Publications

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, wher ...	1995	7676638
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 ...	1996	8760449
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 metho ...	1999	10894633
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 marke ...	1999	10489347
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 tombu ...	2001	18944038
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 ...	2001	11125155
mitochondrial targeting and membrane anchoring of a viral replicase in plant and yeast cells.	replication of the carnation italian ringspot virus genomic rna in plant cells occurs in multivesicular bodies which develop from the mitochondrial outer membrane during infection. orf1 in the viral genome encodes a 36-kda protein, while orf2 codes for the 95-kda replicase by readthrough of the orf1 stop codon. we have shown previously that the n-terminal part of orf1 contains the information leading to vesiculation of mitochondria and that the 36-kda protein localizes to mitochondria. using inf ...	2002	12239325
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 a crystal structure of p19 from the carnation italian ...	2003	14697199
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 interfer ...	2003	12525646
expression of tombusvirus open reading frames 1 and 2 is sufficient for the replication of defective interfering, but not satellite, rna.	yeast cells co-expressing the replication proteins p36 and p95 of carnation italian ringspot virus (cirv) support the rna-dependent replication of several defective interfering (di) rnas derived from either the genome of cirv or the related cymbidium ringspot virus (cymrsv), but not the replication of a satellite rna (sat rna) originally associated with cymrsv. di, but not sat rna, was replicated in yeast cells co-expressing both di and sat rna. using transgenic nicotiana benthamiana plants cons ...	2004	15448375
pelargonium necrotic spot virus: a new member of the genus tombusvirus.	a virus isolate from pelargonium spp., provisionally designated upev (unknown pelargonium virus), had isometric particles 31-33 nm in diameter, with a granular surface structure similar to that of viruses in three genera of family tombusviridae. immunoelectron microscopy proved that upev was serologically distinct from all examined morphologically similar members of the family tombusviridae. the induced cytopathology was characterized by large cytoplasmic virion aggregates and the formation of m ...	2004	15290378
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 consisten ...	2004	15269385
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 germa ...	2004	15593416
introgression of a tombusvirus resistance locus from nicotiana edwardsonii var. columbia to n. clevelandii.	abstract a new variety of nicotiana, n. edwardsonii var. columbia, was evaluated for its capacity to serve as a new source for virus resistance genes. columbia was developed from a hybridization between n. glutinosa and n. clevelandii, the same parents used for the formation of the original n. edwardsonii. however, in contrast to the original n. edwardsonii, crosses between columbia and either of its parents are fertile. thus, the inheritance of virus resistance genes present in n. glutinosa cou ...	2006	18944304
stabilized recombinant suppressors of rna silencing: functional effects of linking monomers of carnation italian ringspot virus p19.	eukaryotes have evolved complex cellular responses to double-stranded rna including the rna silencing pathway. tombusviruses have adapted a mechanism to evade rna silencing that involves a 19 kda dimeric protein (p19) that is a suppressor of rna silencing. in order to develop stabilized p19 proteins, linked versions of p19 from the carnation italian ringspot virus (cirv) were constructed that joined the c-terminus of one subunit to the n-terminus of the second subunit. like the native cirv p19, ...	2007	17976393
effects of ph and salt concentration on the sirna binding activity of the rna silencing suppressor protein p19.	the rna silencing pathway is an important component of the anti-viral immune response in eukaryotes, particularly in plants. in turn, many viruses have evolved mechanisms to evade or suppress this pathway. tombusviruses such as the carnation italian ringspot virus (cirv) express a 19kda protein (p19) that is a suppressor of rna silencing in infected plants. this protein acts as a dimer and binds specifically to short-interfering rna (sirna) through electrostatic interactions between charged resi ...	2007	17559839
