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PubMed1992

Expression of cauliflower mosaic virus ORF II in a baculovirus system.

The cauliflower mosaic virus ORF II encoding the aphid transmission factor (ATF) was mutagenized to introduce a BamHI restriction site upstream from the initiation codon and then cloned into an eukaryotic viral expression vector (Autographa californica nuclear polyhedrosis virus). All recombinant viruses tested in Spodoptera frugiperda (SF21) cells expressed a protein of about 18 kD which comigrated in PAGE with ATF from infected plants. Western blotting using an oligopeptide antiserum to ATF confirmed the identity of the 18-kD protein from infected cells as the product of the ORF II sequences (P18). Subcellular fractionation of cells infected with the recombinant AcMNPV demonstrated that the expressed P18 accumulated intracellularly in an insoluble form. Antiserum was produced in rabbit against the partially purified P18 expressed in SF21 cells. When used to immunogold label ultrathin sections of cauliflower mosaic virus (CaMV)-infected turnip tissue, this antiserum was shown to be highly specific, labelling only the electronlucent inclusion bodies (containing P18) and not other plant cellular components.

Animals↗
PubMed1975

The pathogenesis of cauliflower ear. An experimental study in rabbits.

Appreciating an imcomplete understanding of the pathogenesis of cauliflower ear, an experimental study was designed to demonstrate the pathophysiology of this deformity. The investigation was conducted in 2-month-old rabbits. In one ear a collection of blood was placed under the raised perichondrium which was then sutured back in place and the skin closed. In the other ear an equal amount of blood was deposited between the intact perichondrium and skin. In the first study new cartilage developed under the perichondrium, but in the ear in which the blood was left above the surface of the perichondrium-covered cartilage, complete resorption of the clot occurred. The cauliflower ear was thus shown to be generating cartilage, arising from a layer of raised perichondrium which was further stimulated by a sero-sanguinous medium. The subperichondrial hematoma was extensively invaded by chondroblasts within 2 weeks, and over a period of 4 weeks the new tissue gradually changed into more mature cartilage. It was a consistent finding that the separated perichondrium retracted, thus causing the original cartilage to rise and buckle over the hamatoma, similar to the picture observed in the human pathology.

Animals↗
PubMed1978

Evaluation of potential for aflatoxin occurrence on celery, cauliflower, lettuce, and taro root inoculated with Aspergillus flavus and A. parasiticus.

Culture samples of lettuce, cauliflower, celery, and taro root (Colocasia esculenta) were assayed for the presence of aflatoxin after inoculation with Aspergillus flavus and A. parasiticus. Cultures of A. flavus produced both aflatoxins B1 and G1 on taro root, but produced by B1 on lettuce, cauliflower, and celery. For taro root, the percentage of aflatoxin G1 produced was considerably greater than that of B1. While A. parasiticus did produce mycelia and spores on the lettuce and taro root samples, there were not detectable levels of any aflatoxin produced. All the samples studied were successfully extracted and analyzed qualitatively and quantatively for the presence of aflatoxin by using official AOAC thin layer chromatographic procedures. There is sufficient evidence that Aspergilli can grow on some leafy produce and one strain produced aflatoxins.

Aflatoxins↗
PubMed1983

Observations concerning the discontinuous DNAs of cauliflower mosaic virus.

The double-stranded circular DNA encapsidated within cauliflower mosaic virus (CaMV) particles contains three single-stranded discontinuities, two in one strand and one in the other, so that, upon denaturation, three linear single-stranded DNAs are produced. Here we show that a fourth much smaller single-stranded DNA, termed alpha1, is also present in denatured CaMV DNA preparations. The 5' extremity of alpha1 is identical to that of the alpha-strand, the strand of DNA possessing only one interruption, while its 3' extremity lies just two nucleotides downstream from a major transcription initiation site. We also show that the interrupted strand at each discontinuity sometimes has a single ribonucleotide in place of a deoxyribonucleotide at its 5' extremity. Oligoribonucleotide chains of eight and 10 residues in length have also been detected at the 5' end of one of the discontinuities. These structures are thought to be the vestiges of primers which have not been completely excised prior to encapsidation of the DNA. The possibility that synthesis of the alpha-strand occurs by reverse transcription of viral RNA using initiator tRNA(met) as primer is discussed.

