Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Tombusvirus”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources
PubMed2006

Structures of T=1 and T=3 particles of cucumber necrosis virus: evidence of internal scaffolding.

Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus, of which tomato bushy stunt virus (TBSV) is the type member. The capsid protein for this group of viruses is composed of three major domains: the R domain, which interacts with the RNA genome: the S domain, which forms the tight capsid shell: and the protruding P domain, which extends approximately 40 Angstrom from the surface. Here, we present the cryo-transmission electron microscopy structures of both the T=1 and T=3 capsids to a resolution of approximately 12 Angstrom. The T=3 capsid is essentially identical with that of TBSV, and the T=1 particles are well described by the A subunit pentons from TBSV. Perhaps most notable is the fact that the T=3 particles have an articulated internal structure with two major internal shells, while the internal core of the T=1 particle is essentially disordered. These internal shells of the T=3 capsid agree extremely well in both dimension and character with published neutron-scattering results. This structure, combined with mutagenesis results in the accompanying article, suggests that the R domain forms an internal icosahedral scaffold that may play a role in T=3 capsid assembly. In addition, the N-terminal region has been shown to be involved in chloroplast targeting. Therefore, this region apparently has remarkably diverse functions that may be distributed unevenly among the quasi-equivalent A, B, and C subunits.

Amino Acid Sequence↗
PubMed2005

Kinetics and functional studies on interaction between the replicase proteins of Tomato Bushy Stunt Virus: requirement of p33:p92 interaction for replicase assembly.

The assembly of the functional replicase complex via protein:protein and RNA:protein interactions among the viral-coded proteins, host factors and the viral RNA on cellular membranes is a key step in the replication process of plus-stranded RNA viruses. In this work, we have characterized essential interactions between p33:p33 and p33:p92 replication proteins of Tomato bushy stunt virus (TBSV), a tombusvirus with a non-segmented, plus-stranded RNA genome. Surface plasmon resonance (SPR) measurements with purified recombinant p33 and p92 demonstrate that p33 interacts with p92 in vitro and that the interaction requires the S1 subdomain, whereas the S2 subdomain plays lesser function. Kinetic SPR analyses showed that binding of S1 subdomain to the C-terminal half of p33 takes place with moderate binding affinity in the nanomolar range whereas S2 subdomain binds to p33 with micromolar affinity. Using mutated p33 and p92 proteins, we identified critical amino acid residues within the p33:p92 interaction domain that play essential role in replication and the assembly of the tombusviral replicase. In addition, we show that interaction takes place between replication proteins of TBSV and the closely related Cucumber necrosis virus but not between TBSV and the more distantly related Turnip crinkle virus, suggesting that selective protein interactions might prevent the assembly of chimeric replicases carrying replication proteins from different viruses during mixed infections.

Amino Acid Substitution↗
PubMed1999

Subgenomic mRNA regulation by a distal RNA element in a (+)-strand RNA virus.

Subgenomic (sg) mRNAs are synthesized by (+)-strand RNA viruses to allow for efficient translation of products encoded 3' in their genomes. This strategy also provides a means for regulating the expression of such products via modulation of sg mRNA accumulation. We have studied the mechanism by which sg mRNAs levels are controlled in tomato bushy stunt virus, a small (+)-strand RNA virus which synthesizes two sg mRNAs during infections. Neither the viral capsid nor movement proteins were found to play any significant role in modulating the accumulation levels of either sg mRNA. Deletion analysis did, however, identify a 12-nt-long RNA sequence located approximately 1,000 nt upstream from the site of initiation of sg mRNA2 synthesis that was required specifically for accumulation of sg mRNA2. Further analysis revealed a potential base-pairing interaction between this sequence and a sequence located just 5' to the site of initiation for sg mRNA2 synthesis. Mutant genomes in which this interaction was either disrupted or maintained were analyzed and the results indicated a positive correlation between the predicted stability of the base-pairing interaction and the efficiency of sg mRNA2 accumulation. The functional significance of the long-distance interaction was further supported by phylogenetic sequence analysis which revealed conservation of base-pairing interactions of similar stability and relative position in the genomes of different tombusviruses. It is proposed that the upstream sequence represents a cis-acting RNA element which facilitates sg mRNA accumulation by promoting efficient synthesis of sg mRNA2 via a long-distance RNA-RNA interaction.

