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1.  Endogenous pararetroviral sequences in tomato (Solanum lycopersicum) and related species 
BMC Plant Biology  2007;7:24.
Background
Endogenous pararetroviral sequences (EPRVs) are a recently discovered class of repetitive sequences that is broadly distributed in the plant kingdom. The potential contribution of EPRVs to plant pathogenicity or, conversely, to virus resistance is just beginning to be explored. Some members of the family Solanaceae are particularly rich in EPRVs. In previous work, EPRVs have been characterized molecularly in various species of Nicotiana including N.tabacum (tobacco) and Solanum tuberosum (potato). Here we describe a family of EPRVs in cultivated tomato (Solanum lycopersicum L.) and a wild relative (S.habrochaites).
Results
Molecular cloning and DNA sequence analysis revealed that tomato EPRVs (named LycEPRVs) are most closely related to those in tobacco. The sequence similarity of LycEPRVs in S.lycopersicum and S.habrochaites indicates they are potentially derived from the same pararetrovirus. DNA blot analysis revealed a similar genomic organization in the two species, but also some independent excision or insertion events after species separation, or flanking sequence divergence. LycEPRVs share with the tobacco elements a disrupted genomic structure and frequent association with retrotransposons. Fluorescence in situ hybridization revealed that copies of LycEPRV are dispersed on all chromosomes in predominantly heterochromatic regions. Methylation of LycEPRVs was detected in CHG and asymmetric CHH nucleotide groups. Although normally quiescent EPRVs can be reactivated and produce symptoms of infection in some Nicotiana interspecific hybrids, a similar pathogenicity of LycEPRVs could not be demonstrated in Solanum L. section Lycopersicon [Mill.] hybrids. Even in healthy plants, however, transcripts derived from multiple LycEPRV loci and short RNAs complementary to LycEPRVs were detected and were elevated upon infection with heterologous viruses encoding suppressors of PTGS.
Conclusion
The analysis of LycEPRVs provides further evidence for the extensive invasion of pararetroviral sequences into the genomes of solanaceous plants. The detection of asymmetric CHH methylation and short RNAs, which are hallmarks of RNAi in plants, suggests that LycEPRVs are controlled by an RNA-mediated silencing mechanism.
doi:10.1186/1471-2229-7-24
PMCID: PMC1899175  PMID: 17517142
2.  Widespread Endogenization of Genome Sequences of Non-Retroviral RNA Viruses into Plant Genomes 
PLoS Pathogens  2011;7(7):e1002146.
Non-retroviral RNA virus sequences (NRVSs) have been found in the chromosomes of vertebrates and fungi, but not plants. Here we report similarly endogenized NRVSs derived from plus-, negative-, and double-stranded RNA viruses in plant chromosomes. These sequences were found by searching public genomic sequence databases, and, importantly, most NRVSs were subsequently detected by direct molecular analyses of plant DNAs. The most widespread NRVSs were related to the coat protein (CP) genes of the family Partitiviridae which have bisegmented dsRNA genomes, and included plant- and fungus-infecting members. The CP of a novel fungal virus (Rosellinia necatrix partitivirus 2, RnPV2) had the greatest sequence similarity to Arabidopsis thaliana ILR2, which is thought to regulate the activities of the phytohormone auxin, indole-3-acetic acid (IAA). Furthermore, partitivirus CP-like sequences much more closely related to plant partitiviruses than to RnPV2 were identified in a wide range of plant species. In addition, the nucleocapsid protein genes of cytorhabdoviruses and varicosaviruses were found in species of over 9 plant families, including Brassicaceae and Solanaceae. A replicase-like sequence of a betaflexivirus was identified in the cucumber genome. The pattern of occurrence of NRVSs and the phylogenetic analyses of NRVSs and related viruses indicate that multiple independent integrations into many plant lineages may have occurred. For example, one of the NRVSs was retained in Ar. thaliana but not in Ar. lyrata or other related Camelina species, whereas another NRVS displayed the reverse pattern. Our study has shown that single- and double-stranded RNA viral sequences are widespread in plant genomes, and shows the potential of genome integrated NRVSs to contribute to resolve unclear phylogenetic relationships of plant species.
Author Summary
Eukaryotic genomes contain sequences that have originated from DNA viruses and reverse-transcribing viruses, i.e., retroviruses, pararetroviruses (DNA viruses), and transposons. However, the sequences of non-retroviral RNA viruses, which are unable to convert their genomes to DNA, were until recently considered not to be integrated into eukaryotic nuclear genomes. We present evidence for multiple independent events of horizontal gene transfer from a wide range of RNA viruses, including plus-sense, minus-sense, and double-stranded RNA viruses, into the genomes of distantly related plant lineages. Some non-retroviral integrated RNA viral sequences are conserved across genera within a plant family, whereas others are retained only in a limited number of species in a genus. Integration profiles of non-retroviral integrated RNA viral sequences demonstrate the potential of these sequences to serve as powerful molecular tools for deciphering phylogenetic relationships among related plants. Moreover, this study highlights plants co-opting non-retroviral RNA virus sequences, and provides insights into plant genome evolution and interplay between non-reverse-transcribing RNA viruses and their hosts.
doi:10.1371/journal.ppat.1002146
PMCID: PMC3136472  PMID: 21779172
3.  Cross-Species Functionality of Pararetroviral Elements Driving Ribosome Shunting 
PLoS ONE  2008;3(2):e1650.