Caulimovirus↗
PubMed1983

Electron microscopic studies of the different topological forms of the cauliflower mosaic virus DNA: knotted encapsidated DNA and nuclear minichromosome.

Cauliflower mosaic virus (CaMV) DNA exists under different topological forms in infected plants. First, the population of encapsidated CaMV DNA molecules appears heterogeneous when analysed by gel electrophoresis. The electron microscopic study reported here reveals that CaMV virion DNA contains simple and multiple topological knots. Second, a supercoiled DNA form never found in virions exists as a chromatin-like nucleoprotein complex with nucleosome subunits in the nuclei of infected leaves. The compaction ratio of the minichromosomes is compatible with the nucleosomal structure, the number of nucleosomes (41.0 +/- 2.5) is in keeping with the length of the viral genome.

Cell Nucleus↗
PubMed1984

RNA-dependent DNA polymerase activity in cauliflower mosaic virus-infected plant leaves.

Cauliflower mosaic virus (CaMV) is a plant DNA with an 8-kb circular double-stranded genome. CaMV-specific DNA and RNA molecules present in infected Brassica cells share some structural features with DNAs and RNAs of retroviruses and hepatitis B virus. This led to the hypothesis that CaMV replication occurs via reverse transcription of an RNA intermediate. Here we report the first characterization of a new DNA polymerase activity, specific to CaMV-infected tissues. A subcellular fraction of infected cells shows capacity to copy poly(C) and the heteropolymeric regions of natural mRNAs. Chromatographic isolation of the poly(C)-dependent activity clearly establishes that it is distinct from the classical gamma-like DNA polymerases previously described in plant cells. The significant homology observed between defined regions of the Moloney murine leukemia virus (MMLV) polymerase and CaMV unassigned gene V product favours the idea that the reverse transcriptase-like DNA polymerase detected in infected cells is a virus-encoded enzyme.

Journal Article↗
PubMed1984

A second cauliflower mosaic virus gene product influences the structure of the viral inclusion body.

We have used electron microscopy of thin sections and experiments on isolated viroplasms to compare the properties of four strains of cauliflower mosaic virus (CaMV), three of which were partially or completely deleted in open reading frame (ORF) II. Our results confirm that this gene is required for aphid transmissibility and show that the product of ORF II influences the firmness with which virions are held within the viroplasm. Analysis of the proteins in the viroplasms showed that a mutant with a partial deletion in ORF II produced a protein smaller than the normal ORF product. This smaller protein was non-functional with respect both to aphid transmissibility and properties of the viroplasms.

Journal Article↗
PubMed1984

Initiation of translation of the cauliflower mosaic virus genome from a polycistronic mRNA: evidence from deletion mutagenesis.

Mutants with deletions in the open reading frame (ORF) VII to I region of the cauliflower mosaic virus genome were constructed in vitro and tested for infectivity by inoculation of plants. Sequences within ORF VII and the intergenomic region between ORFs VII and I are not required for viral infectivity. Nevertheless, the location of translation initiation and termination codons within this region governs the infectivity of the virus. Firstly, translation beginning at the initiation codon of ORF VII should reach an in-phase termination codon before the initiation codon of ORF I. Secondly, an initiation codon should be located in the vicinity of the sequence homologous to methionine tRNA, the proposed primer binding site for reverse transcription. Results suggest translation from a polycistronic mRNA by a ;relay race' mechanism whereby ribosomes after passing a termination codon re-initiate protein synthesis at the nearest AUG codon.

Journal Article↗
PubMed1985

Site-specific deletion in cauliflower mosaic virus DNA: possible involvement of RNA splicing and reverse transcription.

A frequent site-specific deletion was observed in the life cycle of cauliflower mosaic virus (S strain). Analysis of the sequence around the deletion site and the parental sequence implied that the deletion was promoted at sequences similar to the donor and acceptor consensus sequences of RNA splicing, designated as the deletion donor and acceptor sequences, respectively. To elucidate the mechanism of this site-specific deletion, point mutations were introduced into the deletion donor sequence (GT to GG or GA transversion). Deletion at the original deletion donor site did not occur in these mutants, instead, new (cryptic) donor sites were activated. All of these activated cryptic sites had sequences similar to the splicing consensus sequence. In all cases except one, the original deletion acceptor site was used. These results can be most readily explained by postulating that the site-specific deletion occurs by reverse transcription of spliced viral RNA. This frequent site-specific deletion was not observed in other strains. For a virus which replicates by reverse transcription, a mechanism to regulate the rate of splicing is required to ensure the intactness of the viral genome. We discuss the possibility that the S strain has a mutation in this regulatory mechanism.