Amino Acid Sequence↗
PubMed1993

Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack.

Expression of viral genes in transgenic plants is a very effective tool for attenuating plant viral infection. Nevertheless, the lack of generality and risk issues related to the expression of viral genes in plants might limit the exploitation of this strategy. Expression in plants of antibodies against essential viral proteins could provide an alternative approach to engineer viral resistance. Recently, expression of complete or engineered antibodies has been successfully achieved in plants. The engineered single-chain Fv antibody scFv (refs 10, 11) is particularly suitable for expression in plants because of its small size and the lack of assembly requirements. Here we present evidence that constitutive expression in transgenic plants of a scFv antibody, directed against the plant icosahedral tombusvirus artichoke mottled crinkle virus, causes reduction of infection incidence and delay in symptom development.

Antibodies, Viral↗
PubMed2003

Recognition of small interfering RNA by a viral suppressor of RNA silencing.

RNA silencing (also known as RNA interference) is a conserved biological response to double-stranded RNA that regulates gene expression, and has evolved in plants as a defence against viruses. The response is mediated by small interfering RNAs (siRNAs), which guide the sequence-specific degradation of cognate messenger RNAs. As a counter-defence, many viruses encode proteins that specifically inhibit the silencing machinery. The p19 protein from the tombusvirus is such a viral suppressor of RNA silencing and has been shown to bind specifically to siRNA. Here, we report the 1.85-A crystal structure of p19 bound to a 21-nucleotide siRNA, where the 19-base-pair RNA duplex is cradled within the concave face of a continuous eight-stranded beta-sheet, formed across the p19 homodimer interface. Direct and water-mediated intermolecular contacts are restricted to the backbone phosphates and sugar 2'-OH groups, consistent with sequence-independent p19-siRNA recognition. Two alpha-helical 'reading heads' project from opposite ends of the p19 homodimer and position pairs of tryptophans for stacking over the terminal base pairs, thereby measuring and bracketing both ends of the siRNA duplex. Our structure provides an illustration of siRNA sequestering by a viral protein.

Amino Acid Sequence↗
PubMed2003

A replication silencer element in a plus-strand RNA virus.

Replication represents a key step in the infectious cycles of RNA viruses. Here we describe a regulatory RNA element, termed replication silencer, that can down-regulate complementary RNA synthesis of a positive-strand RNA virus via an RNA-RNA interaction. This interaction occurs between the 5-nucleotide-long, internally positioned replication silencer and the extreme 3'-terminus of the viral RNA comprising part of the minimal minus-strand initiation promoter. Analysis of RNA synthesis in vitro, using model defective interfering (DI) RNA templates of tomato bushy stunt virus and a partially purified, RNA-dependent RNA polymerase preparation from tombusvirus-infected plants, revealed that this interaction inhibits minus-strand synthesis 7-fold. This functional interaction was supported further by: (i) RNA structure probing; (ii) phylogenetic analysis; (iii) inhibition of activity by short complementary DNAs; and (iv) compensatory mutational analysis. The silencer was found to be essential for accumulation of DI RNAs in protoplasts, indicating that it serves an important regulatory role(s) in vivo. Because similar silencer-promoter interactions are also predicted in other virus genera, this type of RNA-based regulatory mechanism may represent a widely utilized strategy for modulating replication.

Base Sequence↗
PubMed1994

Nucleotide sequence, genomic organization and synthesis of infectious transcripts from a full-length clone of artichoke mottle crinkle virus.

