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Michael Taliansky - One of the best experts on this subject based on the ideXlab platform.
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Umbravirus encoded movement protein induces tubule formation on the surface of protoplasts and binds rna incompletely and non cooperatively
Journal of General Virology, 2001Co-Authors: Kulpash M Nurkiyanova, Eugene V Ryabov, N O Kalinina, Yongchang Fan, Igor A Andreev, A G Fitzgerald, Peter Palukaitis, Michael TalianskyAbstract:Various functions of the cell-to-cell movement protein (MP) of Groundnut rosette virus (GRV) were analysed. The GRV ORF4-encoded protein was shown by immunofluorescence microscopy to generate tubular structures that protrude from the surface of the protoplast. The protein encoded by ORF4 was assessed also for RNA-binding properties. This protein was tagged at its C terminus with six histidine residues, produced in Escherichia coli using an expression vector and purified by affinity chromatography. Gel retardation analysis demonstrated that, in contrast to many other viral MPs, including the 3a MP of Cucumber mosaic virus (CMV), the ORF4-encoded protein bound non-cooperatively to viral ssRNA and formed complexes of low protein:RNA ratios. Competition binding experiments showed that the ORF4-encoded protein bound to both ssRNA and ssDNA without sequence specificity, but did not bind to dsDNA. UV cross-linking and nitrocellulose membrane-retention assays confirmed that both the GRV and the CMV MPs formed complexes with ssRNA and that these complexes showed similar stability in NaCl. Probing the MP–RNA complexes by atomic force microscopy demonstrated that the ORF4-encoded protein bound RNA incompletely, leaving protein-free RNA segments of varying length, while the CMV 3a protein formed highly packed complexes. The significance of the two properties of limited RNA binding and tubule formation of the umbraviral MP is discussed.
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Umbravirus encoded proteins both stabilize heterologous viral rna and mediate its systemic movement in some plant species
Virology, 2001Co-Authors: Eugene V Ryabov, D J Robinson, Michael TalianskyAbstract:The proteins encoded by open reading frame 3 (ORF3) of the Umbraviruses pea enation mosaic virus-2 and tobacco mottle virus, like that of groundnut rosette virus, mediated the movement of viral RNA through the phloem of infected Nicotiana benthamiana or N. clevelandii plants when they were expressed from chimeric tobacco mosaic virus in place of the coat protein. However, these chimeras did not move systemically in N. tabacum. In lysates of N. benthamiana or N. tabacum protoplasts, the chimeric RNAs were more stable than was RNA of tobacco mosaic virus lacking the coat protein gene. The chimeric viruses also protected the latter in trans, suggesting that the ORF3 proteins can increase the stability of heterologous viral RNA. Umbraviral ORF3 proteins contain a conserved arginine-rich domain, and the possible roles of this motif in the functions of the proteins are discussed.
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Umbravirus gene expression helps potato leafroll virus to invade mesophyll tissues and to be transmitted mechanically between plants
Virology, 2001Co-Authors: Eugene V Ryabov, H. Barker, Gillian Fraser, M A Mayo, Michael TalianskyAbstract:Potato leafroll virus (PLRV) was mechanically transmissible when inocula also contained the Umbravirus Pea enation mosaic virus-2 (PEMV-2). In plants infected with PLRV and PEMV-2, PLRV accumulated in clusters of mesophyll cells in both inoculated and systemically infected leaves. No transmissions were obtained by coinoculation with Potato virus Y, Potato virus X (PVX), Tobacco mosaic virus, or Cucumber mosaic virus (CMV), although PLRV was transmissible from mixtures with CMV(ORF4) (a recombinant that contained the movement protein (MP) gene of the Umbravirus Groundnut rosette virus (GRV) in place of the CMV MP gene). In contrast, neither a recombinant PVX that expressed GRV MP nor a mutant of CMV(ORF4), in which the CMV 2b gene was untranslatable, was able to help PLRV transmission. Possibly both a cell-to-cell movement function and counterdefense mechanisms such as those that block posttranscriptional gene silencing are involved in movement of PLRV within plants and its mechanical transmission between plants.