Background
Cauliflower mosaic virus (CaMV) and Rice tungro bacilliform virus (RTBV) belong to distinct genera of pararetroviruses infecting dicot and monocot plants, respectively. In both viruses, polycistronic translation of pregenomic (pg) RNA is initiated by shunting ribosomes that bypass a large region of the pgRNA leader with several short (s)ORFs and a stable stem-loop structure. The shunt requires translation of a 5′-proximal sORF terminating near the stem. In CaMV, mutations knocking out this sORF nearly abolish shunting and virus viability.
Methodology/Principal Findings
Here we show that two distant regions of the CaMV leader that form a minimal shunt configuration comprising the sORF, a bottom part of the stem, and a shunt landing sequence can be replaced by heterologous sequences that form a structurally similar configuration in RTBV without any dramatic effect on shunt-mediated translation and CaMV infectivity. The CaMV-RTBV chimeric leader sequence was largely stable over five viral passages in turnip plants: a few alterations that did eventually occur in the virus progenies are indicative of fine tuning of the chimeric sequence during adaptation to a new host.
Conclusions/Significance
Our findings demonstrate cross-species functionality of pararetroviral cis-elements driving ribosome shunting and evolutionary conservation of the shunt mechanism.
We are grateful to Matthias Müller and Sandra Pauli for technical assistance. This work was initiated at Friedrich Miescher Institute (Basel, Switzerland). We thank Prof. Thomas Boller for hosting the group at the Institute of Botany.
doi:10.1371/journal.pone.0001650
PMCID: PMC2241666  PMID: 18286203
4.  RNA Silencing Is Resistant to Low-Temperature in Grapevine 
PLoS ONE  2013;8(12):e82652.
RNA silencing is a natural defence mechanism against viruses in plants, and transgenes expressing viral RNA-derived sequences were previously shown to confer silencing-based enhanced resistance against the cognate virus in several species. However, RNA silencing was shown to dysfunction at low temperatures in several species, questioning the relevance of this strategy in perennial plants such as grapevines, which are often exposed to low temperatures during the winter season. Here, we show that inverted-repeat (IR) constructs trigger a highly efficient silencing reaction in all somatic tissues in grapevines. Similarly to other plant species, IR-derived siRNAs trigger production of secondary transitive siRNAs. However, and in sharp contrast to other species tested to date where RNA silencing is hindered at low temperature, this process remained active in grapevine cultivated at 4°C. Consistently, siRNA levels remained steady in grapevines cultivated between 26°C and 4°C, whereas they are severely decreased in Arabidopsis grown at 15°C and almost undetectable at 4°C. Altogether, these results demonstrate that RNA silencing operates in grapevine in a conserved manner but is resistant to far lower temperatures than ever described in other species.
doi:10.1371/journal.pone.0082652
PMCID: PMC3869719  PMID: 24376561
5.  A Leafhopper-Transmissible DNA Virus with Novel Evolutionary Lineage in the Family Geminiviridae Implicated in Grapevine Redleaf Disease by Next-Generation Sequencing 
PLoS ONE  2013;8(6):e64194.
A graft-transmissible disease displaying red veins, red blotches and total reddening of leaves in red-berried wine grape (Vitis vinifera L.) cultivars was observed in commercial vineyards. Next-generation sequencing technology was used to identify etiological agent(s) associated with this emerging disease, designated as grapevine redleaf disease (GRD). High quality RNA extracted from leaves of grape cultivars Merlot and Cabernet Franc with and without GRD symptoms was used to prepare cDNA libraries. Assembly of highly informative sequence reads generated from Illumina sequencing of cDNA libraries, followed by bioinformatic analyses of sequence contigs resulted in specific identification of taxonomically disparate viruses and viroids in samples with and without GRD symptoms. A single-stranded DNA virus, tentatively named Grapevine redleaf-associated virus (GRLaV), and Grapevine fanleaf virus were detected only in grapevines showing GRD symptoms. In contrast, Grapevine rupestris stem pitting-associated virus, Hop stunt viroid, Grapevine yellow speckle viroid 1, Citrus exocortis viroid and Citrus exocortis Yucatan viroid were present in both symptomatic and non-symptomatic grapevines. GRLaV was transmitted by the Virginia creeper leafhopper (Erythroneura ziczac Walsh) from grapevine-to-grapevine under greenhouse conditions. Molecular and phylogenetic analyses indicated that GRLaV, almost identical to recently reported Grapevine Cabernet Franc-associated virus from New York and Grapevine red blotch-associated virus from California, represents an evolutionarily distinct lineage in the family Geminiviridae with genome characteristics distinct from other leafhopper-transmitted geminiviruses. GRD significantly reduced fruit yield and affected berry quality parameters demonstrating negative impacts of the disease. Higher quantities of carbohydrates were present in symptomatic leaves suggesting their possible role in the expression of redleaf symptoms.
doi:10.1371/journal.pone.0064194
PMCID: PMC3673993  PMID: 23755117
6.  De Novo Reconstruction of Consensus Master Genomes of Plant RNA and DNA Viruses from siRNAs 
PLoS ONE  2014;9(2):e88513.