The complete nucleotide sequence of the genome of artichoke mottle crinkle virus (AMCV), a member of the tombusvirus group, has been determined. The genome is 4790 nucleotides (nt) in length. A full-length cDNA of the AMCV genome has been cloned in pUC9 downstream of the T7 RNA polymerase promoter. Transcripts were infective when inoculated onto Nicotiana clevelandii and N. benthamiana plants. The AMCV genome contains five open reading frames (ORFs). The first ORF from the 5' terminus (ORF1) encodes a protein with a predicted M(r) of 33K. ORF2 extends through the amber termination codon of ORF1 to yield a polypeptide of predicted M(r) 92K and which is the putative RNA-dependent RNA polymerase. ORF3 codes for the coat protein (41K). Two nested ORFs in different reading frames (ORFs 4 and 5) code for a 22K and a 19K polypeptide respectively. Sequence homologies suggest that the 22K protein could be involved in cell-to-cell movement of virus. ORFs 3, 4 and 5 are translated from two 3' coterminal subgenomic (sg) RNAs, the 5' termini of which have been mapped. The two sg RNAs are 2155 (sg1) and 934 (sg2) nt in length. ORF3 is expressed from sg1 RNA whereas ORFs 4 and 5 are potentially expressed from sg2 RNA. Time course experiments with Cynara scolymus protoplasts indicate that during AMCV infection both positive and negative strands of genomic and sg RNAs are produced and that sg2 RNA is produced before and at a higher level than sg1 RNA.

Amino Acid Sequence↗
PubMed1998

Identification of artichoke mottled crinkle virus (AMCV) proteins required for virus replication: complementation of AMCV p33 and p92 replication-defective mutants.

Mutagenesis of the artichoke mottled crinkle virus (AMCV) genome and complementation studies between replication-defective mutants were undertaken to identify viral protein(s) essential for AMCV replication. Inoculation of Nicotiana benthamiana protoplasts with mutant transcripts revealed that null mutations in ORFs 1 [tA33(-)], 2 [tA92(-)] and 6 [tA7(-)], as well as an ORF 2 mutation [tA92GED] in the GDD motif of the 92 kDa protein, the putative replicase, prevented accumulation of detectable levels of progeny RNA. Conversely, mutations of ORFs 3 [tA41(-)], 4 [tA21(-)] and 5 [tA19(-)] did not substantially affect the accumulation of AMCV genomic and subgenomic RNAs of both positive and negative polarity. Inoculation of N. benthamiana plants with transcripts impaired in replication revealed that tA92(-) and tA7(-) mutants lead to replicating pseudorevertants. Functional analysis of these pseudorevertants showed that: (i) the double stop codon introduced at the end of ORF 1 to prevent the translational readthrough of the 92 kDa protein reverted to a single amber, ochre or opal codon, giving rise to viable genomes; (ii) the putative 7 kDa protein is not essential for genome viability, although the RNA region spanning ORF 6 plays a role in cis in replication. Finally, the two replication-defective mutants tA33(-) and tA92(-) complemented when co-inoculated to N. benthamiana protoplasts, definitively proving that the 33 kDa protein is essential for tombusvirus genome replication. Analysis of viral RNAs from the coinfection experiments showed that tA92(-) was preferentially amplified over tA33(-).

Base Sequence↗
PubMed2000

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.

Cell Membrane↗
PubMed1999

A primary determinant of cap-independent translation is located in the 3'-proximal region of the tomato bushy stunt virus genome.