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satellite rna is essential for encapsidation of groundnut rosette Umbravirus rna by groundnut rosette assistor luteovirus coat protein
Virology, 1999Co-Authors: D J Robinson, Eugene V Ryabov, S K Raj, I M Roberts, Michael TalianskyAbstract:Abstract Groundnut rosette disease is caused by a complex of agents comprising groundnut rosette Umbravirus (GRV), GRV satellite RNA (sat-RNA)groundnut rosette assistor luteovirus (GRAV). Both GRAV and GRV sat-RNA are needed for GRV to be aphid transmissible. To understand the role of GRAVGRV sat-RNA in the aphid transmission of GRV, encapsidation of GRV genomicsatellite RNAs has been studied using transgenic Nicotiana benthamiana plants expressing GRAV coat protein (CP). GRAV CP expressed from a transgene was shown to package GRV genomicsatellite RNAs efficiently, giving a high yield of transcapsidated virus particles. GRV sat-RNA was absolutely essential for this process. GRV genomic RNA was not encapsidated by GRAV CP in the absence of the sat-RNA. Using different mutants of GRV sat-RNA, it was found that some property of full-length satellite RNA molecules, such as size or specific conformation rather than potential open reading frames, was required for the production of virus particles. A correlation between the ability of sat-RNA to stimulate encapsidation of GRV RNA by GRAV CPits capacity to promote aphid transmission of GRV was observed.
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intracellular location of two groundnut rosette Umbravirus proteins delivered by pvx and tmv vectors
Virology, 1998Co-Authors: Eugene V Ryabov, D J Robinson, Karl J Oparka, Simon Santa Cruz, Michael TalianskyAbstract:The proteins encoded by open reading frames (ORF) 3 and 4 of groundnut rosette Umbravirus (GRV) were expressed in Nicotiana benthamiana as fusions with green fluorescent protein (GFP) from modified potato virus X (PVX) and tobacco mosaic virus (TMV) vectors. Regardless of which plant virus vector was used, GFP fused to the ORF3 protein accumulated in large cytoplasmic inclusion bodies and in nucleoli, whereas GFP fused to the ORF4 protein was found in cell walls close to plasmodesmata. Cell-to-cell movement of PVX requires three proteins encoded by the triple gene block (TGB) and also the coat protein (CP). However, when GRV ORF4 was substituted for the PVX CP gene, the hybrid virus was able to move normally in inoculated leaves but not into noninoculated leaves. In contrast, when GRV ORF4 was substituted for the TGB, or for both the TGB and the CP gene, movement of the hybrid viruses was limited to a few epidermal cells neighboring the infection site. Thus, the GRV ORF4 protein can replace the movement proteins of PVX for some of their functions.
Anne E Simon - One of the best experts on this subject based on the ideXlab platform.
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structural analysis and whole genome mapping of a new type of plant virus subviral rna Umbravirus like associated rnas
Viruses, 2021Co-Authors: Jingyuan Liu, Elizabeth Carino, Sayanta Bera, Feng Gao, Jared May, Anne E SimonAbstract:We report the biological and structural characterization of Umbravirus-like associated RNAs (ulaRNAs), a new category of coat-protein dependent subviral RNA replicons that infect plants. These RNAs encode an RNA-dependent RNA polymerase (RdRp) following a −1 ribosomal frameshift event, are 2.7–4.6 kb in length, and are related to Umbraviruses, unlike similar RNA replicons that are related to tombusviruses. Three classes of ulaRNAs are proposed, with citrus yellow vein associated virus (CYVaV) placed in Class 2. With the exception of CYVaV, Class 2 and Class 3 ulaRNAs encode an additional open reading frame (ORF) with movement protein-like motifs made possible by additional sequences just past the RdRp termination codon. The full-length secondary structure of CYVaV was determined using Selective 2’ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) structure probing and phylogenic comparisons, which was used as a template for determining the putative structures of the other Class 2 ulaRNAs, revealing a number of distinctive structural features. The ribosome recoding sites of nearly all ulaRNAs, which differ significantly from those of Umbraviruses, may exist in two conformations and are highly efficient. The 3′ regions of Class 2 and Class 3 ulaRNAs have structural elements similar to those of nearly all Umbraviruses, and all Class 2 ulaRNAs have a unique, conserved 3′ cap-independent translation enhancer. CYVaV replicates independently in protoplasts, demonstrating that the reported sequence is full-length. Additionally, CYVaV contains a sequence in its 3′ UTR that confers protection to nonsense mediated decay (NMD), thus likely obviating the need for Umbravirus ORF3, a known suppressor of NMD. This initial characterization lays down a road map for future investigations into these novel virus-like RNAs.
