Carnation Italian Ringspot Research Articles

The p36 and p95 replicase proteins of Carnation Italian ringspot virus cooperate in stabilizing defective interfering RNA.

The p36 and p95 proteins of Carnation Italian ringspot virus (CIRV), when expressed in Saccharomyces cerevisiae, supported the replication of defective interfering (DI) RNA. Double-label confocal immunofluorescence showed that both proteins localized to mitochondria, independently of each other. DI RNA progeny was localized by in situ hybridization both to mitochondria and to their proximity. Fractionation of cell extracts showed that replicase proteins associated with membranes with a consistent portion of DI RNA. DI RNA transcripts were stabilized more efficiently when co-expressed with both p36 and p95 than with either protein alone. By using the copper-inducible CUP1 promoter, p36 was shown to have an effect on DI RNA stability only above a threshold concentration, suggesting an 'all-or-none' behaviour. Conversely, the stabilizing activity of p95 was proportional to protein concentration in the range examined. Similarly, DI RNA replication level was proportional to p95 concentration and depended on a threshold concentration of p36.

Evaluation of various species demarcation criteria in attempts to classify ten new tombusvirus isolates

The usefulness of various suggested species demarcation criteria was compared in attempts to determine the taxonomic status of ten new tombusvirus isolates. Five of them (Lim 1, 2, 3, 5 and 6) were obtained from different sources of commercially grown statice (Limonium sinuatum), two (Gyp 1 and 2) from different sources of commercially grown Gypsophila paniculata and three from water samples, i.e. from a small river (Schunter) in Northern Germany, from a brook (near Dossenheim) in Southern Germany and from the groundwater in a Limonium production glasshouse in the Netherlands (Lim 4). The immunoelectron microscopical decoration test allowed a quick preliminary assignment of various isolates to several known tombusviruses. A more precise analysis of the relationships was achieved by comparing the deduced amino acid sequences of the coat proteins. Sequence as well as serological data suggested that eight of the isolates should be classified as strains or variants of either Carnation Italian ringspot virus, Grapevine Algerian latent virus, Petunia asteroid mosaic virus or Sikte waterborne virus, respectively, whereas the 9th isolate (Lim 2) appears to represent a distinct new tombusvirus species. The case of the 10th isolate (Lim 5) illustrates the classification problems experienced when the properties of a virus place it close to the more or less arbitrary man-made borderline between virus species and virus strains. The coat protein gene sequences were also determined for some viruses for which these data had not yet been available, i.e. Neckar river virus, Sikte waterborne virus and Eggplant mottled crinkle virus. The sequences of the coat protein gene and also of ORF 1 of the latter virus proved to be almost identical to the corresponding genome regions of the recently described Pear latent virus, which for priority reasons should be renamed. Criteria which have been suggested in addition to serology and sequence comparisons for tombusvirus species demarcation, i.e. differences in natural and in experimental host ranges, in cytopathological features and in coat protein size, appear to be of little value for the classification of new tombusviruses.

Size Selective Recognition of siRNA by an RNA Silencing Suppressor

RNA silencing in plants likely exists as a defense mechanism against molecular parasites such as RNA viruses, retrotransposons, and transgenes. As a result, many plant viruses have adapted mechanisms to evade and suppress gene silencing. Tombusviruses express a 19 kDa protein (p19), which has been shown to suppress RNA silencing in vivo and bind silencing-generated and synthetic small interfering RNAs (siRNAs) in vitro. Here we report the 2.5 Å crystal structure of p19 from the Carnation Italian ringspot virus (CIRV) bound to a 21 nt siRNA and demonstrate in biochemical and in vivo assays that CIRV p19 protein acts as a molecular caliper to specifically select siRNAs based on the length of the duplex region of the RNA.

Replication of Carnation Italian ringspot virus defective interfering RNA in Saccharomyces cerevisiae.

Two plasmids from which the sequences coding for the 36- and 95-kDa proteins of Carnation Italian ringspot virus (CIRV) could be transcribed in vivo in the yeast Saccharomyces cerevisiae under the control of the ADH1 promoter and terminator were constructed. The two proteins, which constitute the viral replicase, were correctly translated and integrated into membranes of the yeast cells. An additional plasmid was introduced in yeasts expressing the CIRV replicase, from which a defective interfering (DI) RNA (DI-7 RNA) could be transcribed under the control of the GAL1 promoter and terminated by the Tobacco ringspot virus satellite ribozyme, which cleaved 19 nucleotides downstream of the 3' end of DI RNA. The DI-7 RNA transcripts were amplified by the viral replicase as demonstrated by the restoration of the authentic 3' end, the requirement of a specific cis-acting signal at this terminus, the preferential accumulation of molecules with the authentic 5' terminus (AGAAA), the synthesis of head-to-tail dimers, the presence of negative strands, and the incorporation of 5-bromo-UTP. Additionally, transformation with a dimeric construct of DI-7 RNA led to the synthesis of monomers, mimicking the activity of the viral replicase in plant cells.

