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At least 19 recordsLinked to original sources
PubMed1999

RT-PCR detection and identification of three species of cucumoviruses with a genus-specific single pair of primers.

Reverse transcription and polymerase chain reaction (RT-PCR) was used for detection and identification of three cucumoviruses (cucumber mosaic virus, CMV; peanut stunt virus, PSV; tomato aspermy virus, TAV) in various plants sources with a single pair of primers, designed as CPTALL-3 and CPTALL-5. The pair of cucumovirus genus-specific primers that flank the coat protein gene were designed and used to amplify a DNA fragment of approximately ranging from 938 to 966 bp. The RT-PCR with the set of primers specifically amplified the target size of DNA fragment in all the tested cucumoviruses (CMV S-IA, S-IB and S-II, PSV and TAV). No DNA product of any length was produced when brome mosaic virus or tobacco mosaic virus RNA was used as templates. The cucumoviruses examined were differentiated by PCR-restriction fragment length polymorphism with different enzymes. This indicates that the designed primers are only specific for the cucumoviruses and useful for reliable information of identification of members of the Cucumovirus genus.

Base Sequence↗
PubMed1995

The conserved, hydrophilic and arginine-rich N-terminal domain of cucumovirus coat proteins contributes to their anomalous electrophoretic mobilities in sodium dodecylsulfate-polyacrylamide gels.

Although the Mr values of the coat proteins (CPs) of several cucumoviruses have been calculated from their deduced amino acid sequences to be approximately 24,000, the experimentally determined M(r) values using the Laemmli SDS-PAGE system were 30,000-31,000. Examination of the amino acid composition revealed that these CPs are neither highly acidic nor highly basic. Post-translational glycosylation or phosphorylation were also ruled out as contributing factors to the observed anomalous electrophoretic mobility because the products of in vitro translation of cucumovirus RNA 4 and in vivo bacterial expression of the cloned CP gene co-migrated with authentic cucumovirus CPs. Comparison of the hydropathy profiles of the CPs revealed the presence in each of a strikingly similar, highly hydrophilic N-terminal domain of 30-32 amino acid residues that contains a cluster of basic amino acids, mainly arginine. Selective chemical cleavage at tryptophan residues in the CPs of cucumoviruses, known to contain single tryptophan residues, yielded two peptides; an N-terminal peptide that contained the conserved hydrophilic domain and a C-terminal peptide. SDS-PAGE analysis showed that the N-terminal, but not the C-terminal, peptide exhibited the anomalous electrophoretic mobility.

Amino Acid Sequence↗
PubMed1995

Role of cucumovirus capsid protein in long-distance movement within the infected plant.

Direct evidence is presented for a host-specific role of the cucumovirus capsid protein in long-distance movement within infected plants. Cucumber (Cucumis sativus L.) is a systemic host for cucumber mosaic cucumovirus (CMV). Tomato aspermy cucumovirus, strain 1 (1-TAV), multiplied to the levels of CMV (i.e., replicated, moved from cell to cell, and formed infectious particles) in the inoculated leaves of cucumbers but was completely unable to spread systemically. The defective long-distance systemic movement of 1-TAV was complemented by CMV in mixed infections. Coinfection of cucumbers with 1-TAV RNA with various combinations of transcripts from full-length cDNA clones of CMV genomic RNA 1, RNA2, and RNA3 showed that CMV RNA3 alone complemented 1-TAV long-distance movement. We obtained mutants containing mutations in the two open reading frames in CMV RNA3 encoding the 3a protein and the capsid protein (CP), both of which are necessary for cell-to-cell movement of CMV. Complementation experiments with mutant CMV RNA3 showed that only 3a protein mutants, i.e., those with an intact CP, complemented the long-distance movement of 1-TAV in cucumbers. Since CMV and TAV have common systemic host plants, the results presented here are strong evidence for an active, host-specific function of the CPs of these two cucumoviruses for long-distance spread in the phloem. The results also suggest that the plasmodesmata in the vascular system and/or at the boundary between the mesophyll and the vascular system, involved in long-distance movement through the phloem, and those in the mesophyll, involved in cell-to-cell movement, differ functionally.

Capsid↗
PubMed1995

Interspecific reassortment of genomic segments in the evolution of cucumoviruses.

