The open reading frame III product of cauliflower mosaic virus is a protein of 15 kDa (p15) that is essential for the virus life cycle. It was shown that the 34 Nterminal amino acids are sufficient to support proteinprotein interaction with the full-length p15 in the yeast two-hybrid system. A corresponding peptide was synthesized and a recombinant p15 was expressed in Escherichia coli and purified. Circular dichroism spectroscopy showed that the peptide and the full-length protein can assume an ␣-helical conformation. Analytical centrifugation allowed to determine that p15 assembles as a rod-shaped tetramer. Oxidative cross-linking of N-terminal cysteines of the peptide generated specific covalent oligomers, indicating that the N terminus of p15 is a coiled-coil that assembles as a parallel tetramer. Mutation of Lys 22 into Asp destabilized the tetramer and put forward the presence of a salt bridge between Lys 22 and Asp 24 in a model building of the stalk. These results suggest a model in which the stalk segment of p15 is located at its N terminus, followed by a hinge that provides the space for presenting the C terminus for interactions with nucleic acids and/or proteins.

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We cloned in Escherichia coli truncated versions of the protein p15 encoded by open reading frame III of caulflower 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.Cauliflower mosaic virus (CaMV) is a 53.8-nm particle (1) that possesses an 8.0-kb, double-stranded DNA genome (2,3) having seven open reading frames (ORFs). The genome is enclosed in an icosahedral capsid composed of two types of viral proteins: the major one (420 subunits per virion) has a molecular mass of 37-42 kDa (4) and the minor one has a molecular mass of 11 kDa (5). It has been shown that the minor capsid protein is encoded by the CaMV ORF III (5). This ORF gives rise to two proteins: p15, consisting of 129 amino acids (6), is the primary translation product (7) and is a non-sequence-specific DNA binding protein able to interact strongly with the CaMV genome (8). The other protein, p1l, corresponds to a processed form of p15. The amino acids are removed from the C terminus by an as yet unidentified protease (5), but the in vivo processing site remains unknown. Only p1l has been found associated with viral particles (5). p1l, unlike p15, does not bind to double-stranded DNA (5).On the basis of these data, we chose to study the influence of the C-terminal region on the interaction between p15 and the CaMV genome. Deletion and point mutation analysis reveal that the C-terminal region of p15 as expected contains a DNA-binding domain and that a proline-rich motif is responsible for the non-sequence-specific recognition between p15 and the viral genome. MATERIALS AND METHODSPlasmids and Construction of Mutants. The plasmid pGM301 consisting of pUC8 containing the CaMV ORF III under control of the lac promoter-operator has been described (9). It allows synthesis of a fusion protein containing the N terminus of p-galactosidase.C-terminal deletion mutants were generated from pGM301 by cleavage at the unique Sal I site, limited BAL-31 digestion, and blunt-end formation with DNA polymerase I large fragment (Klenow), followed by ligation to an 8-bp linker containing a TAG stop codon (GGCTAGCC; Fig. 1A). Plasmids were screened for the linker by Nhe I digestion, and the extent of the deletion was determined by sequencing.For site-directed mutagenesis, pGM301 was digested with EcoRI and the 497-bp fragment containing ORF III was inserted into the EcoRI site of the pSELECT vector (Promega) and transformed into Escherichia coli strain JM109. After infection with the helper phage R408, singlestranded template DNA for mutagenesis was is...

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