Characterization of Distinct Tombusviruses that Cause Diseases of Lettuce and Tomato in the Western United States

A soilborne disease of lettuce, associated with necrosis and dieback, has been found with increasing frequency in California and Arizona over the last 10 years. An isometric virus, serologically related to Tomato bushy stunt virus (TBSV), was consistently isolated from lettuce plants with these disease symptoms. Back-inoculation to healthy lettuce plants and subsequent reisolation of the virus from symptomatic lettuce leaves suggested that this virus was the causal agent of this disease. A tombusvirus was also associated with a necrosis disease of greenhouse-grown tomatoes in Colorado and New Mexico. Complementary DNA representing the 3' end of viral genomic RNAs recovered from diseased lettuce and tomato plants had identical nucleotide sequences. However, these sequences were divergent (12.2 to 17.1%) from sequences of the previously described strains of TBSV, Petunia asteroid mosaic virus (PAMV), Artichoke mottled crinkle virus, and Carnation Italian ringspot virus. Additional tombusvirus isolates were recovered from diseased lettuce and tomato plants and these were most closely related to the TBSV-cherry strain (synonymous with PAMV) and to Cucumber necrosis virus based on comparison of 3'-end sequences (0.1 to 0.6% and 4.8 to 5.1% divergence, respectively). Western blot analysis revealed that the new tombusvirus isolated from diseased lettuce and tomato plants in the western United States is serologically distinct from previously described tombusvirus species and strains. Based on genomic and serological properties, we propose to classify this virus as a new tombusvirus species and name it Lettuce necrotic stunt virus.

The open reading frame 1-encoded ('36K') protein of Carnation Italian ringspot virus localizes to mitochondria.

The localization of the 36 kDa ('36K') protein encoded by open reading frame 1 of Carnation Italian ringspot virus was studied in infected cells and in cells transiently expressing the 36K protein fused to green fluorescent protein (GFP). Subcellular fractionation demonstrated that the 36K protein accumulated in fractions containing mostly mitochondria. Fluorescence microscopy of transiently transformed cells showed that the 36K-GFP fusion protein accumulated in structures which could be stained with the mitochondrial-specific dye MitoTracker. However, these structures were larger than normal mitochondria and were irregular in shape and distribution in the cytoplasm. Electron microscopy showed severe alterations of mitochondria, which were often clumped. The stroma was more electron-opaque, the cristae were irregularly shaped, the intermembrane space was enlarged and the outer membrane was covered with an electron-dense amorphous material whose nature could not be determined. The organelle-targeted 36K protein seems to promote the overgrowth of the mitochondrial outer membrane.

The ORF1 products of tombusviruses play a crucial role in lethal necrosis of virus-infected plants.

Hybrids of cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) tombusviruses were used to identify viral symptom determinants responsible for the generalized necrosis in tombusvirus-infected plants. Surprisingly, symptoms of Nicotiana benthamiana infected with CymRSV/CIRV hybrids were distinctly different. It was demonstrated that not all chimeras expressing wild-type (wt) levels of p19 protein caused systemic necrosis as both parents CymRSV and CIRV did. We showed here that hybrids containing chimeric ORF1 were not able to induce lethal necrosis even if the viral replication of these constructs was not altered significantly. However, if a wt p33 (product of ORF1) of CymRSV was provided in trans in transgenic plants expressing p33 and its readthrough product p92, the lethal necrosis characteristic to tombusvirus infection was restored. In addition, the expression of p33 by a potato virus X viral vector in N. benthamiana caused severe chlorosis and occasionally necrosis, indicating the importance of p33 in wt symptoms of tombusviruses. Thus, our results provide evidence that elicitation of the necrotic phenotype requires the presence of the wt p33 in addition to the p19 protein of tombusviruses.

Expression of a plant virus non-structural protein in Saccharomyces cerevisiae causes membrane proliferation and altered mitochondrial morphology.