Tomato bushy stunt virus (TBSV) is a positive-strand RNA virus and is the prototype member of the genus Tombusvirus. The genomes of members of this genus are not polyadenylated, and prevailing evidence supports the absence of a 5' cap structure. Previously, a 167-nucleotide-long segment (region 3.5) located near the 3' terminus of the TBSV genome was implicated as a determinant of translational efficiency (S.K. Oster, B. Wu and K. A. White, J. Virol. 72:5845-5851, 1998). In the present report, we provide evidence that a 3'-proximal segment of the genome, which includes region 3.5, is involved in facilitating cap-independent translation. Our results indicate that (i) a 5' cap structure can substitute functionally for the absence of region 3.5 in viral and chimeric reporter mRNAs in vivo; (ii) deletion of region 3.5 from viral and chimeric mRNAs has no appreciable effect on message stability; (iii) region 3.5 represents part of a larger 3' proximal element, designated as the 3' cap-independent translational enhancer (3'CITE), that is required for proficient cap-independent translation; (iv) the 3'CITE also facilitates cap-dependent translation; (v) none of the major viral proteins are required for 3'CITE activity; and (vi) no significant 3'CITE-dependent stimulation of translation was observed when mRNAs were tested in vitro in wheat germ extract under various assay conditions. This latter property distinguishes the 3'CITE from other characterized plant viral 3'-proximal cap-independent translational enhancers. Additionally, because the 3'CITE overlaps with cis-acting replication signals, it could potentially participate in regulating the initiation of genome replication.

3' Untranslated Regions↗
PubMed2002

Tomato bushy stunt virus genomic RNA accumulation is regulated by interdependent cis-acting elements within the movement protein open reading frames.

This study on Tomato bushy stunt virus identified and defined three previously unknown regulatory sequences involved in RNA accumulation that are located within the 3'-proximal nested movement protein genes p22 and p19. The first is a 16-nucleotide (nt) element termed III-A that is positioned at the very 3' end of p22 and is essential for RNA accumulation. Approximately 300 nt upstream of III-A resides an approximately 80-nt inhibitory element (IE) that is obstructive to replication only in the absence of a third regulatory element of approximately 30 nt (SUR-III) that is positioned immediately upstream of III-A. Inspection of the nucleotide sequences predicted that III-A and SUR-III can form looped hairpins. A comparison of different tombusviruses showed, in each case, conservation for potential base pairing between the two predicted hairpin-loops. Insertion of a spacer adjacent to the predicted hairpins had no or a minimal effect on RNA accumulation, whereas an insertion in the putative III-A loop abolished genomic RNA multiplication. We conclude that the sequences composing the predicted III-A and SUR-III hairpin-loops are crucial for optimal RNA accumulation and that the inhibitory effect of IE surfaces when the alleged interaction between SUR-III and III-A is disturbed.

Genes, Regulator↗
PubMed2002

Heterologous movement protein strongly modifies the infection phenotype of cucumber mosaic virus.

A hybrid virus (CMVcymMP) constructed by replacing the movement protein (MP) of cucumber mosaic cucumovirus (CMV) with that of cymbidium ringspot tombusvirus (CymRSV) was viable and could efficiently spread both cell to cell and long distance in host plants. The hybrid virus was able to move cell to cell in the absence of functional CP, whereas CP-deficient CMV was restricted to single inoculated cells. In several Chenopodium and Nicotiana species, the symptom phenotype of the hybrid virus infection was clearly determined by the foreign MP gene. In Nicotiana debneyi and Nicotiana tabacum cv. Xanthi, the hybrid virus could move systemically, contrary to CymRSV.

Capsid↗
PubMed2003

A second functional RNA domain in the 5' UTR of the Tomato bushy stunt virus genome: intra- and interdomain interactions mediate viral RNA replication.