Segmented genomes of RNA viruses are thought to evolve and be maintained in analogy to sexual recombination and reassortment in eukaryotic systems. If reassortment among genomes is an important event in cucumoviral evolution, then such events should be detectable among extant viruses. In this study, phylogenetic analyses of cucumoviruses were performed using aligned amino acid sequences. The results reveal different relationships among species when the three genomic segments are compared, suggesting that reassortment events have given rise to extant forms. In addition, we describe a cucumovirus isolate that is composed of genomic segments from two distinct viral species. These results indicate that reassortment events may provide a mechanism for speciation in cucumoviruses.

Amino Acid Sequence↗
PubMed2003

Cucumoviruses.

Research on the molecular biology of cucumoviruses and their plant-virus interactions has been very extensive in the last decade. Cucumovirus genome structures have been analyzed, giving new insights into their genetic variability, evolution, and taxonomy. A new viral gene has been discovered, and its role in promoting virus infection has been delineated. The localization and various functions of each viral-encoded gene product have been established. The particle structures of Cucumber mosaic virus (CMV) and Tomato aspermy virus have been determined. Pathogenicity domains have been mapped, and barriers to virus infection have been localized. The movement pathways of the viruses in some hosts have been discerned, and viral mutants affecting the movement processes have been identified. Host responses to viral infection have been characterized, both temporally and spatially. Progress has been made in determining the mechanisms of replication, gene expression, and transmission of CMV. The pathogenicity determinants of various satellite RNAs have been characterized, and the importance of secondary structure in satellite RNA-mediated interactions has been recognized. Novel plant genes specifying resistance to infection by CMV have been identified. In some cases, these genes have been mapped, and one resistance gene to CMV has been isolated and characterized. Pathogen-derived resistance has been demonstrated against CMV using various segments of the CMV genome, and the mechanisms of some of these forms of resistances have been analyzed. Finally, the nature of synergistic interactions between CMV and other viruses has been characterized. This review highlights these various achievements in the context of the previous work on the biology of cucumoviruses and their interactions with plants.

Biological Evolution↗
PubMed1997

In vivo expression of an overlapping gene encoded by the cucumoviruses.

We recently reported the molecular characterization and functional analysis of an overlapping gene 2b encoded by RNA 2 of the Q strain of cucumber mosaic cucumovirus (Q-CMV). We show here that the homologous gene encoded by the V strain of tomato aspermy cucumovirus (V-TAV) and the WAII strain of CMV (WAII-CMV), which is in a different subgroup to Q-CMV, is also expressed in vivo by demonstrating the accumulation of the mRNA (RNA 4A) and its protein in infected plants. Interestingly, RNA 4A of V-TAV is encapsidated in virions as found previously for Q-CMV whereas WAII-CMV contains very little RNA 4A in virions. As the 2b gene is conserved in all 10 cucumoviral species or strains sequenced to date and the 2b gene is expressed for three of these viruses, we conclude that the 2b gene is a common feature of the Cucumovirus genus.

Base Sequence↗
PubMed1997

Evidence for the occurrence of two distinct subgroups of peanut stunt cucumovirus strains: molecular characterization of RNA3.

Strains of peanut stunt cucumovirus (PSV) were classified into two distinct subgroups, I and II, based on Western and Northern blot analyses using antisera and cloned cDNA probes to strains PSV-ER and PSV-W. These results were corroborated by nucleotide sequence analyses of full-length cDNA clones of RNA3 from representative strains of the two subgroups. Whereas the percentage nucleotide sequence identity between PSV-ER (or PSV-J) and PSV-W RNA3s was determined to be 80%, the corresponding value between strains ER and J was 91%, confirming that strains ER and J belong to the same subgroup (subgroup I) whereas strain W belongs to a separate subgroup (subgroup II). PSV-W and PSV-ER RNA3s are 2173 and 2188 nucleotides long, respectively. Each is dicistronic, encoding a putative movement protein (3a protein) and a coat protein (CP). The intercistronic and 5' untranslated region (UTR) sequences of PSV strains, unlike those of cucumber mosaic cucumovirus (CMV) strains, are highly conserved and thus not useful for distinguishing the two subgroups. However, the 3' UTR sequences of PSV strains, like those of CMV strains, can discriminate between the two subgroups since strains within the same subgroup are 95% identical in their 3' UTRs whereas those in different subgroups are only 74-78% identical. PSV-W and PSV-ER RNA4s were determined to be 994 and 1006 nucleotides long, respectively. PSV 3a and CP genes have higher percentage nucleotide sequence identities to those of tomato aspermy cucumovirus than to those of CMV.

