Q) and its lanthanum complex (QLa(3+)) with very recently isolated plant viral satellite (sat) dsRNA. Comparison of the cumulative binding affinity and the estimated intercalative binding constant pointed towards an additional binding mode of quercetin to exclusively viral dsRNA, which is not recorded for synthetic dsRNAs. The QLa(3+) showed significantly higher affinity toward viral dsRNA than Q and La(3+) alone, most likely as the consequence of quercetin intercalation accompanied by additional electrostatic interaction of La(3+) with the negatively charged viral RNA backbone.

Separation of plant viral satellite double-stranded RNA using high-performance liquid chromatography

High-performance liquid chromatography was developed for further separation of double-stranded (ds) RNAs obtained by CF-11 cellulose chromatography from plants infected with satellite associated cucumber mosaic virus. Fractions separated by monolithic polymer column, especially applicable for nucleic acid analyses, were identified electrophoretically and confirmed with a polymerase chain reaction test. Once standardized, the method has revealed clear evidence of satellite presence without precipitation and electrophoresis. According to demonstrated sensitivity, its application in the preliminary diagnostics of field samples is also predictable. Principally, it can be used as a powerful preparative approach resulting in highly pure satellite dsRNA for further analyses.

Structural Dynamics of Catalytic RNA Highlighted by Fluorescence Resonance Energy Transfer

RNA performs a multitude of essential cellular functions involving the maintenance, transfer, and processing of genetic information. The reason probably is twofold: (a) Life started as a prebiotic RNA World, in which RNA served as the genetic information carrier and catalyzed all chemical reactions required for its proliferation and (b) some of the RNA World functions were conserved throughout evolution because neither DNA nor protein is as adept in fulfilling them. A particular advantage of RNA is its high propensity to form alternative structures as required in subsequent steps of a reaction pathway. Here I describe fluorescence resonance energy transfer (FRET) as a method to monitor a crucial conformational transition on the reaction pathway of the hairpin ribozyme, a small catalytic RNA motif from a self-replicating plant virus satellite RNA and well-studied paradigm of RNA folding. Steady-state FRET measurements in solution allow one to measure the kinetics and requirements of docking of its two independently folding domains; time-resolved FRET reveals the relative thermodynamic stability of the undocked (extended, inactive) and docked (active) ribozyme conformations; while single-molecule FRET experiments will highlight the dynamics of RNA at the individual molecule level. Similar domain docking events are expected to be at the heart of many biological functions of RNA, and the described FRET techniques promise to be adaptable to most of the involved RNA systems.

Imaging of single hairpin ribozymes in solution by atomic force microscopy.

The hairpin ribozyme is a short endonucleolytic RNA motif isolated from a family of related plant virus satellite RNAs. It consists of two independently folding domains, each comprising two Watson-Crick helices flanking a conserved internal loop. The domains need to physically interact (dock) for catalysis of site-specific cleavage and ligation reactions. Using tapping-mode atomic force microscopy in aqueous buffer solution, we were able to produce high quality images of individual hairpin ribozyme molecules with extended terminal helices. Three RNA constructs with either the essential cleavage site guanosine or a detrimental adenosine substitution and with or without a 6-nt insertion to confer flexibility to the interdomain hinge show structural differences that correlate with their ability to form the active docked conformation. The observed contour lengths and shapes are consistent with previous bulk-solution measurements of the transient electric dichroism decays for the same RNA constructs. The active docked construct appears as an asymmetrically docked conformation that might be an indication of a more complicated docking event than a simple collapse around the interdomain hinge.

Bamboo mosaic potexvirus satellite RNA (satBaMV RNA)-encoded P20 protein preferentially binds to satBaMV RNA.

A satellite RNA of 836 nucleotides [excluding the poly(A) tail] depends on the bamboo mosaic potexvirus (BaMV) for its replication and encapsidation. The BaMV satellite RNA (satBaMV) contains a single open reading frame encoding a 20-kDa nonstructural protein (P20). The P20 protein with eight histidine residues at the C terminus was overexpressed in Escherichia coli. Experiments of gel retardation, UV cross-linking, and Northwestern hybridization demonstrated that purified P20 was a nucleic-acid-binding protein. The binding of P20 to nucleic acids was strong and highly cooperative. P20 preferred binding to satBaMV- or BaMV-related sequences rather than to nonrelated sequences. By deletion analysis, the P20 binding sites were mainly located at the 5' and 3' untranslated regions of satBaMV RNA, and the RNA-protein interactions could compete with the poly(G) and, less efficiently, with the poly(U) homopolymers. The N-terminal arginine-rich motif of P20 was the RNA binding domain, as shown by in-frame deletion analysis. This is the first report that a plant virus satellite RNA-encoded nonstructural protein preferentially binds with nucleic acids.

