Mutation of capsid protein phosphorylation sites abolishes cauliflower mosaic virus infectivity

Abstract

The cauliflower mosaic virus (CaMV) capsid protein is derived by bidirectional processing of the precapsid protein (CP56). We expressed several derivatives of CP56 in Escherichia coli and used them as substrates for virus-associated kinase and casein kinase II purified from plant cells. Three serine residues located at the N terminus of the mature viral protein CP44 were identified as phosphorylation targets. A mutation of one of them in the viral context had little or no effect on viral infectivity, but a mutation of all three serines abolished infectivity. The mapping of phosphorylation sites in CP44, but not CP39 or CP37, and immunodetection of the Zn finger motif in CP44 and CP39, but not CP37, support the model that CP39 is produced from CP44 by N-terminal processing and CP37 is produced from CP39 by C-terminal processing. We discuss the possible role of phosphorylation in the processing and assembly of CaMV capsid protein.

FIG. 1.

(A) Schematic representation of the CaMV precapsid (CP56) and capsid proteins. Only the N terminus of CP44 (amino acid 77) is known exactly. The proposed N and C termini for CP39 and CP37 are based on the present study. The positions of the cloned sequences are indicated below. Clones were obtained from PCR amplification as described previously (1). All clones were verified by the dideoxynucleotide sequencing method. NLS, nuclear localization signal; ZnF, zinc finger. (B, upper panel) Phosphorylation assay. CP derivatives were overexpressed in E. coli strain BL21(DE3) cells by using pET3d vector, as described by Chapdelaine and Hohn (1). Bacterial inclusion bodies were isolated, solubilized in 8 M urea, and dialyzed against 10 mM Tris-Cl, pH 7.5. After dialysis, soluble proteins were separated by SDS-PAGE. Overexpressed proteins were visualized in 1 M KCl, electro-eluted in SDS buffer with a Bio-Rad model 422 electro-eluter according to the manufacturer's specifications, and dialyzed against 10 mM Tris-Cl, pH 7.5. Proteins were quantified by staining SDS-PAGE gels with Coomassie blue dye. Reaction mixtures (30 μl each) contained 20 ng of the CP derivatives [except for CP(77-265), of which only 5 ng was used], 15 μg of CaMV particles as a source of virus-associated kinase, and 2.5 μCi of [γ-³²P]ATP (3,000 Ci/mmol) in a solution containing 20 mM Tris-Cl, 10 mM MnCl₂, 0.8 mM dithiothreitol, and 0.25 mM EDTA (16) and were incubated at 37°C for 3 h. Phosphorylation was stopped by the addition of SDS loading buffer, and samples were separated by SDS-12% PAGE. Gels were dried and exposed for autoradiography. (Lower panel) Duplicate gels were transferred onto a nitrocellulose membrane (Schleicher & Schuell) for immunodetection (ECL system; Amersham) with anti-p37 antibodies (1). Note that the (smaller) amount of protein used for CP(77-265) was not sufficient to give a band.

FIG. 2.

Phosphorylation assays of CP(1-265), CP(1-265//S82,86,88A), and CP(99-489) with A. thaliana CK II. Note also the autophosphorylation of CK II α. The phosphorylation assay was carried out as described in the legend for Fig. 1, except that recombinant CK II from A. thaliana was purified and used as described by Klimczak et al. (13).

FIG. 3.

(A) Test for capsid proteins in plants inoculated with CaMV mutants. Results are shown from Western blotting with rabbit anti-CP37 (1) of total viral proteins extracted from plants inoculated with mutant virus DNAs (10). Ten milligrams of total protein was loaded for each sample. (B) Absence of the Zn finger motif in CP37. Results are shown from Western blotting of purified CaMV with rabbit anti-CP37 and anti-Zn finger immunoglobulin G (anti-ZF). For virus purification, 5 g of tissue was homogenized in an extraction buffer (0.2 M Tris-Cl [pH 7.0], 20 mM EDTA, and 1.5 M urea). A one-tenth volume of 20% Triton X-100 was added. Extracts were incubated at 40°C with gentle stirring for 2 h, filtered through four layers of cheesecloth, and cleared by centrifugation at 17,000 × g for 10 min. Supernatants were underlaid with a 15% sucrose cushion, and virus particles were pelleted at 22,000 rpm in an SW27 rotor for 2 h. Pellets were resuspended in 500 μl of storage buffer (0.1 M Tris-Cl [pH 7.4], 2.5 mM MgCl₂). Rabbit anti-Zn finger immunoglobulin G serum was obtained by injection of the peptide NIEGHYANECPN conjugated to bovine serum albumin (Readysysteme AG, Bad Zurzach, Switzerland).

FIG. 4.

Possible Ser/Thr kinase target sites are shown in grey boxes, with those predicted specifically as strong targets for CK II being in white letters. The three serines identified in this study as targets are underlined. Important motifs in the N- and C-terminal regions of CP44, i.e., the nuclear localization signal (RKRK) (14) and the Zn finger motif, are boxed. The N- and presumptive C-terminal sequences of the precursor protein not present in CP44 are shown separately.