Amino Acid Sequence↗
PubMed1995

Characterization of defective RNAs derived from RNA 3 of the Fny strain of cucumber mosaic cucumovirus.

Two defective RNAs (designated D RNA 3 alpha and D RNA 3 beta) were found to be associated with the Fny strain of cucumber mosaic cucumovirus but not with the Sny strain after serial passages in a tobacco host. The D RNAs were derived from RNA 3 by single, in-frame deletions within the 3a open reading frame. A full-length cDNA clone from which biologically active transcripts can be produced in vitro has been constructed for D RNA 3 beta. This transcript can be replicated in tobacco plants infected with subgroup I and II cucumber mosaic cucumovirus strains and with peanut stunt cucumovirus. Translation of D RNA 3 beta in vitro produced a 20-kDa peptide, which was consistent with the predicted coding capacity of the deleted 3a open reading frame. D RNA 3 beta was also associated with polyribosomes isolated from infected tobacco plants. The presence of the D RNAs had no apparent effect upon helper virus yield or symptom production.

Amino Acid Sequence↗
PubMed1998

Involvement of cucumber mosaic cucumovirus RNA2 and RNA3 in viral systemic spread in radish plant.

The genetics of cucumber mosaic cucumovirus (CMV) and the pathogenicity of the virus for Raphanus sativus were analyzed using pseudorecombinants constructed from the infectious transcripts of two naturally occurring strains of cucumber mosaic cucumovirus (CMV-D8 and CMV-Y). CMV-D8, but not CMV-Y, could cause systemic infection of the plant. Viral accumulation and systemic movement in the plants was examined using immuno-tissue blot analysis, dot blot and Northern blot hybridization. Virus was equally distributed and CMV RNAs accumulated to similar levels in the inoculated cotyledons of radish irrespective of the pseudorecombinant, suggesting that there are no apparent differences in the ability of infection and viral accumulation between CMV-D8 and CMV-Y. We found, however, that both RNAs 2 and 3 of CMV-D8 are involved in determining the efficiency for the systemic infection of R. sativus. Co-operated interactions between genetic information of RNAs 2 and 3 would control the efficient translocation of virus from the inoculated leaves to the uninoculated upper leaves of radish plant.

Blotting, Northern↗
PubMed2005

Homology modelling and protein structure based functional analysis of five cucumovirus coat proteins.

Coat proteins (CP) of five cucumovirus isolates, Cucumber mosaic virus (CMV) strains R, M and Trk7, Tomato aspermy virus (TAV) strain P and Peanut stunt virus (PSV) strain Er, were constructed by homology modelling. The X-ray structure of the Fny-CMV CP subunit B was used as a template. Models of cucumovirus CPs were built by the MODELLER program. Model refinements were carried out using the Kollman molecular mechanical force field. Models were analyzed by the PROCHECK programs. Electrostatic potential calculations were applied to all models and functional site search was performed with the PROSITE software, a web based tool for searching biologically significant sites. Symptom determinants published up to the present were compared with the PROSITE hits in the light of 3D models and electrostatic information. In all cases, we analyzed the effect of mutations on the structure, electrostatic potential patterns and function of CPs, respectively. We found that high flexibility of the betaE-alphaEF loop starting with the residue 129 is required, but it is not sufficient for the symptom appearance. Furthermore, phosphorylation of the CP is prospective to be important in the host response mechanism. All analyzed mutations were related to the modifications of the predicted phosphorylation sites. Based on our conclusions we predicted the infectivity of the examined viruses.

Amino Acid Sequence↗
PubMed1999

Molecular evidence for the existence of two distinct subgroups in cucumber mosaic cucumovirus.