Carnation Italian ringspot tombusvirus encodes a protein, referred to as 36K, that possesses a mitochondrial targeting signal and two transmembrane segments which are thought to anchor this protein to the outer membrane of the mitochondrial envelope of infected plant cells. To determine the topology of the virus protein inserted in the cell membrane, as well as the sequence requirements for targeting and insertion, an in vivo system was set up in which this could be analysed in the absence of productive virus infection. The 36K protein was expressed in the yeast Saccharomyces cerevisiae in native form or fused to the green fluorescent protein. Using a fluorescence microscope, large green-fluorescing cytoplasmic aggregates were visible which stained red when cells were treated with the vital stain MitoTracker, which is specific for mitochondria. These aggregates were shown by electron microscopy to be composed of either mitochondria or membranes. The latter type was particularly abundant for the construct in which the green fluorescent protein was fused at the N terminus of the 36K protein. Immunoelectron microscopy demonstrated that the viral protein is present in the anomalous aggregates and Western blot analysis of protein extracts showed 36K to be resistant to alkaline, urea or salt extraction, a property of integral membrane proteins.

Simultaneous detection of five carnation viruses by non-isotopic molecular hybridization

Several viruses, which in some cases can cause severe losses, affect carnation plants. These viruses include carnation mottle virus, carnation etched ring virus (CERV), carnation vein mottle virus, carnation ringspot virus, carnation Italian ringspot virus and carnation latent virus. A non-isotopic molecular hybridization was developed for the detection of these viruses in host plants and the sensitivity of the technique has been compared with enzyme-linked immunosorbent assay and bioassay methods. A procedure was developed to test simultaneously for the five RNA viruses (except CERV). The conditions established for this simultaneous detection did not include the DNA virus CERV due to the necessity of incorporating an additional step of RNase A treatment in the procedure to eliminate background signals. The sensitivity limits obtained for each virus using this multiple detection assay were identical to those obtained with the individual assays. The relative benefits of using this detection procedure for routine diagnosis of carnation viruses are discussed.

Interactions between Tombusviruses and Satellite RNAs of Tomato Bushy Stunt Virus: A Defect in sat RNA B1 Replication Maps to ORF1 of a Helper Virus

The biological properties of two recently described satellite RNAs of tomato bushy stunt virus (TBSV) were analyzed in natural and experimental hosts. Full-length cDNA clones were constructed for sat RNAs B1(822 nt) and B10 (612 nt) and used in inoculations with satellite-free transcripts of different tombusviruses. In all virus-host combinations tested, TBSV sat RNA B10 drastically reduced the accumulation of viral genomic RNA and attenuated symptoms. In contrast, sat RNA B1 caused a less marked reduction of viral RNA level and did not have any effect on symptoms. Experiments with Nicotiana benthamiana protoplasts showed that the differential effects of sat RNAs B1 and B10 on TBSV titer were related to differential abilities to interfere with virus replication. Three tombusviruses tested were able to maintain both sat RNAs in N. benthamiana plants, although carnation Italian ringspot virus (CIRV) was a poor helper for sat RNA B1. Using chimeric viruses, a strong determinant for low sat RNA B1 accumulation was mapped to the 5′-terminal part of the genome of CIRV. The poor helper activity of CIRV was shown to be due to low sat RNA B1 replication. A single-nucleotide mutation in the start codon of CIRV ORF1 restored the ability to replicate sat RNA B1 to high levels. This mutant encodes an ORF1 that is 22 amino acids shorter at the N-terminus than the wild-type virus.

Membrane Targeting Sequences in Tombusvirus Infections

Infection ofNicotiana benthamianacells with cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) viruses results in the formation of conspicuous membranous bodies [multivesicular bodies (MVBs)], which develop from modified peroxisomes or mitochondria, respectively. The organelle targeting signal is located in the proteins of 33 kDa (CymRSV) or 36 kDa (CIRV) encoded by ORF 1, which contain an N-terminal hydrophilic portion followed by two predicted hydrophobic transmembrane segments. Biochemical analysis showed that the 33- and 36-kDa proteins are integral membrane proteins. By exchanging small portions of the ORF 1 sequence between the infectious full-length clones of the two viruses, hybrid constructs were obtained of which thein vitrosynthesized RNA was inoculated toN. benthamianaplants and protoplasts. The structure of infectious clones suggested that both the N-terminal hydrophilic region and the transmembrane segments of the ORF 1-encoded proteins specify which organelle is involved in the synthesis of MVBs. Mutational analysis of the CIRV 36-kDa protein also suggested the presence of an internal mitochondrial targeting sequence similar to that found in several normal host proteins that are synthesized in the cytoplasm and transported to mitochondria. The CymRSV 33-kDa protein did not contain the obvious consensus signals thought to be characteristic of proteins targeted to peroxisomes, and an mitochondrial targeting sequence motif was not evident.