Tomato Cell Death Mediated By Complementary Plant Viral Satellite RNA Sequences

Cell death (necrosis) and severe yellowing (chlorosis) in tomato are induced by cucumber mosaic virus (CMV) supporting particular satellite RNAs. To determine whether CMV RNA sequences also are needed to induce necrosis or chlorosis, tomato seedlings were infected with the potato virus X vector expressing either a necrogenic or chlorosis-inducing satellite RNA of CMV. The infected plants did not develop chlorosis, although they did develop necrosis, but only when all or part of a 335-nucleotide necrogenic satellite RNA was expressed in the (-) polarity; i.e., the strand not packaged in virus particles. Computer-assisted secondary structure analysis suggests that the necrogenicity domain is an octanucleotide loop and adjacent base-paired stem of a thermodynamically stable hairpin structure.

The Hairpin Ribozyme: Discovery, Two-Dimensional Model, and Development for Gene Therapy

This review chronicles the discovery of the hairpin ribozyme, its characterization, and determination of the two-dimensional structure, culminating with its use for human gene therapy as an AIDS therapeutic. The minimal sequence constituting the hairpin ribozyme catalytic domain was identified from a small plant viral satellite RNA. Biochemical characterization showed it to be among the most efficient of all known ribozymes. Mutagenesis determined that the two-dimensional structure had four helices, consisting of 17 Watson-Crick base pairs and one A:G pair for a total of 18 bp. The helices were interspersed with five single-stranded loops. Helices 1 and 2 were located between the ribozyme and substrate, allowing the ribozyme to recognize the substrate. The substrate had a sequence preference of BN*GUC where * is the site of cleavage and N*GUC the substrate loop between these two helices. By using sequences of this type, it was possible to design the ribozyme to base pair with the substrate and cleave heterologous RNA substrates-leading to design of the hairpin ribozyme for gene therapy. The HIV-1 sequence was searched for suitable target sites, and ribozymes were designed, optimized, catalytically characterized, and tested in vivo against HIV-1 targets. Two ribozymes had excellent in vitro catalytic parameters and inhibited in vivo expression of viral proteins by 3-4 logs in tissue culture cells. Viral replication was inhibited as well. They have been developed as human AIDS therapeutics, and will likely be the first ribozymes to be tested as human drugs in clinical trials.

Secondary Structure Probing of Potato Spindle Tuber Viroid (PSTVd) and Sequence Comparison with Other Small Pathogenic RNA Replicons Provides Evidence for Central Non-canonical Base-pairs, Large A-rich Loops, and a Terminal Branch

Using PCR and in vitrotranscription, linear (non-circular) unit-length (+)strand RNA molecules of a lethal PSTVd variant were produced which are able to initiate typical disease symptoms when inoculated into tomato. Non-denaturing gel electrophoresis shows that these transcripts can adopt the same two conformations as circular PSTVd molecules, namely a fast migrating rod-like and a slowly migrating cruciform structure. The rod-like conformer of two end-labelled transcripts was probed with nucleases and dimethyl sulphate, revealing that in solution its right part is identical to computer prediction. In the left part, however, three unique features could be substantiated. (1) In the central region a UV-cross-link able loop is closed and thus contains non-canonical base-pairs (“loop E structure”). (2) Three large “pre-melting loops” are present at 25° to 37°C. The structure of the leftmost one, which is A-rich and conserved in most viroids, correlates with pathogenicity. (3) Two small stem-loops instead of an unbranched structure are found at the left terminus. These hairpins can form in all “large” viroids (≈300 nucleotides or longer), thus placing the dodecamer conserved among these viroids, GGUUCCUGUGGU, within the upper helix and the branch junction. A large viroid from Iresinelacks one of these hairpins, whereas all “small” viroids (≈300 nucleotides or smaller) lack both. In several plant virus satellite RNAs and the newt satellite RNA, the motif GAUUU(U) and dodecamer remnants appear in an equivalent structure comprising two or three hairpins. Using lead- and terbium-induced cleavage of the RNA, metal binding sites were found, mostly in loops. Thus, probing of PSTVd RNA and comparison with other viroids, virusoids, and satellite RNAs contributes to our understanding of the secondary structure of these small pathogenic RNAs.