Virus-infected plants accumulate abundant, 21–24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this ‘siRNA omics’ approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense.
doi:10.1371/journal.pone.0088513
PMCID: PMC3921208  PMID: 24523907
7.  Short ORF-Dependent Ribosome Shunting Operates in an RNA Picorna-Like Virus and a DNA Pararetrovirus that Cause Rice Tungro Disease 
PLoS Pathogens  2012;8(3):e1002568.
Rice tungro disease is caused by synergistic interaction of an RNA picorna-like virus Rice tungro spherical virus (RTSV) and a DNA pararetrovirus Rice tungro bacilliform virus (RTBV). It is spread by insects owing to an RTSV-encoded transmission factor. RTBV has evolved a ribosome shunt mechanism to initiate translation of its pregenomic RNA having a long and highly structured leader. We found that a long leader of RTSV genomic RNA remarkably resembles the RTBV leader: both contain several short ORFs (sORFs) and potentially fold into a large stem-loop structure with the first sORF terminating in front of the stem basal helix. Using translation assays in rice protoplasts and wheat germ extracts, we show that, like in RTBV, both initiation and proper termination of the first sORF translation in front of the stem are required for shunt-mediated translation of a reporter ORF placed downstream of the RTSV leader. The base pairing that forms the basal helix is required for shunting, but its sequence can be varied. Shunt efficiency in RTSV is lower than in RTBV. But in addition to shunting the RTSV leader sequence allows relatively efficient linear ribosome migration, which also contributes to translation initiation downstream of the leader. We conclude that RTSV and RTBV have developed a similar, sORF-dependent shunt mechanism possibly to adapt to the host translation system and/or coordinate their life cycles. Given that sORF-dependent shunting also operates in a pararetrovirus Cauliflower mosaic virus and likely in other pararetroviruses that possess a conserved shunt configuration in their leaders it is tempting to propose that RTSV may have acquired shunt cis-elements from RTBV during their co-existence.
Author Summary
Ribosome shunting, first discovered in plant pararetroviruses, is a translation initiation mechanism that combines 5′ end-dependent scanning and internal initiation and allows a bypass of highly-structured leaders of certain viral and cellular mRNAs. Here we demonstrate that a similar shunt mechanism has been developed by the RNA picorna-like virus RTSV and the DNA pararetrovirus RTBV that form a disease complex in rice. Leader sequences of the RTSV genomic RNA and the RTBV pregenomic RNA possess a conserved shunt configuration with a 5′-proximal short ORF (sORF1) terminating in front of a large stem-loop structure. Like in RTBV and a related pararetrovirus Cauliflower mosaic virus, shunt-mediated translation downstream of the RTSV leader depends on initiation and proper termination of sORF1 translation and on formation of the basal helix of the downstream secondary structure. Given that RTBV-like shunt elements with identical sequence motifs are present in all RTSV isolates but absent in related picorna-like viruses, it is likely that RTSV could have acquired these elements after its encounter with RTBV. Alternatively, the RTSV shunt elements could have evolved independently to adapt to the rice translation machinery. Our study highlights on-going genetic exchange and co-adaptation to the host in emerging viral disease complexes.
doi:10.1371/journal.ppat.1002568
PMCID: PMC3291615  PMID: 22396650
8.  Grapevine leafroll-associated virus 3 
Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management.
doi:10.3389/fmicb.2013.00082
PMCID: PMC3627144  PMID: 23596440
grapevine leafroll disease; GLRaV-3; ampelovirus; Closteroviridae; genetic variants
9.  Evolution of Endogenous Sequences of Banana Streak Virus: What Can We Learn from Banana (Musa sp.) Evolution?▿  
Journal of Virology  2010;84(14):7346-7359.
Endogenous plant pararetroviruses (EPRVs) are viral sequences of the family Caulimoviridae integrated into the nuclear genome of numerous plant species. The ability of some endogenous sequences of Banana streak viruses (eBSVs) in the genome of banana (Musa sp.) to induce infections just like the virus itself was recently demonstrated (P. Gayral et al., J. Virol. 83:6697-6710, 2008). Although eBSVs probably arose from accidental events, infectious eBSVs constitute an extreme case of parasitism, as well as a newly described strategy for vertical virus transmission in plants. We investigated the early evolutionary stages of infectious eBSV for two distinct BSV species—GF (BSGFV) and Imové (BSImV)—through the study of their distribution, insertion polymorphism, and structure evolution among selected banana genotypes representative of the diversity of 60 wild Musa species and genotypes. To do so, the historical frame of host evolution was analyzed by inferring banana phylogeny from two chloroplast regions—matK and trnL-trnF—as well as from the nuclear genome, using 19 microsatellite loci. We demonstrated that both BSV species integrated recently in banana evolution, circa 640,000 years ago. The two infectious eBSVs were subjected to different selective pressures and showed distinct levels of rearrangement within their final structure. In addition, the molecular phylogenies of integrated and nonintegrated BSVs enabled us to establish the phylogenetic origins of eBSGFV and eBSImV.
doi:10.1128/JVI.00401-10
PMCID: PMC2898222  PMID: 20427523
10.  Deep sequencing evidence from single grapevine plants reveals a virome dominated by mycoviruses 
Archives of Virology  2010;156(3):397-403.