Journal Article↗
PubMed1985

Expression of tobacco mosaic virus coat protein by a cauliflower mosaic virus promoter in plants transformed by Agrobacterium.

The 35S transcript promoter of cauliflower mosaic virus directs the synthesis of an abundant RNA in infected leaf tissue that is both a template for reverse transcription during virus replication and also a polycistronic mRNA. The 35S promoter was isolated and linked to a cDNA of the 3' end of tobacco mosaic virus that encodes the coat protein of this single-stranded RNA virus. After transformation of this gene construction into tobacco plants using a disarmed Agrobacterium binary vector, viral coat protein synthesis was detected in transformed leaf tissue. A chimaeric 35S coat protein RNA was detected in transformed callus.

Journal Article↗
PubMed1986

Recombination in a plant virus: template-switching in cauliflower mosaic virus.

A hybrid plasmid, containing tandemly arranged pieces of two different but well-defined cauliflower mosaic virus (CaMV) genomes, was used to study the mechanism by which infectious viral DNA can escape from transforming DNA. Systemic viral infection followed inoculation of Brassica plants with a strain of Agrobacterium tumefaciens containing the hybrid plasmid in its T-DNA. Restriction mapping of uncloned viral DNA from these plants, and sequencing of relevant portions of cloned viral DNA, showed that the majority of viral progeny were probably descendants of DNA produced by transcription/reverse transcription of the viral genome, thus providing further evidence for the hypothesis that this process is normally involved in viral replication. The reverse transcriptase enzyme, which is thought to undergo an intramolecular template switch during viral replication, is shown to move very close to the 5' end of the terminal repeat on the 35S RNA molecule before switching templates. The remaining minority of viral genomes can best be explained as arising from products of recombination between homologous regions of CaMV DNA.

Journal Article↗
PubMed1986

The gene III product (P15) of cauliflower mosaic virus is a DNA-binding protein while an immunologically related P11 polypeptide is associated with virions.

Using electron microscopic immunocytochemical staining we demonstrate that the product of gene III of cauliflower mosaic virus (CaMV) is associated with viral particles. Furthermore, a fusion protein, expressed in bacteria, consisting of the N-terminus of beta-galactosidase and the complete gene III protein of CaMV showed DNA-binding activity. From these two results, we discuss the possible function of this viral polypeptide.

Journal Article↗
PubMed1986

Splicing of an intervening sequence from hybrid cauliflower mosaic viral RNA.

A DNA sequence encompassing intervening sequence 2 of the soybean leghemoglobin gene was inserted into the cauliflower mosaic virus (CaMV) genome. Upon passage through host plants progeny virus slowly accumulated that had lost the intron, apparently by exact splicing of the RNA form of the genome at the exon/intron borders. These findings suggest that reverse transcription from (spliced) RNA to DNA is a normal step in CaMV replication. S1 nuclease mapping revealed a mixed population of unspliced and spliced viral RNAs. The inefficiency of the splicing process is discussed.

Journal Article↗
PubMed1986

Hairpin DNAs of cauliflower mosaic virus generated by reverse transcription in vivo.

Cauliflower mosaic virus (CaMV) is a DNA plant virus which replicates by reverse transcription. During our examination of CaMV replication intermediates by 2-D gel electrophoresis, we have discovered a population of bizarre linear double-stranded hairpin DNAs. The largest hairpin is the size of the CaMV genome; hairpin loop ends of smaller molecules map to several sites around the genome but the open ends are all located close to the origin of reverse transcription at the primer binding site. We believe that the hairpin DNAs are generated in vivo by reverse transcription of CaMV RNA followed by self-primed second strand synthesis. The accumulation of hairpin DNAs in vivo might represent a side reaction of the CaMV reverse transcriptase although an essential role for them in the virus replication cycle cannot be discounted. The structure of the hairpin DNAs provides further evidence for the location of the start site and of the polarity of reverse transcription in CaMV.

Journal Article↗
PubMed1986

The sequence of carnation etched ring virus DNA: comparison with cauliflower mosaic virus and retroviruses.