Plant virus satellite RNAs and their role in engineering resistance to virus diseases

The mechanism of satellite RNA (Sat-RNA) mediated attenuation of virus disease was suggested to be the result of competition between satellite RNAs and their helper viral RNAs for replication. Subcellular distribution of viral coat protein in cucumber mosaic virus (CMV) infected leaf tissues shows that the presence of Sat-RNAs interferes with the movement of CMV coat protein into chloroplants. As a strategy for controlling virus diseases, the expression of Sat-RNAs in transgenic plants confers some extent of resistance that may not be strong enough to protect the plants from natural virus infections. Transgenic plants expressing both the Sat-RNA and the coat protein of CMV exhibit enhanced resistance to the virus.

Tobamovirus helper specificity of satellite tobacco mosaic virus involves a domain near the 5' end of the satellite genome

The molecular basis of the interactions between plant virus satellites and their helper viruses is not understood. The features of the satellite tobacco mosaic virus (STMV) genome that determine tobamovirus helper specificity were investigated using two independent strategies. The first tested the possible significance of regions of nearly identical sequence within the 3'-terminal 150 bases of the genomes of STMV and its natural helper virus, tobacco mild green mosaic virus (TMGMV). A chimeric STMV clone containing the 3' terminus of tobacco mosaic virus (TMV-U1) RNA was infectious in coinfections with TMGMV, but it did not replicate with TMV-U1. In the second strategy, populations of STMV adapted to replication with four alternative helper tobamoviruses were generated by serial passage in tobacco. RNase protection analyses of these RNA populations showed that in all cases there had been a genetic change 50 to 60 bases from the 5' terminus of the STMV genome. Similar changes were detected in several progenies of STMV clones replicated with TMV-U1, indicating that change at this site was essential for replication with a helper virus other than TMGMV. Sequence analyses of the changes at this 'helper adaptation domain' showed consistently the deletion of a single G from five consecutive Gs at bases 61 to 65. Infectivity experiments with STMV clones containing this G deletion showed that this change alone was not sufficient to modify helper specificity, so additional factors which remain to be identified must also be involved. Nevertheless, these experiments show the ability of STMV populations to undergo rapid, reproducible evolution by both selection of pre-existing variants and de novo mutation, and constitute the first molecular demonstration that the helper virus acts as a selection pressure on the evolution of satellite populations.

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

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

A rapid and versatile method for cloning viroids or other circular plant pathogenic RNAs

We surveyed the occurrence of unique restriction sites on the cDNAs of viroids, virusoids, and plant viral satellite RNAs that have a circular RNA as an intermediate of replication and found that four such sites would linearize their circular cDNAs. A rapid and simple method was then developed for cloning a naturally occurring viroid from Nematanthus wettsteinii plants. First-strand cDNA was synthesized using random hexanucleotide DNA primers and M-MuLV reverse transcriptase (Superscript RT). Second-strand DNA was synthesized by employing the replacement synthesis method using Escherichia coli RNase H, E. coli DNA polymerase I, E. coli DNA ligase, and β-NAD +. The circular double-stranded DNA was analyzed for the presence of commonly available, unique restriction sites and subsequently linearized with a selected restriction enzyme. The linear cDNA was ligated to dephosphorylated plasmid vector pGEM 3Z f(+) and cloned in E. coli strain DH5α. This cDNA cloning procedure is suitable for cloning sequence variants of well-characterized viroids, virusoids, certain plant viral satellite RNAs, and new such pathogens of unknown sequence.

A plant virus satellite RNA exhibits a significant sequence complementarity to a chloroplast tRNA.

A plant virus satellite RNA exhibits a significant sequence complementarity to a chloroplast tRNA.

A single nucleotide change within a plant virus satellite RNA alters the host specificity of disease induction

Some RNA plant viruses contain satellite RNAs which are dependent upon their associated virus for replication and encapsidation. Some cucumber mosaic virus satellite RNAs induce chlorosis on any of several host plants, including either tobacco or tomato. The exchange of sequence domains between cDNA clones of chlorosis-inducing and non-pathogenic satellite RNAs delimited the chlorosis domain for both tobacco and tomato plants to the same region. Site-directed mutagenesis of one nucleotide (149) within this domain changed the host plant specificity for a chlorotic response to satellite RNA infection from tomato to tobacco. Within the chlorosis domain, three conserved nucleotides are either deleted or altered in all satellite RNAs that do not induce chlorosis. Deletion of one of these nucleotides (153) did not affect satellite RNA replication but rendered it non-pathogenic. Thus, two single nucleotides have been identified which play central roles in those interactions between the virus, its satellite RNA and the host plant, and together result in a specific disease state.