Infectious full-length cDNA clones from the genomic RNAs of a subgroup II cucumber mosaic cucumovirus strain (Trk7) were obtained. Sequence analysis of the whole genome revealed strong homology (99%) to the genome of Q-CMV, the only subgroup II strain whose entire genomic nucleotide sequence had been available in the database, and an overall 75% homology to those of subgroup I strains. We provide sequence comparisons of different parts of 1a, 2a and 2b proteins of Cucumovirus species, and propose phylogenetic trees based on these protein sequences.

Amino Acid Sequence↗
PubMed2004

Compatibility of the movement protein and the coat protein of cucumoviruses is required for cell-to-cell movement.

For the cell-to-cell movement of cucumoviruses both the movement protein (MP) and the coat protein (CP) are required. These are not reversibly exchangeable between Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV). The MP of CMV is able to function with the TAV CP (chimera RT), but TAV MP is unable to promote the cell-to-cell movement in the presence of CMV CP (chimera TR). To gain further insight into the non-infectious nature of the TR recombinant, RNA 3 chimeras were constructed with recombinant MPs and CPs. The chimeric MP and one of the CP recombinants were infectious. The other recombinant CP enabled virus movement only after the introduction of two point mutations (Glu-->Lys and Lys-->Arg at aa 62 and 65, respectively). The mutations served to correct the CP surface electrostatic potential that was altered by the recombination. The infectivity of the TR virus on different test plants was restored by replacing the sequence encoding the C-terminal 29 aa of the MP with the corresponding sequence of the CMV MP gene or by exchanging the sequence encoding the C-terminal 15 aa of the CP with the same region of TAV. The analysis of the recombinant clones suggests a requirement for compatibility between the C-terminal 29 aa of the MP and the C-terminal two-thirds of the CP for cell-to-cell movement of cucumoviruses.

Amino Acid Substitution↗
PubMed2003

Association of cucumovirus and potyvirus with betelvine (Piper betle L.) as evidenced by ELISA and RT-PCR.

An attempt was made to detect various viruses of Piper betle grown at Mahoba and Banthara in India. DAC-ELISA and RT-PCR tests were performed in leaf sap samples of betelvine for detection of a cucumovirus (Cucumber mosaic virus) and potyvirus (Bean yellow mosaic virus) using specific antibodies and universal primers of respective viruses. DAC-ELISA could detect only CMV. However, RT-PCR detected both cucumovirus and potyvirus infection in betelvine samples. Association of CMV with betelvine was observed for the first time in the present study.

Antibodies, Viral↗
PubMed1995

Cucumber mosaic cucumovirus antibodies from a synthetic phage display library.

Antibody fragments (scFv) that bind specifically to particles of cucumber mosaic cucumovirus (CMV) were obtained from a library which encodes a diverse array of synthetic antibody fragments, each displayed on the surface of filamentous bacteriophage. After four rounds of selection and enrichment, several clones were obtained which produced scFv that bound specifically to purified particles of CMV in ELISA. BstNI digestion of phagemid DNA resulted in the same restriction pattern for all clones. The nucleotide sequences of three of the clones showed that they belonged to the human VH1 family and that they had a complementarity determining region loop of 7 amino acids. Phage-displayed antibodies and soluble scFv secreted by these clones reacted with particles of CMV in sap from infected plants in ELISA. In immunoblotting tests, soluble scFv preparations reacted with SDS-denatured coat protein extracted from purified preparations of CMV isolates belonging to either subgroup I or II and also with protein extracted by SDS treatment of seeds harvested from naturally infected lupin plants. The results demonstrate the feasibility, and potential applicability, of recombinant antibody methods in plant pathology.

Amino Acid Sequence↗
PubMed1997

Structural analysis of a necrogenic strain of cucumber mosaic cucumovirus satellite RNA in planta.

