Cucumber mosaic virus as carrier of a hepatitis C virus-derived epitope

Cucumber mosaic virus (CMV) is a three component isodiametric plant virus which is common worldwide and has an extremely wide host range. A pseudorecombinant was made, derived from the RNA3 component of the CMV-S strain, carrying the coat protein (CP) gene, and the RNA1,2 components of the CMV-D strain. This system developed mild mosaic and vein clearing in Xanthi tobacco three weeks after inoculation. The CP gene was then engineered in three different positions, to encode a Hepatitis C virus (HCV) epitope. The selected peptide was the so-called R9 mimotope, a synthetic surrogate derived from a consensus profile of many hypervariable region 1 (HVR1) sequences of the putative HCV envelope protein E2. Serum samples from 60 patients with chronic hepatitis C displayed a significant immunoreactivity to crude plant extracts infected with the chimeric CMV. These results suggest that further investigation should be made into a possible vaccine function for the CMV-HCV mimotope system.     

<Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> as carrier of a hepatitis C virus-derived epitope

Summary. Cucumber mosaic virus (CMV) is a three component isodiametric plant virus which is common worldwide and has an extremely wide host range. A pseudorecombinant was made, derived from the RNA3 component of the CMV-S strain, carrying the coat protein (CP) gene, and the RNA1,2 components of the CMV-D strain. This system developed mild mosaic and vein clearing in Xanthi tobacco three weeks after inoculation. The CP gene was then engineered in three different positions, to encode a Hepatitis C virus (HCV) epitope. The selected peptide was the so-called R9 mimotope, a synthetic surrogate derived from a consensus profile of many hypervariable region 1 (HVR1) sequences of the putative HCV envelope protein E2. Serum samples from 60 patients with chronic hepatitis C displayed a significant immunoreactivity to crude plant extracts infected with the chimeric CMV. These results suggest that further investigation should be made into a possible vaccine function for the CMV-HCV mimotope system.     

Molecular characterization of a strain of cucumber mosaic virus based on coat protein and movement protein genes

Cucumber mosaic virus (CMV) A strain (CMV-A) isolated from Amaranthus tricolor was partially characterized at molecular level. Complete coat protein (CP) and movement protein (MP) ORFs were cloned and sequenced. The 657 bp region of CP gene and the 840 bp region of MP gene encode 218 and 276 amino acids, respectively. CP, at nucleotide level, showed 90-98% sequence identity with the CMV subgroup I and less than 80% with the CMV subgroup II, it showed at amino acid level 92-96% identity with the subgroup I and 74-87% with the subgroup II. The nucleotide and amino acid sequence identities of MP ranged in 91-94% and 92-96%, respectively with the subgroup I but in 81-83% with the subgroup II. Phylogenetic trees generated from nucleotide and amino acid sequences of both CP and MP genes identified the virus strain as a member of the subgroup IB. CMV-A CP also displayed a remarkably higher homology with Indian strains of CMV than with other CMV strains and formed a separate cluster within the subgroup IB.     

Comparative studies on tomato aspermy and cucumber mosaic viruses. III. Further studies on relationship and construction of a virus from parts of the two viral genomes

Although the V strain of tomato aspermy virus (TAV) and the Q strain of cucumber mosaic virus (CMV) do not show any detectable serological relationship or nucleotide base sequence homology, it was possible to construct a pseudo-recombinant virus from parts of their RNA genomes. The pseudo-recombinant from the two largest RNA molecules of TAV (T₁ and T₂) when combined with the third largest molecule of CMV (C₃) formed a stable virus whose coat protein was like that of CMV, but it induced symptoms on several plants indistinguishable from those produced by TAV. It is concluded that TAV and CMV have functionally divided genomes and that the coat protein cistron is located on RNA component 3, whereas the cistron or cistrons determining host reactions are located on either RNA component 1 or 2, or on both 1 and 2.     

The cognate coat protein is required for cell-to-cell movement of a chimeric brome mosaic virus mediated by the cucumber mosaic virus movement protein

Cucumber mosaic cucumovirus (CMV) and brome mosaic bromovirus (BMV) have many similarities, including the three-dimensional structure of virions, genome organizations, and requirement of the coat protein (CP) for cell-to-cell movement. We have shown that a chimeric BMV with the CMV 3a movement protein (MP) gene instead of its own cannot move from cell to cell in Chenopodium quinoa, a common permissive host for both BMV and CMV. Another chimeric BMV was constructed by replacing both MP and CP genes of BMV with those of CMV (MP/CP-chimera) and tested for its infectivity in C. quinoa, to determine whether the CMV CP has some functions required for the CMV MP-mediated cell-to-cell movement and to exhibit functional difference between CPs of BMV and CMV. Cell-to-cell movement of the MP/CP-chimera occurred, and small local lesions were induced on the inoculated leaves. A frameshift mutation introduced in the CMV CP gene of the MP/CP-chimera resulted in a lack of cell-to-cell movement of the chimeric virus. These results indicate that the viral movement mediated by the CMV MP requires its cognate CP. Deletion of the amino-terminal region in CMV CP, which is not obligatory for CMV movement, also abolished cell-to-cell movement of the MP/CP-chimera. This may suggest some differences in cell-to-cell movement of the MP/CP-chimera and CMV. On the other hand, the sole replacement of BMV CP gene with that of CMV abolished viral cell-to-cell movement, suggesting a possibility that the viral movement mediated by the BMV MP may also require its cognate CP. Functional compatibility between MP and CP in viral cell-to-cell movement is discussed.     