NICOTIANA BENTHAMIANA PLANTS TRANSFORMED WITH THE COMPLETE PRE-READTHROUGH DOMAIN OR THE N-PROXIMAL REGION OF THE REPLICASE GENE FROM CYMBIDIUM RINGSPOT VIRUS RNA ARE RESISTANT TO VIRUS INFECTION

Nicotiana benthamiana plants were transformed with sequences derived from the 5’-terminal open reading frames (ORF 1 and 2) of the cymbidium ringspot tombusvirus (CymRSV). These sequences included the pre-readthrough region of the replicase or its N-terminal region, the readthrough domain, and the full-length replicase gene in which the stop codon of ORF 1 was abolished. Some lines transformed with the first two sequences showed complete resistance when challenged with CymRSV. Experiments using protoplasts from resistant lines showed that the resistance operates at the single cell level by abolishing viral replication. Lines resistant to CymRSV were susceptible to two other tombusviruses, artichoke mottled crinkle and carnation Italian ringspot. These data and the lack of correlation between transgene expression and resistance would suggest this to be a form of RNA-mediated resistance. Since resistant lines were only found among those transformed with sequences involved in the build-up of the virus replication complex, it is suggested that resistance may be due to the interference of these sequences with those of the infecting virus.

The 5'-terminal region of a tombusvirus genome determines the origin of multivesicular bodies.

Multivesicular bodies (MVB) are membranous cytoplasmic inclusions that are invariably associated with tombusvirus infections regardless of the virus species, the host, or the tissue examined. MVB are virus-induced structures since they are absent from tissues of healthy plants and are always present both in infected plants and protoplasts. MVB derive from peroxisomes in cells infected by a number of tombusviruses including cymbidium ringspot virus (CymRSV) and from mitochondria in cells infected by another tombusvirus, carnation Italian ringspot virus (CIRV). By using common restriction sites in full-length infectious clones, hybrid clones of these two viruses were constructed. In addition, a mutant of CIRV was prepared in which the protein encoded by the first open reading frame was shortened by 22 amino acids. All mutant transcripts were viable and infected Nicotiana benthamiana plants. Infected leaf tissue samples were collected, processed for thin sectioning, and observed in the electron microscope. The origin of MVB was shown to be under the control of the 5' region of the viral genome. A sequence as short as about 600 nucleotides in ORF 1 contained the determinants for formation of MVB from peroxisomes or mitochondria.

Characterization of Resistance to Cymbidium Ringspot Virus in Transgenic Plants Expressing a Full-Length Viral Replicase Gene

Transgenic Nicotiana benthamiana plants expressing the replicase gene of cymbidium ringspot tombusvirus (CymRSV) were tested for the biological activity of the transgene. Protoplasts from one resistant and three susceptible transgenic lines were transfected with in vitro synthesized defective interfering (DI) RNA, a small subviral molecule which requires only the virus replicase for replication. Only protoplasts of susceptible transgenic lines were able to sustain the replication of DI RNA, whereas protoplasts of the resistant line were not. The transgene of the resistant line was recovered from transgenic cells, cloned, and shown to be intact and functional, which suggests that this is not a case of dysfunctional replicase conferring resistance to transgenic plants as a "dominant negative mutant." Plants of the transgenic line resistant to CymRSV were shown to be susceptible to two other tombusviruses, artichoke mottled crinkle and carnation Italian ringspot virus. Since the resistance is specific and inversely correlated to the level of expression of the transgene, it appears to be a form of RNA-mediated resistance.

Physicochemical studies on tomato bushy stunt virus variants

AbstractTomato bushy stunt virus particles have previously been physically and chemically well characterized. Different strains, however, are known to have different host plants and symptoms, and they can be distinguished serologically. In an attempt to correlate this behaviour with physicochemical properties, we have performed a comparative study on isolates of four of the main variants of this virus (Type strain, petunia asteroid mosaic virus, pelargonium leaf curl virus, and carnation Italian ringspot virus) with regard to sedimentation velocity characteristics, translation diffusivity (from quasi‐elastic light scattering measurements), and molecular weight (from both low‐speed sedimentation equilibrium and the Svedberg equation).It is demonstrated that there are no clear differences between the four strains, particularly insofar as their sedimentation characteristics (both velocity and low‐speed equilibrium). There is some suggestion from diffusion measurements that the Type strain virus particles may have a higher mass, although the presence of some aggregation phenomena may provide an alternative explanation for the diffusion data.