The 5' untranslated regions (UTRs) of (+)-strand RNA viruses play a variety of roles in the reproductive cycles of these infectious agents. Tomato bushy stunt virus (TBSV) belongs to this class of RNA virus and is the prototype member of the genus Tombusvirus. Previous studies have demonstrated that a T-shaped domain (TSD) forms in the 5' half of the TBSV 5' UTR and that it plays a central role in viral RNA replication. Here we have extended our structure-function analysis to the 3' half of the 5' UTR. Investigation of this region in the context of a model viral replicon (i.e., a TBSV-derived defective interfering [DI] RNA) revealed that this segment contains numerous functionally relevant structural features. In vitro solution structure probing along with comparative and computer-aided RNA secondary structure analyses predicted the presence of a simple stem loop (SL5) followed by a more complex downstream domain (DSD). Both structures were found to be essential for efficient DI RNA accumulation when tested in a plant protoplast system. For SL5, maintenance of the base of its stem was the principal feature required for robust in vivo accumulation. In the DSD, both helical and unpaired regions containing conserved sequences were necessary for efficient DI RNA accumulation. Additionally, optimal DI RNA accumulation required a TSD-DSD interaction mediated by a pseudoknot. Modifications that reduced accumulation did not appreciably affect DI RNA stability in vivo, indicating that the DSD and SL5 act to facilitate viral RNA replication.

5' Untranslated Regions↗
PubMed1998

Analysis of RNA-dependent RNA polymerase structure and function as guided by known polymerase structures and computer predictions of secondary structure.

RNA-dependent RNA polymerases (RdRps) function as the catalytic subunit of the viral replicase required for the replication of all positive strand RNA viruses. The vast majority of RdRps have been identified solely on the basis of sequence similarity. Structural studies of RdRps have lagged behind those of the DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, and reverse transcriptases until the recent report of the partial crystal structure of the poliovirus RdRp, 3Dpol [Hansen, J. L., et al. (1997). Structure 5, 1109-1122]. We seek to address whether all RdRps will have structures similar to those found in the poliovirus polymerase structure. Therefore, the PHD method of Rost and Sander [Rost, B., and Sander, C. (1993a). J. Mol. Biol. 232, 584-599; Rost, B., and Sander, C. (1994). Protein 19, 55-77] was used to predict the secondary structure of the RdRps from six different viral families: bromoviruses, tobamoviruses, tombusvirus, leviviruses, hepatitis C-like viruses, and picornaviruses. These predictions were compared with the known crystal structure of the poliovirus polymerase. The PHD method was also used to predict picornavirus structures in places in which the poliovirus crystal structure was disordered. All five families and the picornaviruses share a similar order of secondary structure elements present in their polymerase proteins. All except the leviviruses have the unique region observed in the poliovirus 3Dpol that is suggested to be involved in polymerase oligomerization. These structural predictions are used to explain the phenotypes of a collection of mutations that exist in several RNA polymerases. This analysis will help to guide further characterization of RdRps.

Amino Acid Sequence↗
PubMed1999

Both RNA rearrangement and point mutation contribute to repair of defective chimeric viral genomes to form functional hybrid viruses in plants.

The putative movement protein gene (p27) plus 5' and 3' flanking sequences of cucumber leaf spot aureusvirus (CLSV) was inserted into an infectious cucumber necrosis tombusvirus (CNV) cDNA clone containing a deletion in the cell-to-cell movement protein gene. Approximately 5% of plants inoculated with synthetic transcripts of two such defective chimeric CNV/CLSV cDNA clones developed systemic symptoms 7-19 days postinoculation. Reverse transcription-polymerase chain reaction and sequence analysis of virus obtained from systemically infected leaves indicated that both point mutation and RNA rearrangement (deletion) contributed to the formation of movement competent CNV/CLSV hybrid viruses. The hybrid viruses were found to accumulate to high levels in infected plants, to form stable virions, and to be mechanically transmissible. In addition, a hybrid virus that lacked 50 amino acids at the carboxyl-terminal region of CLSV p27 was still capable of facilitating CNV movement. These data provide experimental evidence for the role of CLSV p27 in viral cell-to-cell movement and demonstrate that p27 can enable efficient movement of the CNV genome. Moreover, the data show that RNA rearrangements known to occur during CNV RNA replication can contribute to rapid evolution of the CNV genome.

Amino Acid Sequence↗
PubMed2005

Viral RNA replication in association with cellular membranes.