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the multifunctional long distance movement protein of pea enation mosaic virus 2 protects viral and host transcripts from nonsense mediated decay
Mbio, 2020Co-Authors: Philip Z Johnson, Muhammad Ilyas, Anne E SimonAbstract:ABSTRACT The nonsense-mediated decay (NMD) pathway presents a challenge for RNA viruses with termination codons that precede extended 3′ untranslated regions (UTRs). The Umbravirus Pea enation mosaic virus 2 (PEMV2) is a nonsegmented, positive-sense RNA virus with an unusually long 3′ UTR that is susceptible to NMD. To establish a systemic infection, the PEMV2 long-distance movement protein p26 was previously shown to both stabilize viral RNAs and bind them for transport through the plant’s vascular system. The current study demonstrated that p26 protects both viral and nonviral messenger RNAs from NMD. Although p26 localizes to both the cytoplasm and nucleolus, p26 exerts its anti-NMD effects exclusively in the cytoplasm independently of long-distance movement. Using a transcriptome-wide approach in the model plant Nicotiana benthamiana, p26 protected a subset of cellular NMD target transcripts, particularly those containing long, structured, GC-rich 3′ UTRs. Furthermore, transcriptome sequencing (RNA-seq) revealed that the NMD pathway is highly dysfunctional during PEMV2 infection, with 1,820 (48%) of NMD targets increasing in abundance. Widespread changes in the host transcriptome are common during plant RNA virus infections, and these results suggest that, in at least some instances, virus-mediated NMD inhibition may be a major contributing factor. IMPORTANCE Nonsense-mediated decay (NMD) represents an RNA regulatory pathway that degrades both natural and faulty messenger RNAs with long 3′ untranslated regions. NMD targets diverse families of RNA viruses, requiring that viruses counteract the NMD pathway for successful amplification in host cells. A protein required for long-distance movement of Pea enation mosaic virus 2 (PEMV2) is shown to also protect both viral and host mRNAs from NMD. RNA-seq analyses of the Nicotiana benthamiana transcriptome revealed that PEMV2 infection significantly impairs the host NMD pathway. RNA viruses routinely induce large-scale changes in host gene expression, and, like PEMV2, may use NMD inhibition to alter the host transcriptome in an effort to increase virus amplification.
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unusual dicistronic expression from closely spaced initiation codons in an Umbravirus subgenomic rna
Nucleic Acids Research, 2018Co-Authors: Feng Gao, Olga M Alekhina, Konstantin S Vassilenko, Anne E SimonAbstract:Translation commencing at closely spaced initiation codons is common in RNA viruses with limited genome space. In the subgenomic RNA (sgRNA) of Pea enation mosaic virus 2, two closely spaced, out-of-frame start codons direct synthesis of movement/stability proteins p26 and p27. Efficient translation from AUG26/AUG27 is dependent on three 3'-proximal cap-independent translation enhancers (3'CITEs), whereas translation of the genomic (gRNA) requires only two. Contrary to strictly scanning-dependent initiation at the gRNA, sequence context of AUG26/AUG27 does not conform with Kozak requirements and insertion of efficient upstream AUGs had pronounced effects for AUG26 but only moderate effects for AUG27. Insertion of a hairpin within an extended 5' UTR did not significantly impact translation from AUG26/AUG27. Furthermore, AUG27 repressed translation from upstream AUG26 and this effect was mitigated when inter-codon spacing was reduced. Addition of a stable hairpin to the very 5' end of the sgRNA severely restricted translation, testifying that this 3'CITE-driven initiation is 5' end-dependent. Similar to gRNA, sgRNA reporter transcripts were nearly exclusively associated with light polysomes and 3'CITE-promoted long-distance interaction connecting the sgRNA ends affected the number of templates translated and not the initiation rate. We propose a non-canonical, 3'CITE-driven mechanism for efficient dicistronic expression from Umbravirus sgRNAs.
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RNA virus evasion of nonsense-mediated decay
PLOS Pathogens, 2018Co-Authors: Xuefeng Yuan, Erika Sawicki, Anne E SimonAbstract:Nonsense-mediated decay (NMD) is a host RNA control pathway that removes aberrant transcripts with long 3’ untranslated regions (UTRs) due to premature termination codons (PTCs) that arise through mutation or defective splicing. To maximize coding potential, RNA viruses often contain internally located stop codons that should also be prime targets for NMD. Using an agroinfiltration-based NMD assay in Nicotiana benthamiana, we identified two segments conferring NMD-resistance in the carmovirus Turnip crinkle virus (TCV) genome. The ribosome readthrough structure just downstream of the TCV p28 termination codon stabilized an NMD-sensitive reporter as did a frameshifting element from Umbravirus Pea enation mosaic virus. In addition, a 51-nt unstructured region (USR) at the beginning of the TCV 3’ UTR increased NMD-resistance 3-fold when inserted into an unrelated NMD-sensitive 3’ UTR. Several additional carmovirus 3’ UTRs also conferred varying levels of NMD resistance depending on the construct despite no sequence similarity in the analogous region. Instead, these regions displayed a marked lack of RNA structure immediately following the NMD-targeted stop codon. NMD-resistance was only slightly reduced by conversion of 19 pyrimidines in the USR to purines, but resistance was abolished when a 2-nt mutation was introduced downstream of the USR that substantially increased the secondary structure in the USR through formation of a stable hairpin. The same 2-nt mutation also enhanced the NMD susceptibility of a subgenomic RNA expressed independently of the genomic RNA. The conserved lack of RNA structure among most carmoviruses at the 5’ end of their 3’ UTR could serve to enhance subgenomic RNA stability, which would increase expression of the encoded capsid protein that also functions as the RNA silencing suppressor. These results demonstrate that the TCV genome has features that are inherently NMD-resistant and these strategies could be widespread among RNA viruses and NMD-resistant host mRNAs with long 3’ UTRs.