Structural studies of plant viral RNA molecules have been based on in vitro chemical and enzymatic modification. That approach, along with mutational analysis, has proven valuable in predicting structural models for some plant viruses such as tobacco mosaic tobamovirus and brome mosaic bromovirus. However, in planta conditions may be dramatically different from those found in vitro. In this study we analyzed the structure of cucumber mosaic cucumovirus satellite RNA (sat RNA) strain D4 in vivo and compared it to the structures found in vitro and in purified virions. Following a methodology developed to determine the structure of 18S rRNA within intact plant tissues, different patterns of adenosine and cytosine modification were found for D4-sat RNA molecules in vivo, in vitro, and in virions. This chemical probing procedure identifies adenosine and cytosine residues located in unpaired regions of the RNA molecules. Methylation data, a genetic algorithm in the STAR RNA folding program, and sequence alignment comparisons of 78 satellite CMV RNA sequences were used to identify several helical regions located at the 5' and 3' ends of the RNA molecule. Data from previous mutational and sequence comparison studies between satellite RNA strains inducing necrosis in tomato plants and those strains not inducing necrosis allowed us to identify one helix and two tetraloop regions correlating with the necrogenicity syndrome.

Base Sequence↗
PubMed1999

Recombination between genomic RNAs of two cucumoviruses under conditions of minimal selection pressure.

Recombination is considered to play a key role in RNA virus evolution; however, little is known about its occurrence under natural conditions. We inoculated tobacco plants with wild-type strains of two closely related cucumovirus species: cucumber mosaic virus (CMV) and tomato aspermy virus (TAV). RNA from the inoculated leaves of doubly-infected plants was tested for the presence of recombination events in an 0.8-kb central portion of the viral RNA3. Using a sensitive and specific RT-PCR procedure, we amplified recombinant segments of RNA3 in 3 of 82 tobacco plants infected with both viruses. In each plant in which recombinant segments were amplified, several different crossover sites were observed, all of which were located within a short stretch of high sequence similarity. Two plants had both CMV-TAV and TAV-CMV recombinants. In all cases, precise homologous recombination had occurred. To the best of our knowledge, this is the first report of interspecific recombination between wild-type plant RNA viruses under conditions of minimal selection pressure in favor of the recombinants.

Base Sequence↗
PubMed1998

Circular dichroism studies of CMV-D and CMV-S: two strains of cucumber mosaic cucumovirus with a different biological behaviour.

Cucumber mosaic cucumovirus is a plant virus in which a typical satellite RNA system is present, displaying a dualistic biological behaviour. In fact, it has been shown that satRNA is able either to aggravate or attenuate the viral disease symptomatology with a modulating capability going from death of the host plant to a surprising absence of symptoms. D-satRNA and S-satRNA have been considered the prototype necrogenic and non necrogenic satRNAs respectively. On the basis of circular dichroism spectroscopy, it is suggested that the different biological behaviours can be explained by taking into account the different capabilities exerted by S- and D-satRNAs in inducing structuring effects onto CMV-S and CMV-D genomic RNAs.

Circular Dichroism↗
PubMed2003

Cucumovirus- and bromovirus-encoded movement functions potentiate cell-to-cell movement of tobamo- and potexviruses.

Cucumber mosaic virus (CMV, a cucumovirus) and Brome mosaic virus (BMV, a bromovirus) require the coat protein (CP) in addition to the 3a movement protein (MP) for cell-to-cell movement, while Cowpea chlorotic mottle virus (CCMV, a bromovirus) does not. Using bombardment-mediated transcomplementation assays, we investigated whether the movement functions encoded by these viruses potentiate cell-to-cell movement of movement-defective Tomato mosaic virus (ToMV, a tobamovirus) and Potato virus X (PVX, a potexvirus) mutants in Nicotiana benthamiana. Coexpression of CMV 3a and CP, but neither protein alone, complemented the defective movement of ToMV and PVX. A C-terminal deletion in CMV 3a (3a Delta C33) abolished the requirement of CP in transporting the ToMV genome. The action of 3a Delta C33 was inhibited by coexpression of wild-type 3a. These findings were confirmed in tobacco with ToMV-CMV chimeric viruses. Either BMV 3a or CCMV 3a alone efficiently complemented the movement-defective phenotype of the ToMV mutant. Therefore, every 3a protein examined intrinsically possesses the activity required to act as MP. In transcomplementation of the PVX mutant, the activities of BMV 3a, CCMV 3a, and CMV 3a Delta C33 were very low. The activities of the bromovirus 3a proteins were enhanced by coexpression of the cognate CP but the activity of CMV 3a Delta C33 was not. Based on these results, possible roles of cucumo- and bromovirus CPs in cell-to-cell movement are discussed.

Base Sequence↗