All plus-strand RNA viruses replicate in association with cytoplasmic membranes of infected cells. The RNA replication complex of many virus families is associated with the endoplasmic reticulum membranes, for example, picorna-, flavi-, arteri-, and bromoviruses. However, endosomes and lysosomes (togaviruses), peroxisomes and chloroplasts (tombusviruses), and mitochondria (nodaviruses) are also used as sites for RNA replication. Studies of individual nonstructural proteins, the virus-specific components of the RNA replicase, have revealed that the replication complexes are associated with the membranes and targeted to the respective organelle by the ns proteins rather than RNA. Many ns proteins have hydrophobic sequences and may transverse the membrane like polytopic integral membrane proteins, whereas others interact with membranes monotopically. Hepatitis C virus ns proteins offer examples of polytopic transmembrane proteins (NS2, NS4B), a "tip-anchored" protein attached to the membrane by an amphipathic alpha-helix (NS5A) and a "tail-anchored" posttranslationally inserted protein (NS5B). Semliki Forest virus nsP1 is attached to the plasma membrane by a specific binding peptide in the middle of the protein, which forms an amphipathic alpha-helix. Interaction of nsP1 with membrane lipids is essential for its capping enzyme activities. The other soluble replicase proteins are directed to the endo-lysosomal membranes only as part of the initial polyprotein. Poliovirus ns proteins utilize endoplasmic reticulum membranes from which vesicles are released in COPII coats. However, these vesicles are not directed to the normal secretory pathway, but accumulate in the cytoplasm. In many cases the replicase proteins induce membrane invaginations or vesicles, which function as protective environments for RNA replication.

Alphavirus↗
PubMed2004

A whole genome perspective on the phylogeny of the plant virus family Tombusviridae.

Most current classifications of viruses are based on single gene analysis of capsid protein or polymerase. The comparison of entire genomes is a more balanced approach that should provide a more complete picture of relatedness. We have used a singular value decomposition (SVD)-based analysis to generate phylogenetic trees using whole genome protein sequences from a family of single-stranded RNA plant viruses. Our dataset includes the 26 species of the family Tombusviridae, 25 of which have complete genome sequences cataloged in GenBank. The resulting phylogenetic tree agrees well with current taxonomic classifications, but with significant exceptions. One previously unassigned virus within this family, Maize necrotic streak virus, is definitively placed within the genus Tombusvirus by this analysis. In addition, the analysis defines two distinct subsets within the genus Necrovirus. Future datasets will be expanded to include other icosahedral positive strand RNA plant viruses, and then perhaps all positive strand RNA plant viruses.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and complete nucleotide sequence of galinsoga mosaic virus genomic RNA.

The complete genomic sequence of galinsoga mosaic virus (GaMV) was determined. The genome consists of 3,803 nucleotides and has five open reading frames (ORFs). The 5' ORF (ORF 1) encodes a protein with predicted molecular mass of 23 kDa and readthrough of its amber stop codon probably yields a 82 kDa protein (ORF 2). ORFs 3 and 4 encode two polypeptides with molecular masses of 8 and 7 kDa, respectively. ORF 5 encodes the 36 kDa capsid protein. Amino acid sequence comparisons revealed that the nonstructural proteins encoded by ORFs 1, 3, and 4 were more similar to the corresponding gene products of tobacco necrosis virus, strain A, than to those of carmoviruses. Conversely, the coat protein was more similar to that of tombusviruses. The readthrough region of the viral replicase (ORF 2) had high sequence homology with that of carmo-, tombus-, and necroviruses. Computer analysis of the protein encoded by ORF 1 as well as of the corresponding product of turnip crinkle (TCV) and melon necrotic spot (MNSV) carmoviruses revealed the presence of a sequence with local hydrophobicity and hydrophobic moment characteristic of mitochondrial targeting sequence which may explain the origin of the carmovirus-induced multivesicular bodies from mitochondria.

Amino Acid Sequence↗