Sequence of a cauliflower mosaic virus strain infecting solanaceous plants.

The complete nucleotide sequence (8031 bp) of the DNA of cauliflower mosaic virus (CaMV) strain B29 is reported. This strain is unusual, since it infects both cruciferous and solanaceous plants. So far, from data of sequence comparisons between B29 and other CaMV strains there is no evidence for any obvious correlation between host range and distinct sequence features.

The full-length product of cauliflower mosaic virus open reading frame III is associated with the viral particle.

The gene III product (P15) of cauliflower mosaic virus (CaMV) is a DNA binding protein in which the DNA binding activity is located on its C-terminal part. In previous work, a C-terminal processed form of P15 (P11) was detected in purified viral particles as a minor component. The full-length P15 was shown to be present and to be matured, possibly by a cysteine proteinase, in CaMV replication complexes isolated from infected turnip leaves. In this paper, we have shown that a virion-enriched fraction obtained from such replication complexes by size exclusion chromatography contained most of the P15 in its uncleaved form and was enriched in the activity responsible for its proteolysis. This enabled us to characterize better the proteinase activity (temperature and pH optimum; effect of specific inhibitors) responsible for P15 cleavage and to confirm that it corresponds to a cysteine proteinase. Based upon these observations, a purification procedure for CaMV particles was devised which impaired the cleavage of P15 into P11 and allowed the isolation of virions containing almost exclusively the noncleaved form. This finding supports our hypothesis that the CaMV gene III product could be involved in the folding of the viral genome during encapsidation.

Identification of C-terminal amino acid residues of cauliflower mosaic virus open reading frame III protein responsible for its DNA binding activity.

We cloned in Escherichia coli truncated versions of the protein p15 encoded by open reading frame III of cauliflower mosaic virus. We then compared the ability of the wild-type p15 (129 amino acids) and the deleted p15 to bind viral double-stranded DNA genome. Deletions of > 11 amino acids in the C-terminal proline-rich region resulted in loss of DNA binding activity of wild-type p15. Moreover, a point mutation of the proline at position 118 sharply reduced the interaction between the viral protein and DNA. These results suggest that cauliflower mosaic virus p15 belongs to the family of DNA binding proteins having a proline-rich motif involved in interaction with double-stranded DNA.

Processing of the minor capsid protein of the cauliflower mosaic virus requires a cysteine proteinase.

The major capsid protein of the cauliflower mosaic virus (CaMV) is processed in vivo. The viral aspartic proteinase that catalyses this maturation has been characterized previously and is coded by the CaMV gene V. This virus has a second capsid protein, a minor component, encoded by gene III. This protein, P3, is also processed at its C-terminus in vivo. To determine whether P3 is matured by the CaMV proteinase P5, we expressed, in Saccharomyces cerevisiae, P3, P5 and a fusion protein P7-P4, containing potential sites of cleavage. P5 was found to be involved in maturation of P7-P4 but did not cleave P3. The latter result was confirmed by experiments carried out with an in vitro translation system (the reticulocyte lysate) and with preparations of replication complexes purified from infected plants. Moreover, [N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leu cyl]-amido(4-guanido)butane, a specific inhibitor of cysteine proteinases, inhibited the maturation of P3, suggesting that the two CaMV capsid proteins are not processed by the same proteolytic event.

The cauliflower mosaic virus reverse transcriptase is not produced by the mechanism of ribosomal frameshifting in Saccharomyces cerevisiae.

The capsid protein and the reverse transcriptase of cauliflower mosaic virus (CaMV) are encoded by two genes (ORF IV and ORF V) that lie in different translation reading frames. A comparison can be drawn between the synthesis of both CaMV proteins and the fusion protein in a yeast retrotransposon, Ty, resulting from a +1 frameshifting event which fuses two out-of-phase ORFs encoding the structural protein and the reverse transcriptase of Ty. For this reason, we constructed a yeast expression vector containing CaMV ORF VII fused to CaMV ORF III by a fragment of 452 bp including the overlapping region of ORF IV and ORF V, ORF VII and ORF III being used as reporter genes. We characterized two proteins (22 and 50 kDa) synthesized from this plasmid in the yeast expression system. We demonstrated that the 50-kDa polypeptide is not synthesized from a +1 frameshifting event but is probably a dimeric form of the 22-kDa protein. From this result we conclude that the CaMV reverse transcriptase is not produced by a mechanism of ribosomal frameshifting.