Natural Occurrence of Carnation Italian Ringspot Virus in a Cherry Tree

AbstractA virus isolate from a sweet cherry tree was identified by means of the immunoelectron microscopical decoration test and the agar gel double diffusion test as carnation Italian ringspot virus (CIRV). By means of ISEM and ELISA the virus could be detected directly in some leaves of this tree which were distorted and showed necrosis on the main veins, but not in symptomless leaves. This finding and the recently reported isolation of the virus from a brook in a forested area in Germany suggest that CIRV has a wider host range and is more widely spread in nature than originally thought.ZusammenfassungNatürliches Vorkommen von Carnation Italian Ringspot Virus in einem Süßkirschen‐BaumEin Virusisolat aus einem Kirschbaum wurde mit Hilfe des immunelektronenmikroskopischen Dekorationstestes und des Agargeldoppeldiffusionstestes als carnation Italian ringspot virus (CIRV) identifiziert. Mit Hilfe von ELISA und ISEM konnte das Virus auch direkt in Blättern von Süßkirschen nachgewiesen werden, die Nekrosen an der Mittelader aufwiesen und eine verzogene Spreite hatten. In symptomlosen Blättern verlief der Virusnachweis negativ. Die Identifizierung des CIRV in einem Kirschbaum und seine kürzlich berichtete Isolierung aus einem Waldbach deuten darauf hin, daß sein Wirtspflanzenkreis und seine Verbreitung in der Natur weiter sind, als ursprünglich angenommen wurde.

Isolation of Carnation Ringspot Virus from a Canal Near a Sewage Plant: cDNA Hybridization Analysis, Serology and Cytopathology

AbstractA small water sample of only 60 ml from the Oker Aue canal near Braunschweig was ultracentrifuged. The resuspended pellet was rubbed on Chenopodium quinoa leaves which responded with the formation of almost 200 local lesions. The virus causing these lesions was identified as carnation ringspot virus (CarRSV) by means of dot blot hybridization using random‐primed cDNAs to the viral nucleic acids and by means of serology. Northern blot analysis revealed that the two RNA species of the virus which consisted of c. 3.7 and 1.5 Kb, respectively, have little or no base sequence homology. In immunoelectrophoresis at pH 7.0 the virus migrated towards the cathode. The isometric particles were 33 nm in diameter, round to slightly angular in outline, and showed a distinct granular or knobly surface structure. Virus particles occurred in the cytoplasm, nuclei, and vacuoles of infected cells which in addition contained amorphous granules in the cytoplasm and/or proliferated endoplasmic reticulum. Heavily, affected cells were necrotized and contained large virus particle aggregates which sometimes were crystallized.CarRSV is the third carnation virus, after carnation mottle and carnation Italian ringspot viruses, which was identified in a natural water.

Isolation of Carnation Italian Ringspot Virus from a Creek in a Forested Area South West of Bonn

AbstractDuring a survey of plant viruses in small ponds and creeks in a forested area near Bad Münstereifel about 30 km south‐west of Bonn several viruses were isolated. One of these was identified as carnation Italian ringspot virus which previously has been isolated only twice from carnations originating from Italy and USA and which infected carnations only with difficulty. The role of this virus in the forest ecosystem is so far unknown.

Serological Relationships among Tombusviruses

Summary Serological differentiation indices (SDIs) based on serum titrations in agar-gel double diffusion tests were determined for all ten definitive tombusviruses known at present, namely artichoke mottled crinkle, carnation Italian ringspot, cymbidium ringspot, eggplant mottled crinkle, Moroccan pepper, pelargonium leaf curl, petunia asteroid mosaic, the BS3 and type strains of tomato bushy stunt, and tombus Neckar viruses. More than 2000 titrations using 222 antisera from 36 rabbits were done. The SDIs ranged from 1 to >9, and all intermediate values were found. Simple trigonometry and computer methods were used to classify the results. There was no correlation between the serological relatedness of the particles of these viruses and the homology of their genome nucleic acids previously assessed by hybridization tests. In this respect they resemble the tymoviruses but not the tobamoviruses. The electrophoretic migration of the particles of 12 tombusviruses was studied in 1% agarose containing 20 mm-phosphate buffer pH 7.0. Some serologically indistinguishable tombusvirus strains migrated at different rates.

Ultrastructure and Origin of Cytoplasmic Multivesicular Bodies Induced by Carnation Italian Ringspot Virus

SUMMARY Ultrastructural investigations of different hosts infected with the tombusvirus carnation Italian ringspot have shown that the multivesicular bodies typical of tombusvirus infections develop from modified mitochondria. Digestion with RNase in high- or low-salt medium demonstrated that multivesicular bodies contain RNA, some of which is double-stranded and, possibly, of viral origin. Cytochrome oxidase was identified in multivesicular bodies by cytochemical tests.