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multiple cis acting elements modulate programmed 1 ribosomal frameshifting in pea enation mosaic virus
Nucleic Acids Research, 2016Co-Authors: Anne E SimonAbstract:: Programmed -1 ribosomal frameshifting (-1 PRF) is used by many positive-strand RNA viruses for translation of required products. Despite extensive studies, it remains unresolved how cis-elements just downstream of the recoding site promote a precise level of frameshifting. The Umbravirus Pea enation mosaic virus RNA2 expresses its RNA polymerase by -1 PRF of the 5'-proximal ORF (p33). Three hairpins located in the vicinity of the recoding site are phylogenetically conserved among Umbraviruses. The central Recoding Stimulatory Element (RSE), located downstream of the p33 termination codon, is a large hairpin with two asymmetric internal loops. Mutational analyses revealed that sequences throughout the RSE and the RSE lower stem (LS) structure are important for frameshifting. SHAPE probing of mutants indicated the presence of higher order structure, and sequences in the LS may also adapt an alternative conformation. Long-distance pairing between the RSE and a 3' terminal hairpin was less critical when the LS structure was stabilized. A basal level of frameshifting occurring in the absence of the RSE increases to 72% of wild-type when a hairpin upstream of the slippery site is also deleted. These results suggest that suppression of frameshifting may be needed in the absence of an active RSE conformation.
Gustaaf A De Zoeten - One of the best experts on this subject based on the ideXlab platform.
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mutational evidence that the vpg is involved in the replication and not the movement of pea enation mosaic virus 1
Journal of General Virology, 2000Co-Authors: Jihad S Skaf, Marilou H Schultz, Hisae Hirata, Gustaaf A De ZoetenAbstract:Pea enation mosaic disease is caused by an obligatory association between the enamovirus Pea enation mosaic virus-1 (PEMV-1) and the Umbravirus Pea enation mosaic virus-2(PEMV-2). Encapsidated RNAs 1 and 2 are covalently linked to a 3138 Da VPg encoded by the RNA of PEMV-1. To determine the role of the VPg in the pathogenicity of PEMV (PEMV-1+PEMV-2), the infectivity of clones with mutations in key amino acids in the VPg was evaluated in protoplasts and in plants. Using quantitative, real-time RT–PCR, we concluded that the inability of certain mutants to infect plants was due to their replicative (and not their movement) incompetence. Mutant clones that produced delayed and less severe infections accumulated 10- to 100-fold less RNA-1 compared to WT-RNA-1 both in plants and in protoplasts. The RNAs of clones that produced WT-like infections accumulated to levels similar to those of WT-PEMV. Also, we demonstrate that the severity of symptoms produced by WT-PEMV is proportional to the amount of RNA-1 that accumulates in infected plants and seems to be independent of the amount of RNA-2. A dual role for the VPg in the pathogenicity of PEMV is proposed.
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mutational evidence that the vpg is involved in the replication and not the movement of pea enation mosaic virus 1
Journal of General Virology, 2000Co-Authors: Jihad S Skaf, Marilou H Schultz, Hisae Hirata, Gustaaf A De ZoetenAbstract:Pea enation mosaic disease is caused by an obligatory association between the enamovirus Pea enation mosaic virus-1 (PEMV-1) and the Umbravirus Pea enation mosaic virus-2(PEMV-2). Encapsidated RNAs 1 and 2 are covalently linked to a 3138 Da VPg encoded by the RNA of PEMV-1. To determine the role of the VPg in the pathogenicity of PEMV (PEMV-1+PEMV-2), the infectivity of clones with mutations in key amino acids in the VPg was evaluated in protoplasts and in plants. Using quantitative, real-time RT–PCR, we concluded that the inability of certain mutants to infect plants was due to their replicative (and not their movement) incompetence. Mutant clones that produced delayed and less severe infections accumulated 10- to 100-fold less RNA-1 compared to WT-RNA-1 both in plants and in protoplasts. The RNAs of clones that produced WT-like infections accumulated to levels similar to those of WT-PEMV. Also, we demonstrate that the severity of symptoms produced by WT-PEMV is proportional to the amount of RNA-1 that accumulates in infected plants and seems to be independent of the amount of RNA-2. A dual role for the VPg in the pathogenicity of PEMV is proposed.