We have characterized the virome in single grapevines by 454 high-throughput sequencing of double-stranded RNA recovered from the vine stem. The analysis revealed a substantial set of sequences similar to those of fungal viruses. Twenty-six putative fungal virus groups were identified from a single plant source. These represented half of all known mycoviral families including the Chrysoviridae, Hypoviridae, Narnaviridae, Partitiviridae, and Totiviridae. Three of the mycoviruses were associated with Botrytis cinerea, a common fungal pathogen of grapes. Most of the rest appeared to be undescribed. The presence of viral sequences identified by BLAST analysis was confirmed by sequencing PCR products generated from the starting material using primers designed from the genomic sequences of putative mycoviruses. To further characterize these sequences as fungal viruses, fungi from the grapevine tissue were cultured and screened with the same PCR probes. Five of the mycoviruses identified in the total grapevine extract were identified again in extracts of the fungal cultures.
Electronic supplementary material
The online version of this article (doi:10.1007/s00705-010-0869-8) contains supplementary material, which is available to authorized users.
doi:10.1007/s00705-010-0869-8
PMCID: PMC3044836  PMID: 21140178
11.  A Stretch of 11 Amino Acids in the βB-βC Loop of the Coat Protein of Grapevine Fanleaf Virus Is Essential for Transmission by the Nematode Xiphinema index▿ †  
Journal of Virology  2010;84(16):7924-7933.
Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV) from the genus Nepovirus, family Secoviridae, cause a severe degeneration of grapevines. GFLV and ArMV have a bipartite RNA genome and are transmitted specifically by the ectoparasitic nematodes Xiphinema index and Xiphinema diversicaudatum, respectively. The transmission specificity of both viruses maps to their respective RNA2-encoded coat protein (CP). To further delineate the GFLV CP determinants of transmission specificity, three-dimensional (3D) homology structure models of virions and CP subunits were constructed based on the crystal structure of Tobacco ringspot virus, the type member of the genus Nepovirus. The 3D models were examined to predict amino acids that are exposed at the external virion surface, highly conserved among GFLV isolates but divergent between GFLV and ArMV. Five short amino acid stretches that matched these topographical and sequence conservation criteria were selected and substituted in single and multiple combinations by their ArMV counterparts in a GFLV RNA2 cDNA clone. Among the 21 chimeric RNA2 molecules engineered, transcripts of only three of them induced systemic plant infection in the presence of GFLV RNA1. Nematode transmission assays of the three viable recombinant viruses showed that swapping a stretch of (i) 11 residues in the βB-βC loop near the icosahedral 3-fold axis abolished transmission by X. index but was insufficient to restore transmission by X. diversicaudatum and (ii) 7 residues in the βE-αB loop did not interfere with transmission by the two Xiphinema species. This study provides new insights into GFLV CP determinants of nematode transmission.
doi:10.1128/JVI.00757-10
PMCID: PMC2916547  PMID: 20519403
12.  Characterization of microRNAs Identified in a Table Grapevine Cultivar with Validation of Computationally Predicted Grapevine miRNAs by miR-RACE 
PLoS ONE  2011;6(7):e21259.
Background
Alignment analysis of the Vv-miRNAs identified from various grapevine cultivars indicates that over 30% orthologous Vv-miRNAs exhibit a 1–3 nucleotide discrepancy only at their ends, suggesting that this sequence discrepancy is not a random event, but might mainly derive from divergence of cultivars. With advantages of miR-RACE technology in determining precise sequences of potential miRNAs from bioinformatics prediction, the precise sequences of vv-miRNAs predicted computationally can be verified with miR-RACE in a different grapevine cultivar. This presents itself as a new approach for large scale discovery of precise miRNAs in different grapevine varieties.
Methodology/Principal Findings
Among 88 unique sequences of Vv-miRNAs from bioinformatics prediction, 83 (96.3%) were successfully validated with MiR-RACE in grapevine cv. ‘Summer Black’. All the validated sequences were identical to their corresponding ones obtained from deep sequencing of the small RNA library of ‘Summer Black’. Quantitative RT-PCR analysis of the expressions levels of 10 Vv-miRNA/target gene pairs in grapevine tissues showed some negative correlation trends. Finally, comparison of Vv-miRNA sequences with their orthologs in Arabidopsis and study on the influence of divergent bases of the orthologous miRNAs on their targeting patterns in grapevine were also done.
Conclusion
The validation of precise sequences of potential Vv-miRNAs from computational prediction in a different grapevine cultivar can be a new way to identify the orthologous Vv-miRNAs. Nucleotide discrepancy of orthologous Vv-miRNAs from different grapevine cultivars normally does not change their target genes. However, sequence variations of some orthologous miRNAs in grapevine and Arabidopsis can change their targeting patterns. These precise Vv-miRNAs sequences validated in our study could benefit some further study on grapevine functional genomics.
doi:10.1371/journal.pone.0021259
PMCID: PMC3145640  PMID: 21829435
13.  Deep Sequencing of Viroid-Derived Small RNAs from Grapevine Provides New Insights on the Role of RNA Silencing in Plant-Viroid Interaction 
PLoS ONE  2009;4(11):e7686.
