Biochemical characterization of the helper component of Cauliflower mosaic virus

Abstract

The helper component of Cauliflower mosaic virus is encoded by viral gene II. This protein (P2) is dispensable for virus replication but required for aphid transmission. The purification of P2 has never been reported, and hence its biochemical properties are largely unknown. We produced the P2 protein via a recombinant baculovirus with a His tag fused at the N terminus. The fusion protein was purified by affinity chromatography in a soluble and biologically active form. Matrix-assisted laser desorption time-of-flight mass spectrometry demonstrated that P2 is not posttranslationally modified. UV circular dichroism revealed the secondary structure of P2 to be 23% alpha-helical. Most alpha-helices are suggested to be located in the C-terminal domain. Using size exclusion chromatography and aphid transmission testing, we established that the active form of P2 assembles as a huge soluble oligomer containing 200 to 300 subunits. We further showed that P2 can also polymerize as long paracrystalline filaments. We mapped P2 domains involved in P2 self-interaction, presumably through coiled-coil structures, one of which is proposed to form a parallel trimer. These regions have previously been reported to also interact with viral P3, another protein involved in aphid transmission. Possible interference between the two types of interaction is discussed with regard to the biological activity of P2.

FIG. 1

Modifications introduced in P2. The P2 protein is schematically represented at the top. The empty boxes correspond to the predicted helices α1 and α2. The amino acid sequence of the C-terminal domain of P2 encompassing the α1 and α2 regions is listed below the diagram of P2. Hydrophobic residues at positions possibly involved in coiled-coil formation are underlined. The four mutations engineered in mod constructs as well as the four amino acids inserted in +4 constructs are also indicated. The bottom line represents the complete amino acid sequence of HP2Cter.

FIG. 2

Purification of HP2 and P2H. P2 was fused to a His tag at either its N (HP2) or C (P2H) terminus and produced in Sf9 cells via a baculovirus recombinant. (A) Purified HP2 (lane 2) and P2H (lane 3) were analyzed by SDS–12% PAGE and stained with Coomassie blue. A crude extract from insect cells infected with a P2-encoding baculovirus recombinant was loaded in lane 1. (B and C) The same proteins as in panel A were transferred onto nitrocellulose membranes and immunodetected with a P2 antiserum (B) or subjected to a P3 binding assay as described in Materials and Methods (C). The positions of molecular weight markers (in thousands) are shown on the left.

FIG. 3

UV-CD spectroscopy and MALDI-TOF mass spectrometry of HP2. (A) Purified HP2 was dialyzed in DB5 buffer and analyzed at a concentration of 5.1 μM by far-UV-CD spectroscopy in which two successive scans were averaged. (B) Purified HP2 was subjected to MALDI-TOF mass spectrometry. For each peak, the mass in daltons is indicated. The spectrum shows the presence of the trimer (3M), the dimer (2M), the monomer (M), and multicharged ions (3M/2 and M/2).

FIG. 4

Size exclusion chromatography of HP2. The elution profile of soluble HP2 at a concentration of 1.12 mg/ml was recorded in Superose 6 (prep grade) (Pharmacia) as described in Materials and Methods. The column was calibrated using, as standard molecular mass markers, purified CaMV virions (20,000 kDa), thyroglobulin (669 kDa), ferritin (443 kDa), aldolase (160 kDa), bovine serum albumin (66 kDa), and cytochrome c (12.4 kDa). The position of the elution peak of each marker protein is indicated above the graph together with the corresponding molecular mass (in kilodaltons).

FIG. 5

Electron microscopy of HP2 polymers. (A and B) Paracrystal bundles were sedimented as described in the text, trapped on a microscopy grid, and negatively stained with 2% ammonium molybdate. (C and D) Purified soluble HP2 was prepared as described in the text, and the protein present in the resulting solution was trapped on a microscopy grid and stained similarly. Bars, 30 nm (A), 100 nm (C), and 12 nm (B and D).

FIG. 6

P2-P2 interactions. (A) Schematic outline of native, truncated, or mutated versions of P2 fused to GST as described in Materials and Methods. The GST protein is represented as an oval (not to scale), whereas the lines correspond to the amino acid sequence of P2; helices α1 and α2 are symbolized by boxes. The four point mutations in α1 are represented by vertical lines in GST-P2mod. The 4-amino-acid insertion in α2 is represented by a inverted V in GST-P2 + 4. (B to F) Fusion proteins produced in E. coli were separated by SDS-PAGE and stained with Coomassie blue (B) or transferred onto nitrocellulose membranes (C to F). Numbers in panel A correspond to lanes in panels B to F; 10 μg of total protein was loaded in each lane. The membranes were probed with periplasmic extracts from bacteria producing P2::PhoA (C), P2 + 4::PhoA (D), P2mod::PhoA (E), and P2Cter::PhoA (F). In panel F, lanes 1 and 2 are from the same gel as lanes 3 to 9, for which the contrast was augmented in order to visualize the weaker signals in lanes 6 and 9. The positions of molecular weight markers (in thousands) are indicated on the right.

FIG. 7

Characterization of the C-terminal domain of P2. (A) Purified HP2Cter was dialyzed in DB5 buffer and analyzed at a concentration of 9.9 μM by far-UV-CD spectroscopy in which two successive scans were averaged. (B) A similarly purified sample was subjected to MALDI-TOF mass spectrometry. For each peak, the mass (in daltons) is indicated. The spectrum shows the presence of the trimer (3M), the dimer (2M), the monomer (M), and multicharged ions (3M/2). (C) Purified HP2Cter (10 μg) was visualized on a Coomassie blue-stained SDS–15% polyacrylamide gel, either before cross-linking or after 5 and 10 min of cross-linking with 0.5% glutaraldehyde (lanes 1, 2, and 3, respectively). (D) The pep(α1) peptide (approximately 5 μg), was loaded on an SDS-Tricine gel and stained with Coomassie blue either untreated (lane 1) or cross-linked with sulfo-GMBS at two different concentrations (5 and 10 mM in lanes 2 and 3, respectively). Pep(α1) peptide was also subjected to disulfide oxidative cross-linking for 1 min (lane 4) or 5 min (lane 5). The positions of molecular weight markers (in thousands) in panels C and D are indicated on the left. Numbers on the right indicate the positions of the monomeric (bands 1), dimeric (bands 2), and trimeric (bands 3) forms of HP2Cter (C) and pep(α1) (D).