Cauliflower mosaic virus coat protein is phosphorylated ... - Europe PMC

Proc. Nati. Acad. Sci. USA

Vol. 84, pp. 1824-1828, April 1987 Biochemistry

Cauliflower mosaic virus coat protein is phosphorylated in vitro by a virion-associated protein kinase (ADP/casein kinase/plant virus/retrold virus/reverse transcription)

Jose MARTINEZ-IZQUIERDO AND THOMAS HOHN Friedrich Miescher-Institut, P.O. Box 2543, CH-4002 Basel, Switzerland

Communicated by Diter von Wettstein, November 21, 1986 (received for review October 20, 1986)

branes at 40 V (constant voltage) overnight (18). Treatment of the nitrocellulose membranes was as described (19). Rabbit anti-p37 serum diluted 1:1000 with the milk-based solution A (19) and 1% goat preimmune serum and peroxidase-coupled goat anti-rabbit IgG (Bio-Rad) was used. Protein Kinase Assay. The standard in vitro reaction mixture contained 15 pug of viral particles, 50 mM Tris HCl (pH 7.4), 0.6 mM MnCI2 (unless otherwise specified), 1 mM dithiothreitol, 2 AtM ATP, and 2.5 ACi of [y32P]ATP (5000 Ci/mmol; 1 Ci = 37 GBq; Amersham) in a final volume of 50 Al. The reaction mixture was incubated at 370C for 30 min (unless otherwise specified), and the reaction was terminated by the addition of 16.7 /l of 5 x NaDodSO4/PAGE sample buffer (46) and a 4-min incubation at 90'C. After electrophoresis, phosphorylated proteins were visualized by staining and autoradiography. For exact quantitation, the individual bands were cut out, and radioactivity was measured in a liquid scintillation counter. Phosphoamino Acid Analysis. Analysis was as described (20).

A protein kinase has been found to be assoABSTRACT ciated with particles of the plant virus cauliflower mosaic virus. This protein kinase can phosphorylate endogenous viral capsid proteins in vitro and exchange substrates with casein kinase type H. The activity is not affected by cAMP but is enhanced considerably by ADP. The cofactor is either Mn2+ or Mg2+, and the phosphate donor is either ATP or GTP. Serine and threonine residues are phosphorylated.

The structural and functional proteins of various animal viruses, including members of the Retroviridae and Hepadnaviridae families, are phosphorylated by virus-associated protein kinases (1-12). In contrast, among the plant viruses, only cauliflower mosaic virus (CaMV) contains phosphorylated proteins (13), but little is known about the protein kinase responsible. Various properties of CaMV are related to those of animal retroviruses (14). CaMV is a double-stranded DNA virus that replicates through an RNA intermediate using a reverse transcriptase, which is closely related to the same enzyme from retroviruses. In this report we show that a protein kinase is associated firmly with CaMV particles and can act on endogenous and exogenous substrates. The activity is characterized biochemically and compared to other known protein kinases. Similar results have been independently described by another group

RESULTS CaMV Structural Proteins and in Vitro Phosphorylation. The NaDodSO4/PAGE analysis of CaMV particles reveals a complex pattern of major and minor protein bands (21, 22). The pattern shown in Fig. 1, lane 1, is obtained only when fresh virus preparations are used. When stored preparations are used (e.g., for 3 weeks at 40C), smaller-sized bands predominate (including bands at 32 and 27 kDa) resembling those in ref. 23, which indicate proteolytic activity. Antiserum prepared against the major 37-kDa protein species (anti-p37) was used to detect related coat protein species by immunoblotting (Fig. 1, lane 2). Major bands at 37 and 44 kDa (p37 and p44, respectively) as well as minor bands at 40, 57, 80, 90, and 96 kDa (p40, p57, p80, p90, and p96, respectively) were detected on the immunoblots, suggesting that these protein species may indeed share common sequences. Most likely p37 and p44 (and perhaps p40) are derived from p57 by proteolytic cleavage, and p80 and p96 are dimers of p37 and of p44, respectively (21, 24). When viral particles were incubated with [y-32P]ATP in the presence of Mn2+ or Mg2+, they were phosphorylated. The labeled phosphate incorporated was resistant to DNases and RNases but sensitive to proteinase K. Electrophoresis revealed that the phosphorylated proteins were coat protein species identified as pp44 (75%), pp57 (8%), and pp96 (15%) in Fig. 1, lane 3. Proteins p37, p40, and p80 remained essentially unphosphorylated, although p37 represents 60% of the capsid protein. This phosphorylation pattern corresponds to the one obtained from viral particles labeled in vivo with 32p (ref. 13 and unpublished data) and strengthens the

(15). MATERIALS AND METHODS Virus Isolation. CaMV, strain CM4-184 (16) was propagated in turnips (Brassica rapa, Just Right). Viral particles free of inclusion bodies were isolated as described (17). Purification of Viral Capsid Proteins and Antisera Production. CaMV particles were subjected to preparative NaDodS04/PAGE (46). After electrophoresis the gel was soaked in 0.5 M KCl at 40C, and the opaque gel bands were excised and washed three times with 0.5 M NaCl. Pieces of gel (1 cm3) were placed in the elution chamber of an ISCO electrophoretic sample concentrator. Elution of p37 protein was at 3 W for 15 hr in 0.05% NaDodSO4/0.1 M glycine/0.0125 M Tris HCl, pH 8.3. After completion of extraction, the 200-gl sample was collected, and the sample well was washed twice with 100 /l of water; sample and washings were pooled and stored at -20'C. Anti-p37 sera was raised in rabbits by i.m. injection of 100 ,tg of purified p37 emulsified in Freund's complete adjuvant. Further booster doses were given under similar conditions (in Freund's incomplete adjuvant) after 4, 7, 9, and 10 weeks. Serum samples were collected 10 days after the last injection. Immunoblotting. Proteins were electrotransferred from NaDodSO4/polyacrylamide gels to nitrocellulose memThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviations: CaMV, cauliflower mosaic virus; ORF, open reading frame.

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pp96 (labeled pair). The stability of the label to prolonged incubation in the presence of a large excess of unlabeled ATP (Fig. 2A) indicates that the incorporated radioactivity was due to de novo phosphorylation rather than to a phosphate exchange reaction.