Background
Viroids 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 Findings
To 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/Significance
These 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.
doi:10.1371/journal.pone.0007686
PMCID: PMC2767511  PMID: 19890399
14.  Discovery of Replicating Circular RNAs by RNA-Seq and Computational Algorithms 
PLoS Pathogens  2014;10(12):e1004553.
Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR). However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA (AHVd-like RNA), respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small RNAs.
Author Summary
Viroids are a unique class of subviral pathogens found in plants, and they are difficult to identify since they are free circular non-coding RNAs and often replicate to low levels in host cells. We previously described the computational algorithm PFOR that discovers viroids by analyzing total small RNAs of the infected plants obtained by next-generation sequencing platforms. However, the algorithm written in PERL language is very slow, and viroid identification depends on the in vivo accumulation of extensively overlapping sets of small RNAs to target viroids. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was significantly faster than PFOR. We also describe a simple computational program that after incorporation into PFOR2 is capable of identifying viroids from deep sequencing of long RNAs instead of small RNAs. Moreover, we report the identification of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA by the computational approach. Since our new algorithms do not depend on the analysis of viroid-derived small RNAs produced in vivo, it is possible to discover viroids in a wide range of host species including plants, invertebrates and vertebrates.
doi:10.1371/journal.ppat.1004553
PMCID: PMC4263765  PMID: 25503469
15.  Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems 
PLoS Pathogens  2010;6(2):e1000764.
We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.
Author Summary
Short RNAs derived from invading viruses with RNA genomes are important components of antiviral immunity in plants, worms and flies. The regulated generation of these short RNAs, and their engagement by the immune apparatus, is essential for inhibiting viral growth in these organisms. Mammals have the necessary protein components to generate these viral-derived short RNAs (“vsRNAs”), raising the question of whether vsRNAs in mammals are a general feature of infections with RNA viruses. Our work with Hepatitis C, Polio, Dengue, Vesicular Stomatitis, and West Nile viruses in a broad host repertoire demonstrates the generality of RNA virus-derived vsRNA production, and the ability of the cellular short RNA apparatus to engage these vsRNAs in mammalian cells. Detailed analyses of vsRNA and host-derived short RNA populations demonstrate both common and virus-specific features of the interplay between viral infection and short RNA populations. The vsRNA populations described in this work represent a novel dimension in both viral pathogenesis and host response.
doi:10.1371/journal.ppat.1000764
PMCID: PMC2820531  PMID: 20169186
16.  Deconstruction of the (Paleo)Polyploid Grapevine Genome Based on the Analysis of Transposition Events Involving NBS Resistance Genes 
PLoS ONE  2012;7(1):e29762.
Plants have followed a reticulate type of evolution and taxa have frequently merged via allopolyploidization. A polyploid structure of sequenced genomes has often been proposed, but the chromosomes belonging to putative component genomes are difficult to identify. The 19 grapevine chromosomes are evolutionary stable structures: their homologous triplets have strongly conserved gene order, interrupted by rare translocations. The aim of this study is to examine how the grapevine nucleotide-binding site (NBS)-encoding resistance (NBS-R) genes have evolved in the genomic context and to understand mechanisms for the genome evolution. We show that, in grapevine, i) helitrons have significantly contributed to transposition of NBS-R genes, and ii) NBS-R gene cluster similarity indicates the existence of two groups of chromosomes (named as Va and Vc) that may have evolved independently. Chromosome triplets consist of two Va and one Vc chromosomes, as expected from the tetraploid and diploid conditions of the two component genomes. The hexaploid state could have been derived from either allopolyploidy or the separation of the Va and Vc component genomes in the same nucleus before fusion, as known for Rosaceae species. Time estimation indicates that grapevine component genomes may have fused about 60 mya, having had at least 40–60 mya to evolve independently. Chromosome number variation in the Vitaceae and related families, and the gap between the time of eudicot radiation and the age of Vitaceae fossils, are accounted for by our hypothesis.
doi:10.1371/journal.pone.0029762
PMCID: PMC3256180  PMID: 22253773
17.  A Single Banana Streak Virus Integration Event in the Banana Genome as the Origin of Infectious Endogenous Pararetrovirus▿  
Journal of Virology  2008;82(13):6697-6710.