Carnation etched ring virus (CERV) DNA comprises 7932 bp. CERV primer binding sites and overall genome organization are similar to those of the related cauliflower mosaic virus (CaMV). The six open reading frames of CERV showed amino acid homology (50-80%) with CaMV ORFs I-VI; no homologues of CaMV ORFs VII or VIII were found. CERV ORFs 1-5 interface each other with the sequence ATGA. The comparison of CERV ORF5 with CaMV ORFV highlighted regions which show homologies to retrovirus gag/pol protease, RNase H and DNA polymerase domains; the possibility that the DNA polymerase domain comprises two subdomains, operating off different templates, is discussed. Both CERV and CaMV ORFs I have sequence homology to tobacco mosaic virus P30 and plastocyanin.

Journal Article↗
PubMed1988

In vitro expression of cauliflower mosaic virus genes.

The eight major open reading frames (ORFs) of cauliflower mosaic virus (CaMV) have been cloned for in vitro transcription and translation. All the ORFs could be translated. Using antisera against either purified virus or specific gene products, the translation products were screened by immunoprecipitation. The products of ORFs III, IV and V were confirmed as components of the virions. Molecular weights of primary translation products were determined and compared with those found in vivo. A further series of constructs was designed to test whether translation of adjacent ORFs is coupled in a relay-race fashion as proposed on the basis of earlier in vivo mutagenesis studies. Downstream genes on dicistronic RNAs could be translated, although inefficiently. In view of the similarity between the arrangement of the CaMV coat protein and reverse transcriptase genes and the corresponding genes of retroviruses, we asked whether the CaMV reverse transcriptase could be expressed in vitro as a fusion protein, e.g. by ribosomal frame shifting. No such fusion was observed, suggesting that the polymerase gene is translated from its own ATG.

Journal Article↗
PubMed1989

Cauliflower mosaic virus produces an aspartic proteinase to cleave its polyproteins.

Cauliflower mosaic virus (CaMV), a plant pararetrovirus, produces polyproteins from its adjacent genes for the coat protein (ORF IV) and for enzymatic functions (ORF V). The N-terminal domain of the latter gene includes a sequence showing homology to the active site of other retroviral and acid proteases. We have now shown that this domain does indeed produce a functional aspartic protease that can process both the polyproteins. Mutations in the putative active site abolished virus infectivity. In transient expression studies in protoplasts, the N-terminal domain of ORF V was able to free active CAT enzyme from a precursor containing an N-terminal fusion of a portion of ORF IV. The junction between the two domains of this artificial polyprotein comprised sequences from the ORF IV product that had previously been shown to include a proteolytic processing site. The protease mutants were not able to free active CAT enzyme from this precursor. Direct analysis of cleavage at the same site in the ORF IV product using proteins expressed in Escherichia coli revealed the expected products. In vitro translation of a synthetic transcript covering ORF V was used to study the autocatalytic cleavage of the ORF product. Pulse-chase experiments showed that the 80 kd initial translation product was processed to yield a N-terminal doublet of polypeptides of 22 and 20 kd apparent mol. wt, which cover the protease domain. The mutants in the active site were not processed.

Amino Acid Sequence↗
PubMed1990

The reverse transcriptase gene of cauliflower mosaic virus is translated separately from the capsid gene.

Cauliflower mosaic virus (CaMV) possesses start codons at the beginning of its reverse transcriptase (RT) gene (ORF V) suggesting that, unlike in retroviruses and retrotransposons, it is translated independently from the capsid gene (ORF IV). To test this hypothesis a mutational analysis of the CaMV ORF IV/V overlapping region was performed. Mutants in which both ORFs are separated by stop codons in all three reading frames are viable and stable, while mutations affecting the first two AUG codons of ORF V are either lethal or unstable, giving rise to true and second site reversions. Mutants in which the AUG codons were replaced by ACG or AAG reverted only slowly and ACG could direct the synthesis of small amounts of reporter enzyme in transfected plant protoplasts, showing that this codon can act in plant cells as a weak start codon. CaMV has apparently developed a strategy for translation of the RT gene different from that in retroviruses and retrotransposons, but similar to that of hepadnaviruses, another group of pararetroviruses. The separate translation of the RT gene as a common feature of pararetroviruses might reflect the difference in their life cycle in comparison with retroviruses.

Base Sequence↗