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expression and suppression of circulative aphid transmission in pea enation mosaic virus
Journal of General Virology, 1997Co-Authors: S A Demler, Jihad S Skaf, D G Ruckerfeeney, Gustaaf A De ZoetenAbstract:Pea enation mosaic virus (PEMV) is composed of two autonomously replicating virus RNAs related to the genomic RNAs of viruses in the genera Luteovirus and Umbravirus. The transmission of PEMV resembles that of its luteovirus relatives in utilizing circulative aphid transmission. However, unlike its luteovirus counterparts, PEMV can also be mechanically transmitted. Prolonged mechanical passage of PEMV can lead to the loss of aphid transmissibility, a trait that is mirrored by specific changes in the PEMV virion composition. These changes were used to examine the virus contribution to vector transmission and the mechanisms by which it is regulated. Using a local lesion isolation technique, one aphid transmissible and two aphid non-transmissible isolates of PEMV were compared. Structural analysis of a 54 kDa minor structural subunit unique to the aphid transmissible isolate demonstrated that it was a fusion of the 21 kDa virus coat protein and a 33 kDa protein encoded immediately downstream of the 21 kDa ORF, consistent with the formation of the 54 kDa subunit by translational readthrough. Genetic analyses utilizing exchanges between infectious in vitro transcripts of each isolate demonstrated that although the 33 kDa protein was non-essential for infection, its presence was mandatory for aphid transmission, and that specific changes within the 33 kDa ORF were sufficient to confer or abolish aphid transmission. This study also demonstrates that isolates of PEMV exist as mixtures of aphid transmissible and non-transmissible genotypes, and provides insight into the mechanisms used by this virus to down-regulate aphid transmission in response to a specific selection pressure.
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the satellite rnas associated with the groundnut rosette disease complex and pea enation mosaic virus sequence similarities and ability of each other s helper virus to support their replication
Journal of General Virology, 1996Co-Authors: S A Demler, Gustaaf A De Zoeten, D G Rucker, D J Robinson, Angelika Ziegler, A F MurantAbstract:Pea enation mosaic virus (PEMV) and the causal agents of groundnut rosette disease are diverse examples of disease complexes involving two RNA species, one of which is related to the genomes of luteoviruses and the other to those of Umbraviruses. In both complexes, these viral RNA components may be supplemented with satellite RNAs that are dependent on the Umbravirus component for replication and systemic movement, and on the luteovirus component for encapsidation and vector transmission. Sequence analysis identified regions of similarity between the satellites of groundnut rosette virus (GRV) and PEMV, particularly at the 5′ and 3′ termini and around duplicate sequence repeats present in each satellite RNA. The Umbravirus GRV and the Umbravirus-like PEMV RNA-2 were each able to support the replication and systemic spread of homologous and heterologous satellites. The presence of the PEMV satellite in infections with GRV had no effect on symptom expression in Nicotiana spp. or in Arachis hypogaea. Likewise, in Pisum sativum, the GRV satellite had no effect on the symptoms induced by PEMV. However, the intense yellow blotch symptoms induced in Nicotiana benthamiana by the YB3 GRV satellite in conjunction with GRV were also manifested when PEMV was the helper. Although PEMV RNA-1 was capable of supporting the encapsidation and aphid transmission of the GRV satellite, no evidence was obtained that the essential role of the GRV satellite in the aphid transmission of GRV could be supplied by the PEMV satellite. These data further strengthen the hypothesis of an evolutionary relationship between PEMV and the luteovirus-Umbravirus complexes.
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replication of the satellite rna of pea enation mosaic virus is controlled by rna 2 encoded functions
Journal of General Virology, 1994Co-Authors: S A Demler, D G Rucker, L Nooruddin, Gustaaf A De ZoetenAbstract:The helper virus mediating replication of the satellite RNA (RNA 3) of pea enation mosaic virus (PEMV) consists of two autonomously replicating, taxonomically unrelated viral RNAs with ties to the luteovirus (RNA 1) and the newly proposed Umbravirus (RNA 2) genera. The following study dissects the relative contribution of each of the genomic RNAs of PEMV to the subsistence and dissemination of this satellite RNA. Infectivity assays in a pea protoplast system demonstrate that RNA 2 alone is responsible for the replication of RNA 3, an observation that is supported in part by shared regions of sequence homology at the 5′ and 3′ termini of both RNAs. In pea seedlings, infectivity assays also demonstrated that the presence of RNA 2 alone is necessary for the systemic invasion of RNA 3. In contrast, the luteovirus-like phase of PEMV (RNA 1) is solely responsible for the encapsidation and aphid transmission of both RNA 2 and the satellite RNA. In a manner comparable to several other virus-satellite systems, the satellite of PEMV also displays a differential response in its capacity to attenuate symptom expression in selected host species. Thus, the satellite RNA of PEMV exists in a trilateral arrangement with its host and two viral RNAs, comparable in many respects to the satellite-virus-host interaction occurring with ground-nut rosette disease.