Sequencing of plant nuclear genomes reveals the widespread presence of integrated viral sequences known as endogenous pararetroviruses (EPRVs). Banana is one of the three plant species known to harbor infectious EPRVs. Musa balbisiana carries integrated copies of Banana streak virus (BSV), which are infectious by releasing virions in interspecific hybrids. Here, we analyze the organization of the EPRV of BSV Goldfinger (BSGfV) present in the wild diploid M. balbisiana cv. Pisang Klutuk Wulung (PKW) revealed by the study of Musa bacterial artificial chromosome resources and interspecific genetic cross. cv. PKW contains two similar EPRVs of BSGfV. Genotyping of these integrants and studies of their segregation pattern show an allelic insertion. Despite the fact that integrated BSGfV has undergone extensive rearrangement, both EPRVs contain the full-length viral genome. The high degree of sequence conservation between the integrated and episomal form of the virus indicates a recent integration event; however, only one allele is infectious. Analysis of BSGfV EPRV segregation among an F1 population from an interspecific genetic cross revealed that these EPRV sequences correspond to two alleles originating from a single integration event. We describe here for the first time the full genomic and genetic organization of the two EPRVs of BSGfV present in cv. PKW in response to the challenge facing both scientists and breeders to identify and generate genetic resources free from BSV. We discuss the consequences of this unique host-pathogen interaction in terms of genetic and genomic plant defenses versus strategies of infectious BSGfV EPRVs.
doi:10.1128/JVI.00212-08
PMCID: PMC2447048  PMID: 18417582
18.  Intracellular Hepadnavirus Nucleocapsids Are Selected for Secretion by Envelope Protein-Independent Membrane Binding 
Journal of Virology  2000;74(24):11472-11478.
Hepadnaviruses are DNA viruses but, as pararetroviruses, their morphogenesis initiates with the encapsidation of an RNA pregenome, and these viruses have therefore evolved mechanisms to exclude nucleocapsids that contain incompletely matured genomes from participating in budding and secretion. We provide here evidence that binding of hepadnavirus core particles from the cytosol to their target membranes is a distinct step in morphogenesis, discriminating among different populations of intracellular capsids. Using the duck hepatitis B virus (DHBV) and a flotation assay, we found about half of the intracellular capsids to be membrane associated due to an intrinsic membrane-binding affinity. In contrast to free cytosolic capsids, this subpopulation contained largely mature, double-stranded DNA genomes and lacked core protein hyperphosphorylation, both features characteristic for secreted virions. Against expectation, however, the selective membrane attachment observed did not require the presence of the large DHBV envelope protein, which has been considered to be crucial for nucleocapsid-membrane interaction. Furthermore, removal of surface-exposed phosphate residues from nonfloating capsids by itself did not suffice to confer membrane affinity and, finally, hyperphosphorylation was absent from nonenveloped nucleocapsids that were released from DHBV-transfected cells. Collectively, these observations argue for a model in which nucleocapsid maturation, involving the viral genome, capsid structure, and capsid dephosphorylation, leads to the exposure of a membrane-binding signal as a step crucial for selecting the matured nucleocapsid to be incorporated into the capsid-independent budding of virus particles.
PMCID: PMC112426  PMID: 11090143
19.  Virus-Derived Gene Expression and RNA Interference Vector for Grapevine 
Journal of Virology  2012;86(11):6002-6009.
The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests.
doi:10.1128/JVI.00436-12
PMCID: PMC3372183  PMID: 22438553
20.  Correction: High throughput approaches reveal splicing of primary microRNA transcripts and tissue specific expression of mature microRNAs in Vitis vinifera 
BMC Genomics  2010;11:109.
The version of this article published in BMC Genomics 2009, 10:558, contains data in Table 1 which are now known to be unreliable, and an illustration, in Figure 1, of unusual miRNA processing events predicted by these unreliable data. In this full-length correction, new data replace those found to be unreliable, leading to a more straightforward interpretation without altering the principle conclusions of the study. Table 1 and associated methods have been corrected, Figure 1 deleted, supplementary file 1 added, and modifications made to the sections "Deep sequencing of small RNAs from grapevine leaf tissue" and "Microarray analysis of miRNA expression". The editors and authors regret the inconvenience caused to readers by premature publication of the original paper.
Background
MicroRNAs are short (~21 base) single stranded RNAs that, in plants, are generally coded by specific genes and cleaved specifically from hairpin precursors. MicroRNAs are critical for the regulation of multiple developmental, stress related and other physiological processes in plants. The recent annotation of the genome of the grapevine (Vitis vinifera L.) allowed the identification of many putative conserved microRNA precursors, grouped into multiple gene families.
Results
Here we use oligonucleotide arrays to provide the first indication that many of these microRNAs show differential expression patterns between tissues and during the maturation of fruit in the grapevine. Furthermore we demonstrate that whole transcriptome sequencing and deep-sequencing of small RNA fractions can be used both to identify which microRNA precursors are expressed in different tissues and to estimate genomic coordinates and patterns of splicing and alternative splicing for many primary miRNA transcripts.