cymbidium ringspot virus harnesses rna silencing to control the accumulation of virus parasite satellite rna.	cymbidium ringspot virus (cymrsv) satellite rna (satrna) is a parasitic subviral rna replicon that replicates and accumulates at the cost of its helper virus. this 621-nucleotide (nt) satrna species has no sequence similarity to the helper virus, except for a 51-nt-long region termed the helper-satellite homology (hsh) region, which is essential for satrna replication. we show that the accumulation of satrna strongly depends on temperature and on the presence of the helper virus p19 silencing su ...	2008	18815304
localization of the carnation italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the tom complex.	carnation italian ringspot virus (cirv) is a positive-strand rna virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral rna replication. while the membrane-bound components of the cirv replication complex, including a 36-kd rna-binding protein (p36), are known to be essential for these changes in ...	2008	18811953
context-influenced cap-independent translation of tombusvirus mrnas in vitro.	tomato bushy stunt virus (tbsv) possesses a positive-strand rna genome that is not 5'-capped or 3'-polyadenylated. previous analysis revealed that the tbsv genome contains a 3'-cap-independent translational enhancer (3'cite) in its 3'-untranslated region (3'utr) that facilitates translation of viral mrnas in vivo. a long-range 5'-3' rna-rna interaction between the 3'cite and the 5'utr of viral mrnas is necessary for function, and this rna bridge has been proposed to mediate delivery of translati ...	2008	18775547
inhibition of 3' modification of small rnas in virus-infected plants require spatial and temporal co-expression of small rnas and viral silencing-suppressor proteins.	plant viruses are inducers and targets of rna silencing. viruses counteract with rna silencing by expressing silencing-suppressor proteins. many of the identified proteins bind sirnas, which prevents assembly of silencing effector complexes, and also interfere with their 3' methylation, which protects them against degradation. here, we investigated the 3' modification of silencing-related small rnas in nicotiana benthamiana plants infected with viruses expressing rna silencing suppressors, the p ...	2008	18539609
new isolates of carnation italian ringspot virus differ from the original one by having replication-associated proteins with a typical tombusvirus-like n-terminus and by inducing peroxisome- rather than mitochondrion-derived multivesicular bodies.	five new isolates of carnation italian ringspot virus (cirv) from cherry trees, gypsophila and surface water differ from the original carnation isolate (cirv-car) and also from pelargonium necrotic spot virus (pelnsv) by having an orf 1/orf1-rt with a typical tombusvirus-like 5'end and by inducing the formation of peroxisome- rather than mitochondrion-derived multivesicular bodies (mvbs). this supports with natural isolates earlier conclusions reached by others with artificially produced hybrid ...	2009	19768632
studies of a viral suppressor of rna silencing p19-cfp fusion protein: a fret-based probe for sensing double-stranded fluorophore tagged small rnas.	eukaryotes have evolved complex cellular responses to double-stranded rna. one response that is highly conserved across many species is the rna silencing pathway. tombusviruses have evolved a mechanism to evade the rna silencing pathway that involves a small protein, p19, that acts as a suppressor of rna silencing. this protein binds specifically to small-interfering rnas (sirnas) with nanomolar affinity in a sequence-independent manner and with size selectivity. here we demonstrate a new approa ...	2009	19491057
cysteine residues of carnation italian ringspot virus p19 suppressor of rna silencing maintain global structural integrity and stability for sirna binding.	carnation italian ringspot virus (cirv) has evolved a protein called p19 that acts as a suppressor of rna silencing in the host cell and aids in viral persistence. this protein has been shown to be sensitive to cysteine alkylation resulting in a reduction in its ability to bind to short-interfering rna (sirna). to determine the sites within the protein that are sensitive to alkylation, we systematically tested the functional role of each cysteine residue using site-directed mutagenesis. variants ...	2009	19341824
properties of a novel satellite rna associated with tomato bushy stunt virus infections.	the biological and molecular properties of a novel satellite rna (satrna l) associated with tomato bushy stunt virus (tbsv) are described. satrna l consisted of a linear single-stranded rna of 615 nt, lacked significant open reading frames (orfs) and had no sequence identity with the helper genome other than in the 5'-proximal 7 nt and in a central region that is also conserved in all tombusvirus genomic, defective interfering and satellite rnas. secondary-structure analysis showed the presence ...	2010	20484559