D J Robinson - One of the best experts on this subject based on the ideXlab platform.
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Umbravirus encoded proteins both stabilize heterologous viral rna and mediate its systemic movement in some plant species
Virology, 2001Co-Authors: Eugene V Ryabov, D J Robinson, Michael TalianskyAbstract:The proteins encoded by open reading frame 3 (ORF3) of the Umbraviruses pea enation mosaic virus-2 and tobacco mottle virus, like that of groundnut rosette virus, mediated the movement of viral RNA through the phloem of infected Nicotiana benthamiana or N. clevelandii plants when they were expressed from chimeric tobacco mosaic virus in place of the coat protein. However, these chimeras did not move systemically in N. tabacum. In lysates of N. benthamiana or N. tabacum protoplasts, the chimeric RNAs were more stable than was RNA of tobacco mosaic virus lacking the coat protein gene. The chimeric viruses also protected the latter in trans, suggesting that the ORF3 proteins can increase the stability of heterologous viral RNA. Umbraviral ORF3 proteins contain a conserved arginine-rich domain, and the possible roles of this motif in the functions of the proteins are discussed.
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satellite rna is essential for encapsidation of groundnut rosette Umbravirus rna by groundnut rosette assistor luteovirus coat protein
Virology, 1999Co-Authors: D J Robinson, Eugene V Ryabov, S K Raj, I M Roberts, Michael TalianskyAbstract:Abstract Groundnut rosette disease is caused by a complex of agents comprising groundnut rosette Umbravirus (GRV), GRV satellite RNA (sat-RNA)groundnut rosette assistor luteovirus (GRAV). Both GRAV and GRV sat-RNA are needed for GRV to be aphid transmissible. To understand the role of GRAVGRV sat-RNA in the aphid transmission of GRV, encapsidation of GRV genomicsatellite RNAs has been studied using transgenic Nicotiana benthamiana plants expressing GRAV coat protein (CP). GRAV CP expressed from a transgene was shown to package GRV genomicsatellite RNAs efficiently, giving a high yield of transcapsidated virus particles. GRV sat-RNA was absolutely essential for this process. GRV genomic RNA was not encapsidated by GRAV CP in the absence of the sat-RNA. Using different mutants of GRV sat-RNA, it was found that some property of full-length satellite RNA molecules, such as size or specific conformation rather than potential open reading frames, was required for the production of virus particles. A correlation between the ability of sat-RNA to stimulate encapsidation of GRV RNA by GRAV CPits capacity to promote aphid transmission of GRV was observed.
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intracellular location of two groundnut rosette Umbravirus proteins delivered by pvx and tmv vectors
Virology, 1998Co-Authors: Eugene V Ryabov, D J Robinson, Karl J Oparka, Simon Santa Cruz, Michael TalianskyAbstract:The proteins encoded by open reading frames (ORF) 3 and 4 of groundnut rosette Umbravirus (GRV) were expressed in Nicotiana benthamiana as fusions with green fluorescent protein (GFP) from modified potato virus X (PVX) and tobacco mosaic virus (TMV) vectors. Regardless of which plant virus vector was used, GFP fused to the ORF3 protein accumulated in large cytoplasmic inclusion bodies and in nucleoli, whereas GFP fused to the ORF4 protein was found in cell walls close to plasmodesmata. Cell-to-cell movement of PVX requires three proteins encoded by the triple gene block (TGB) and also the coat protein (CP). However, when GRV ORF4 was substituted for the PVX CP gene, the hybrid virus was able to move normally in inoculated leaves but not into noninoculated leaves. In contrast, when GRV ORF4 was substituted for the TGB, or for both the TGB and the CP gene, movement of the hybrid viruses was limited to a few epidermal cells neighboring the infection site. Thus, the GRV ORF4 protein can replace the movement proteins of PVX for some of their functions.