Conclusions
Our results show that many microRNAs are differentially expressed in different tissues and during fruit maturation in the grapevine. Furthermore, the demonstration that whole transcriptome sequencing can be used to identify candidate splicing events and approximate primary microRNA transcript coordinates represents a significant step towards the large-scale elucidation of mechanisms regulating the expression of microRNAs at the transcriptional and post-transcriptional levels.
doi:10.1186/1471-2164-11-109
PMCID: PMC2831844  PMID: 20152027
21.  Transfection of Diaporthe perjuncta with Diaporthe RNA Virus 
Diaporthe perjuncta is a pathogen of grapevines worldwide. A positive-strand RNA virus, Diaporthe RNA virus (DaRV), occurs in hypovirulent isolates of this fungus. A virus-free isolate from a South African grapevine was transfected with in vitro-transcribed positive strands of DaRV. Based on reverse transcription-PCR and partial sequence analysis, the transfected virus was identified as DaRV. The in vitro-transcribed RNA transcripts used to transfect fungal spheroplasts contained parts of the vector at their distal ends. These vector sequences were separated from the DaRV genome during replication in the new host. The transfected isolate had morphological features that differed from those of the isogenic virus-free strain, including production of a yellow pigment, a decreased growth rate, and lack of sporulation. An apple-based pathogenicity test did not reveal any differences in virulence between the virus-free and DaRV-transfected isolates. This study showed that virus-free fungal hosts can be successfully transfected with viruses other than the Cryphonectria parasitica hypovirus.
doi:10.1128/AEM.69.7.3952-3956.2003
PMCID: PMC165159  PMID: 12839766
22.  Hypothesis for heritable, anti-viral immunity in crustaceans and insects 
Biology Direct  2009;4:32.
Background
It is known that crustaceans and insects can persistently carry one or more viral pathogens at low levels, without signs of disease. They may transmit them to their offspring or to naïve individuals, often with lethal consequences. The underlying molecular mechanisms have not been elucidated, but the process has been called viral accommodation. Since tolerance to one virus does not confer tolerance to another, tolerance is pathogen-specific, so the requirement for a specific pathogen response mechanism (memory) was included in the original viral accommodation concept. Later, it was hypothesized that specific responses were based on the presence of viruses in persistent infections. However, recent developments suggest that specific responses may be based on viral sequences inserted into the host genome.
Presentation of the hypothesis
Non-retroviral fragments of both RNA and DNA viruses have been found in insect and crustacean genomes. In addition, reverse-transcriptase (RT) and integrase (IN) sequences are also common in their genomes. It is hypothesized that shrimp and other arthropods use these RT to recognize "foreign" mRNA of both RNA and DNA viruses and use the integrases (IN) to randomly insert short cDNA sequences into their genomes. By chance, some of these sequences result in production of immunospecific RNA (imRNA) capable of stimulating RNAi that suppresses viral propagation. Individuals with protective inserts would pass these on to the next generation, together with similar protective inserts for other viruses that could be amalgamated rapidly in individual offspring by random assortment of chromosomes. The most successful individuals would be environmentally selected from billions of offspring.
Conclusion
This hypothesis for immunity based on an imRNA generation mechanism fits with the general principle of invertebrate immunity based on a non-host, "pattern recognition" process. If proven correct, understanding the process would allow directed preparation of vaccines for selection of crustacean and insect lines applicable in commercial production species (e.g., shrimp and bees) or in control of insect-borne diseases. Arising from a natural host mechanism, the resulting animals would not be artificially, genetically modified (GMO).
Reviewers
This article was reviewed by Akria Shibuya, Eugene V. Koonin and L. Aravind.
doi:10.1186/1745-6150-4-32
PMCID: PMC2757015  PMID: 19725947
23.  The ancient Virus World and evolution of cells 
Biology Direct  2006;1:29.
Background
Recent advances in genomics of viruses and cellular life forms have greatly stimulated interest in the origins and evolution of viruses and, for the first time, offer an opportunity for a data-driven exploration of the deepest roots of viruses. Here we briefly review the current views of virus evolution and propose a new, coherent scenario that appears to be best compatible with comparative-genomic data and is naturally linked to models of cellular evolution that, from independent considerations, seem to be the most parsimonious among the existing ones.
Results
Several genes coding for key proteins involved in viral replication and morphogenesis as well as the major capsid protein of icosahedral virions are shared by many groups of RNA and DNA viruses but are missing in cellular life forms. On the basis of this key observation and the data on extensive genetic exchange between diverse viruses, we propose the concept of the ancient virus world. The virus world is construed as a distinct contingent of viral genes that continuously retained its identity throughout the entire history of life. Under this concept, the principal lineages of viruses and related selfish agents emerged from the primordial pool of primitive genetic elements, the ancestors of both cellular and viral genes. Thus, notwithstanding the numerous gene exchanges and acquisitions attributed to later stages of evolution, most, if not all, modern viruses and other selfish agents are inferred to descend from elements that belonged to the primordial genetic pool. In this pool, RNA viruses would evolve first, followed by retroid elements, and DNA viruses. The Virus World concept is predicated on a model of early evolution whereby emergence of substantial genetic diversity antedates the advent of full-fledged cells, allowing for extensive gene mixing at this early stage of evolution. We outline a scenario of the origin of the main classes of viruses in conjunction with a specific model of precellular evolution under which the primordial gene pool dwelled in a network of inorganic compartments. Somewhat paradoxically, under this scenario, we surmise that selfish genetic elements ancestral to viruses evolved prior to typical cells, to become intracellular parasites once bacteria and archaea arrived at the scene. Selection against excessively aggressive parasites that would kill off the host ensembles of genetic elements would lead to early evolution of temperate virus-like agents and primitive defense mechanisms, possibly, based on the RNA interference principle. The emergence of the eukaryotic cell is construed as the second melting pot of virus evolution from which the major groups of eukaryotic viruses originated as a result of extensive recombination of genes from various bacteriophages, archaeal viruses, plasmids, and the evolving eukaryotic genomes. Again, this vision is predicated on a specific model of the emergence of eukaryotic cell under which archaeo-bacterial symbiosis was the starting point of eukaryogenesis, a scenario that appears to be best compatible with the data.