Stability and competitiveness of interviral recombinant RNAs derived from a chimeric cucumovirus

We previously described interviral recombinant RNAs derived from a chimeric virus having RNAs 1 and 2 of cucumber mosaic virus (CMV) with RNA 3 from the related tomato aspermy virus (TAV) and the 2b gene from either TAV or another strain of CMV. Here, we show that these interviral recombinant RNAs 3 were stable in the infected plants and could co-exist with their wild-type parental viral RNAs in the same plants, but their de novo generations were inhibited in the presence of the wild-type parental viral RNAs. The recombinant viral genomes did not prevent the replication of other viral RNAs or vice versa, but one of the interviral recombinant viruses induced different symptoms in Physalis floridana from those induced by the parental chimeric virus without the interviral RNA 3 recombinant. Factors such as the nature of the 2b gene and/or the presence or absence of competing wild-type parental RNAs influenced the generation of the recombinant RNAs described. Our data provide additional mechanistic insight into generation, stabilization and competition of recombinant viral RNA in infected host plants.     

Cell-to-cell movement of Alfalfa mosaic virus can be mediated by the movement proteins of Ilar-, bromo-, cucumo-, tobamo- and comoviruses and does not require virion formation

RNA 3 of Alfalfa mosaic virus (AMV) encodes the movement protein (MP) and coat protein (CP). Chimeric RNA 3 with the AMV MP gene replaced by the corresponding MP gene of Prunus necrotic ringspot virus, Brome mosaic virus, Cucumber mosaic virus or Cowpea mosaic virus efficiently moved from cell-to-cell only when the expressed MP was extended at its C-terminus with the C-terminal 44 amino acids of AMV MP. MP of Tobacco mosaic virus supported the movement of the chimeric RNA 3 whether or not the MP was extended with the C-terminal AMV MP sequence. The replacement of the CP gene in RNA 3 by a mutant gene encoding a CP defective in virion formation did not affect cell-to-cell transport of the chimera's with a functional MP. A GST pull-down technique was used to demonstrate for the first time that the C-terminal 44 amino acids of the MP of a virus belonging to the family Bromoviridae interact specifically with AMV virus particles. Together, these results demonstrate that AMV RNA 3 can be transported from cell-to-cell by both tubule-forming and non-tubule-forming MPs if a specific MP-CP interaction occurs.     

RNAs 4A and 5 are present in tomato aspermy virusand both subgroups of cucumber mosaic virus

Summary.  Primer extension analysis was used to determine the presence of RNAs 4A and 5 in tomato aspermy virus (TAV) and both subgroups of cucumber mosaic virus (CMV). RNAs 4A and 5 were detected in TAV and all CMV strains (representative of both subgroups) that were analysed, except for one subgroup I CMV strain which lacked detectable RNA 5. In subgroup II CMV strains the RNA 5 population was found to consist of two sequence variants. Comparison of the RNA 5 sequences from TAV and CMV indicated that TAV and subgroup II CMV RNA 5 share a much greater degree of sequence similarity than either has with subgroup I RNA 5. RNA 4A and the encoded 2b protein appear to be unique to the cucumovirus genus of the tripartite viruses, which share an otherwise common genome structure, and may have played a role in the evolutionary origin of this genus. Accepted November 15, 1996 Received May 27, 1996     

Molecular organization and stabilizing forces of simple RNA viruses. V. The role of lysyl residues in the stabilization of cucumber mosaic virus strain S.