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the satellite rnas associated with the groundnut rosette disease complex and pea enation mosaic virus sequence similarities and ability of each other s helper virus to support their replication
Journal of General Virology, 1996Co-Authors: S A Demler, Gustaaf A De Zoeten, D G Rucker, D J Robinson, Angelika Ziegler, A F MurantAbstract:Pea enation mosaic virus (PEMV) and the causal agents of groundnut rosette disease are diverse examples of disease complexes involving two RNA species, one of which is related to the genomes of luteoviruses and the other to those of Umbraviruses. In both complexes, these viral RNA components may be supplemented with satellite RNAs that are dependent on the Umbravirus component for replication and systemic movement, and on the luteovirus component for encapsidation and vector transmission. Sequence analysis identified regions of similarity between the satellites of groundnut rosette virus (GRV) and PEMV, particularly at the 5′ and 3′ termini and around duplicate sequence repeats present in each satellite RNA. The Umbravirus GRV and the Umbravirus-like PEMV RNA-2 were each able to support the replication and systemic spread of homologous and heterologous satellites. The presence of the PEMV satellite in infections with GRV had no effect on symptom expression in Nicotiana spp. or in Arachis hypogaea. Likewise, in Pisum sativum, the GRV satellite had no effect on the symptoms induced by PEMV. However, the intense yellow blotch symptoms induced in Nicotiana benthamiana by the YB3 GRV satellite in conjunction with GRV were also manifested when PEMV was the helper. Although PEMV RNA-1 was capable of supporting the encapsidation and aphid transmission of the GRV satellite, no evidence was obtained that the essential role of the GRV satellite in the aphid transmission of GRV could be supplied by the PEMV satellite. These data further strengthen the hypothesis of an evolutionary relationship between PEMV and the luteovirus-Umbravirus complexes.
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complete nucleotide sequence and organization of the rna genome of groundnut rosette Umbravirus
Journal of General Virology, 1996Co-Authors: Michael Taliansky, D J Robinson, A F MurantAbstract:Complementary DNA clones representing the entire genome of groundnut rosette Umbravirus (GRV) were obtained and sequenced. GRV RNA comprises 4019 nucleotides and contains four large open reading frames (ORFs). The second ORF from the 5′ end includes sequences that encode motifs characteristic of viral RNA-dependent RNA polymerases and is probably expressed by a -1 frameshift mechanism as a fusion protein with the product of the 5′-most ORF. The other two ORFs are almost completely overlapping in different reading frames, and are probably expressed from subgenomic RNA. One of the putative products has significant sequence similarity with viral movement proteins. None of the putative proteins encoded by GRV RNA seems to be a structural protein. In genome organization and in the amino acid sequences of its potential products, the RNA of GRV is similar tot hat of carrot mottle mimic Umbravirus, and to the Umbravirus-like RNA-2 of pea enation mosaic virus.
Eugene V Ryabov - One of the best experts on this subject based on the ideXlab platform.
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Umbravirus encoded movement protein induces tubule formation on the surface of protoplasts and binds rna incompletely and non cooperatively
Journal of General Virology, 2001Co-Authors: Kulpash M Nurkiyanova, Eugene V Ryabov, N O Kalinina, Yongchang Fan, Igor A Andreev, A G Fitzgerald, Peter Palukaitis, Michael TalianskyAbstract:Various functions of the cell-to-cell movement protein (MP) of Groundnut rosette virus (GRV) were analysed. The GRV ORF4-encoded protein was shown by immunofluorescence microscopy to generate tubular structures that protrude from the surface of the protoplast. The protein encoded by ORF4 was assessed also for RNA-binding properties. This protein was tagged at its C terminus with six histidine residues, produced in Escherichia coli using an expression vector and purified by affinity chromatography. Gel retardation analysis demonstrated that, in contrast to many other viral MPs, including the 3a MP of Cucumber mosaic virus (CMV), the ORF4-encoded protein bound non-cooperatively to viral ssRNA and formed complexes of low protein:RNA ratios. Competition binding experiments showed that the ORF4-encoded protein bound to both ssRNA and ssDNA without sequence specificity, but did not bind to dsDNA. UV cross-linking and nitrocellulose membrane-retention assays confirmed that both the GRV and the CMV MPs formed complexes with ssRNA and that these complexes showed similar stability in NaCl. Probing the MP–RNA complexes by atomic force microscopy demonstrated that the ORF4-encoded protein bound RNA incompletely, leaving protein-free RNA segments of varying length, while the CMV 3a protein formed highly packed complexes. The significance of the two properties of limited RNA binding and tubule formation of the umbraviral MP is discussed.