Conclusion
The existence of several genes that are central to virus replication and structure, are shared by a broad variety of viruses but are missing from cellular genomes (virus hallmark genes) suggests the model of an ancient virus world, a flow of virus-specific genes that went uninterrupted from the precellular stage of life's evolution to this day. This concept is tightly linked to two key conjectures on evolution of cells: existence of a complex, precellular, compartmentalized but extensively mixing and recombining pool of genes, and origin of the eukaryotic cell by archaeo-bacterial fusion. The virus world concept and these models of major transitions in the evolution of cells provide complementary pieces of an emerging coherent picture of life's history.
Reviewers
W. Ford Doolittle, J. Peter Gogarten, and Arcady Mushegian.
doi:10.1186/1745-6150-1-29
PMCID: PMC1594570  PMID: 16984643
24.  Artificial MicroRNA-Mediated Virus Resistance in Plants▿  
Journal of Virology  2007;81(12):6690-6699.
RNA silencing in plants is a natural defense system against foreign genetic elements including viruses. This natural antiviral mechanism has been adopted to develop virus-resistant plants through expression of virus-derived double-stranded RNAs or hairpin RNAs, which in turn are processed into small interfering RNAs (siRNAs) by the host's RNA silencing machinery. While these virus-specific siRNAs were shown to be a hallmark of the acquired virus resistance, the functionality of another set of the RNA silencing-related small RNAs, microRNAs (miRNAs), in engineering plant virus resistance has not been extensively explored. Here we show that expression of an artificial miRNA, targeting sequences encoding the silencing suppressor 2b of Cucumber mosaic virus (CMV), can efficiently inhibit 2b gene expression and protein suppressor function in transient expression assays and confer on transgenic tobacco plants effective resistance to CMV infection. Moreover, the resistance level conferred by the transgenic miRNA is well correlated to the miRNA expression level. Comparison of the anti-CMV effect of the artificial miRNA to that of a short hairpin RNA-derived small RNA targeting the same site revealed that the miRNA approach is superior to the approach using short hairpin RNA both in transient assays and in transgenic plants. Together, our data demonstrate that expression of virus-specific artificial miRNAs is an effective and predictable new approach to engineering resistance to CMV and, possibly, to other plant viruses as well.
doi:10.1128/JVI.02457-06
PMCID: PMC1900123  PMID: 17344304
25.  3'-coterminal subgenomic RNAs and putative cis-acting elements of Grapevine leafroll-associated virus 3 reveals 'unique' features of gene expression strategy in the genus Ampelovirus 
Virology Journal  2010;7:180.
Background
The family Closteroviridae comprises genera with monopartite genomes, Closterovirus and Ampelovirus, and with bipartite and tripartite genomes, Crinivirus. By contrast to closteroviruses in the genera Closterovirus and Crinivirus, much less is known about the molecular biology of viruses in the genus Ampelovirus, although they cause serious diseases in agriculturally important perennial crops like grapevines, pineapple, cherries and plums.
Results
The gene expression and cis-acting elements of Grapevine leafroll-associated virus 3 (GLRaV-3; genus Ampelovirus) was examined and compared to that of other members of the family Closteroviridae. Six putative 3'-coterminal subgenomic (sg) RNAs were abundantly present in grapevine (Vitis vinifera) infected with GLRaV-3. The sgRNAs for coat protein (CP), p21, p20A and p20B were confirmed using gene-specific riboprobes in Northern blot analysis. The 5'-termini of sgRNAs specific to CP, p21, p20A and p20B were mapped in the 18,498 nucleotide (nt) virus genome and their leader sequences determined to be 48, 23, 95 and 125 nt, respectively. No conserved motifs were found around the transcription start site or in the leader sequence of these sgRNAs. The predicted secondary structure analysis of sequences around the start site failed to reveal any conserved motifs among the four sgRNAs. The GLRaV-3 isolate from Washington had a 737 nt long 5' nontranslated region (NTR) with a tandem repeat of 65 nt sequence and differed in sequence and predicted secondary structure with a South Africa isolate. Comparison of the dissimilar sequences of the 5'NTRs did not reveal any common predicted structures. The 3'NTR was shorter and more conserved. The lack of similarity among the cis-acting elements of the diverse viruses in the family Closteroviridae is another measure of the complexity of their evolution.
Conclusions
The results indicate that transcription regulation of GLRaV-3 sgRNAs appears to be different from members of the genus Closterovirus. An analysis of the genome sequence confirmed that GLRaV-3 has an unusually long 5'NTR of 737 nt compared to other monopartite members of the family Closteroviridae, with distinct differences in the sequence and predicted secondary structure when compared to the corresponding region of the GLRaV-3 isolate from South Africa.
doi:10.1186/1743-422X-7-180
PMCID: PMC2922190  PMID: 20682046

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