Experiments have been carried out to identify the chemical groups responsible for the stabilization of CMV (strain S).At pH 10.3 CMV-protein and CMV-RNA reassociated into nucleoproteins that possess no specific viruslike structure. In rate zonal centrifugation the nucleoproteins sedimented as three zones trailing the virus marker. The nucleoproteins were isolated and analyzed for RNA component contents with polyacrylamide electrophoresis. In order of increasing sedimentation rates, they contained RNA components 4, 3, and 1 + 2. Separate treatment of CMV-protein and CMV-RNA at pH 10.3, followed by neutralization, had no effect on their capability to reassemble normally at pH 7. The nucleoproteins formed at pH 10.3 could be dissociated by 2.0 M LiCl. The protein reisolated in this manner could also be reassembled at pH 7 with CMV-RNA to form normal virions. These experiments suggest that the aborted reassembly at alkaline pH is caused mainly by a deprotonation of protein amino-acid residues and RNA bases with a pK around 10, and that reprotonation can reverse any pH-induced conformational perturbation of CMV-protein.Reaction of CMV with the lysyl-specific reagent trinitrobenzene-sulfonic acid (TNBS) led to structural transformation of the virions into slower sedimenting trinitrophenylated (TNP)-nucleoproteins with undefined structures. These nucleoproteins also contained CMV-RNA components 4, 3, and 1 + 2 in order of increasing sedimentation rates. The time-course of the TNBS reaction and the structural transformation suggested that approximately 3 of 15 lysyl residues in each subunit are essential to the stability of the virion. Protein could easily be prepared from 3TNP-CMV by means of 2.0 M LiCl dissociation. The yellow color and the spectral properties provided direct identification of CMV-protein as the site of trinitrophenylation. CMV with nine TNP substituents per subunit failed to dissociate under the influence of 2.0 M LiCl. Reassembly at pH 7.2 failed when 3TNP-CMV-protein was used as protein constituent.Reaction of the D-strain of CMV with TNBS was much slower. The structural transformation occurred at a higher TNP substitution level, and was more gradual than with CMV-S. This observation is in agreement with the generally greater stability of CMV-D.Of several bromoviruses tested, the reactivity with TNBS decreased in the following order: BBMV > CCMV > BMV. All but BMV degraded in their entirety under the conditions investigated.     

RNA2 of TRV SYM breaks the rules for tobravirus genome structure

Currently, all of the RNA2 molecules described for all of the more than thirty sequenced isolates of the three tobraviruses, Tobacco rattle virus (TRV), Pea early-browning virus (PEBV) and Pepper ringspot virus (PepRSV), have the virus coat protein (CP) gene located in the 5' proximal position. However, sequencing of the RNA2 of the SYM isolate of TRV revealed that this isolate has a unique genome structure in which the virus CP gene is located in the central region of RNA2 downstream of three completely novel open reading frames (ORFN1, ORFN2 and ORFN3). An infectious clone of SYM RNA2 was constructed and mutations were introduced separately into each of the novel genes to interrupt their translation. However, none of the mutations resulted in any noticeable change in the ability of TRV RNA1 or RNA2 to replicate and move systemically in the leaves or roots of infected plants. In addition, individual expression of the novel ORFs either from a Potato virus X (PVX) vector or from a binary plasmid in Agrobacterium tumefaciens did not reveal any potential function.     

In vitro evidence for RNA binding properties of the coat protein of prunus necrotic ringspot ilarvirus and their comparison to related and unrelated viruses

Summary.  The RNA binding properties of the prunus necrotic ringspot virus (PNRSV) coat protein (CP) were demonstrated by northwestern and dot-blot analyses. The capability to bind PNRSV RNA 4 was compared with viruses representing three different interactions prevailing in the assembly and architecture of virions. The results showed that cucumber mosaic virus (CMV) and PNRSV CPs, which stabilise their virions mainly through RNA-protein interactions bound PNRSV RNA 4 even at very high salt concentrations. The CP of cherry leaf roll nepovirus, whose virions are predominantly stabilised by protein-protein interactions did not bind even at the lowest salt concentration tested. Finally the CP of carnation mottle carmovirus, that has an intermediate position in which both RNA-protein and protein-protein interactions are equally important showed a salt-dependent RNA binding. Received July 30, 1998 Accepted November 23, 1998     

Molecular evidence and sequence analysis of a natural reassortant between <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> subgroup IA and II strains

Cucumber mosaic virus (CMV) is a tripartite RNA virus and has been divided into three subgroups, named IA, IB, and II. Some studies have found a few natural reassortants between CMV subgroups, although reassortment between CMV subgroups is infrequent. In our present work, a CMV reassortant, named CMV-Tsh, was obtained from a tomato plant. The complete sequence of CMV-Tsh genomic RNAs has been determined and analyzed. The results of sequence comparisons and phylogenetic analyses revealed that CMV-Tsh RNAs 1 and 3 are derived from one or two CMV subgroup II strain(s), while RNA2 is derived from a CMV subgroup IA strain. A PCR and restriction enzyme analysis-based method was developed to analyze the possibility of mixed infection by CMV strains of different subgroup in the CMV-Tsh-infected tomato plant. The results of the restriction enzyme analysis proved that CMV-Tsh is the unique strain in the tomato plant. Taken together, CMV-Tsh is a natural reassortant having CMV subgroup IA RNA2 and subgroup II RNAs 1 and 3. The GenBank Accession numbers of the sequences reported in this paper are EF202595- EF202597.