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Umbravirus encoded proteins both stabilize heterologous viral rna and mediate its systemic movement in some plant species
Virology, 2001Co-Authors: Eugene V Ryabov, D J Robinson, Michael TalianskyAbstract:The proteins encoded by open reading frame 3 (ORF3) of the Umbraviruses pea enation mosaic virus-2 and tobacco mottle virus, like that of groundnut rosette virus, mediated the movement of viral RNA through the phloem of infected Nicotiana benthamiana or N. clevelandii plants when they were expressed from chimeric tobacco mosaic virus in place of the coat protein. However, these chimeras did not move systemically in N. tabacum. In lysates of N. benthamiana or N. tabacum protoplasts, the chimeric RNAs were more stable than was RNA of tobacco mosaic virus lacking the coat protein gene. The chimeric viruses also protected the latter in trans, suggesting that the ORF3 proteins can increase the stability of heterologous viral RNA. Umbraviral ORF3 proteins contain a conserved arginine-rich domain, and the possible roles of this motif in the functions of the proteins are discussed.
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Umbravirus gene expression helps potato leafroll virus to invade mesophyll tissues and to be transmitted mechanically between plants
Virology, 2001Co-Authors: Eugene V Ryabov, H. Barker, Gillian Fraser, M A Mayo, Michael TalianskyAbstract:Potato leafroll virus (PLRV) was mechanically transmissible when inocula also contained the Umbravirus Pea enation mosaic virus-2 (PEMV-2). In plants infected with PLRV and PEMV-2, PLRV accumulated in clusters of mesophyll cells in both inoculated and systemically infected leaves. No transmissions were obtained by coinoculation with Potato virus Y, Potato virus X (PVX), Tobacco mosaic virus, or Cucumber mosaic virus (CMV), although PLRV was transmissible from mixtures with CMV(ORF4) (a recombinant that contained the movement protein (MP) gene of the Umbravirus Groundnut rosette virus (GRV) in place of the CMV MP gene). In contrast, neither a recombinant PVX that expressed GRV MP nor a mutant of CMV(ORF4), in which the CMV 2b gene was untranslatable, was able to help PLRV transmission. Possibly both a cell-to-cell movement function and counterdefense mechanisms such as those that block posttranscriptional gene silencing are involved in movement of PLRV within plants and its mechanical transmission between plants.
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satellite rna is essential for encapsidation of groundnut rosette Umbravirus rna by groundnut rosette assistor luteovirus coat protein
Virology, 1999Co-Authors: D J Robinson, Eugene V Ryabov, S K Raj, I M Roberts, Michael TalianskyAbstract:Abstract Groundnut rosette disease is caused by a complex of agents comprising groundnut rosette Umbravirus (GRV), GRV satellite RNA (sat-RNA)groundnut rosette assistor luteovirus (GRAV). Both GRAV and GRV sat-RNA are needed for GRV to be aphid transmissible. To understand the role of GRAVGRV sat-RNA in the aphid transmission of GRV, encapsidation of GRV genomicsatellite RNAs has been studied using transgenic Nicotiana benthamiana plants expressing GRAV coat protein (CP). GRAV CP expressed from a transgene was shown to package GRV genomicsatellite RNAs efficiently, giving a high yield of transcapsidated virus particles. GRV sat-RNA was absolutely essential for this process. GRV genomic RNA was not encapsidated by GRAV CP in the absence of the sat-RNA. Using different mutants of GRV sat-RNA, it was found that some property of full-length satellite RNA molecules, such as size or specific conformation rather than potential open reading frames, was required for the production of virus particles. A correlation between the ability of sat-RNA to stimulate encapsidation of GRV RNA by GRAV CPits capacity to promote aphid transmission of GRV was observed.
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intracellular location of two groundnut rosette Umbravirus proteins delivered by pvx and tmv vectors
Virology, 1998Co-Authors: Eugene V Ryabov, D J Robinson, Karl J Oparka, Simon Santa Cruz, Michael TalianskyAbstract:The proteins encoded by open reading frames (ORF) 3 and 4 of groundnut rosette Umbravirus (GRV) were expressed in Nicotiana benthamiana as fusions with green fluorescent protein (GFP) from modified potato virus X (PVX) and tobacco mosaic virus (TMV) vectors. Regardless of which plant virus vector was used, GFP fused to the ORF3 protein accumulated in large cytoplasmic inclusion bodies and in nucleoli, whereas GFP fused to the ORF4 protein was found in cell walls close to plasmodesmata. Cell-to-cell movement of PVX requires three proteins encoded by the triple gene block (TGB) and also the coat protein (CP). However, when GRV ORF4 was substituted for the PVX CP gene, the hybrid virus was able to move normally in inoculated leaves but not into noninoculated leaves. In contrast, when GRV ORF4 was substituted for the TGB, or for both the TGB and the CP gene, movement of the hybrid viruses was limited to a few epidermal cells neighboring the infection site. Thus, the GRV ORF4 protein can replace the movement proteins of PVX for some of their functions.