protein mediated mirna detection and sirna enrichment using p19.	p19 rna binding protein from the carnation italian ringspot virus (cirv) is an rna-silencing suppressor that binds small interfering rna (sirna) with high affinity. we created a bifunctional p19 fusion protein with an n-terminal maltose binding protein (mbp), for protein purification, and a c-terminal chitin binding domain (cbd) to bind p19 to chitin magnetic beads. the fusion protein binds dsrnas in the size range of 20-23 nucleotides, but does not bind ssrna or dsdna. relative affinities of th ...	2010	20569217
viral rnai suppressor reversibly binds sirna to outcompete dicer and risc via multiple turnover.	rna interference is a conserved gene regulatory mechanism employed by most eukaryotes as a key component of their innate immune response to viruses and retrotransposons. during viral infection, the rnase-iii-type endonuclease dicer cleaves viral double-stranded rna into small interfering rnas (sirnas) 21-24 nucleotides in length and helps load them into the rna-induced silencing complex (risc) to guide the cleavage of complementary viral rna. as a countermeasure, many viruses have evolved viral ...	2011	21354178
electronic detection of microrna at attomolar level with high specificity.	small rna (19-23 nucleotides) molecules play an important role in gene regulation, embryonic differentiation, hematopoiesis, and a variety of cancers. here, we present an ultrasensitive, extremely specific, label-free, and rapid electronic detection of micrornas (mirnas) using a carbon nanotubes field-effect transistor functionalized with the carnation italian ringspot virus p19 protein biosensor. mirna-122a was chosen as the target, which was first hybridized to a probe molecule. the probe-mirn ...	2013	23909395
ph-sensitive residues in the p19 rna silencing suppressor protein from carnation italian ringspot virus affect sirna binding stability.	tombusviruses, such as carnation italian ringspot virus (cirv), encode a protein homodimer called p19 that is capable of suppressing rna silencing in their infected hosts by binding to and sequestering short-interfering rna (sirna) away from the rna silencing pathway. p19 binding stability has been shown to be sensitive to changes in ph but the specific amino acid residues involved have remained unclear. using constant ph molecular dynamics simulations, we have identified key ph-dependent residu ...	2013	23450521
tombusvirus y-shaped translational enhancer forms a complex with eif4f and can be functionally replaced by heterologous translational enhancers.	certain plus-strand rna plant viruses that are uncapped and nonpolyadenylated rely on rna elements in their 3' untranslated region, termed 3'-cap-independent translational enhancers (3'cites), for efficient translation of their proteins. here, we have investigated the properties of the y-shaped class of 3'cite present in the tombusvirus carnation italian ringspot virus (cirv). while some types of 3'cite have been found to function through recruitment of translation initiation factors to the vira ...	2013	23192876
a unique n-terminal sequence in the carnation italian ringspot virus p36 replicase-associated protein interacts with the host cell escrt-i component vps23.	like most positive-strand rna viruses, infection by plant tombusviruses results in extensive rearrangement of specific host cell organelle membranes that serve as the sites of viral replication. the tombusvirus tomato bushy stunt virus (tbsv) replicates within spherules derived from the peroxisomal boundary membrane, a process that involves the coordinated action of various viral and cellular factors, including constituents of the endosomal sorting complex required for transport (escrt). escrt i ...	2014	24672030
enrichment of phosphatidylethanolamine in viral replication compartments via co-opting the endosomal rab5 small gtpase by a positive-strand rna virus.	positive-strand rna viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. these viruses require host factors and various lipids to form viral replication complexes (vrcs). the vrcs built by tomato bushy stunt virus (tbsv) are enriched with phosphatidylethanolamine (pe) through a previously unknown pathway. to unravel the mechanism of pe enrichment within the tbsv replication compartment, in this paper, the a ...	2016	27760128
heterologous expression of carnation italian ringspot virus p36 protein enhances necrotic cell death in response to acetic acid in saccharomyces cerevisiae.	a universal feature of the replication of positive-strand rna viruses is the association with intracellular membranes. carnation italian ringspot virus (cirv) replication in plants occurs in vesicles derived from the mitochondrial outer membrane. the product encoded by cirv orf1, p36, is required for targeting the virus replication complex to the outer mitochondrial membrane both in plant and yeast cells. here the yeast saccharomyces cerevisiae was used as a model host to study the effect of cir ...	2017	27637297