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Phosphorylation of the Budgerigar Fledgling Disease Virus Major Capsid Protein Vp1t

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Phosphorylation of the Budgerigar Fledgling Disease Virus Major Capsid Protein Vp1t JOURNAL OF VIROLOGY, JUIY 1992, p. 4551-4555 Vol. 66, No. 7 0022-538X/92/074551-05$02.00/0 Copyright © 1992, American Society for Microbiology Phosphorylation of the Budgerigar Fledgling Disease Virus Major Capsid Protein VP1t JOHN I. HAYNES II AND RICHARD A. CONSIGLI* Division of Biology, Section of Virology and Oncology, Ackert Hall, Kansas State University, Manhattan, Kansas 66506-4901 Received 19 February 1992/Accepted 30 March 1992 The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyoma- virus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32p; to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein. Budgerigar fledgling disease virus (BFDV) has been iso- murine counterpart revealed 47% identity between the VP1 lated from fledgling budgerigars (Melopsittacus undulatus) proteins and 26.5 and 29.5% identity between the VP2 and showing lesions in numerous tissues, including skin, feather, VP3 proteins, respectively (35). follicle, kidney, uropygial gland, crop, liver, heart, bone Previous studies (1, 2, 4, 15, 24-26, 32, 41) have demon- marrow, spleen, and brain (3, 9, 10, 17, 33). BFDV, the first strated that the major capsid protein of the mouse polyoma- reported nonmammalian polyomavirus, can cause mortality virus is differentially modified after translation in several rates of up to 100% in budgerigar aviaries (3, 5, 9, 10, 18, 33). ways, including phosphorylation (1, 2, 4, 15, 24, 32). These Its classification as a polyomavirus is based on electron modifications are believed to contribute to the existence of at microscopic studies of its morphology, its resistance to least six species of the protein, which can be separated by organic solvents, its ability to replicate in the nuclei of isoelectric focusing (4). In the murine polyomavirus, these infected cells, the buoyant density of virions (1.34 g/ml), and species have been designated A (most basic) to F (most the characteristics of its DNA, which is supercoiled, with a acidic). Two-dimensional analysis of SV40 VP1 also re- molecular weight of 330,000 (5, 11, 22). In addition, Lehn vealed the presence of several different isoelectric forms of and Muller (22) were able to demonstrate transformation of this protein (31). SV40 VP1, like polyomavirus VP1, was nonpermissive primary hamster embryo fibroblasts by shown to be differentially phosphorylated (32). It was thus of BFDV. interest to determine whether VP1 of the avian polyomavirus Elucidation of the complete nucleotide sequence (35) of BFDV also undergoes modification by phosphorylation. We BFDV revealed similarities with both simian virus 40 (SV40) also wished to investigate the possible existence of multiple and murine polyomavirus. The DNA contains 4,980 bp and, isoelectric species of BFDV VP1 which may be differentially like those of the related viruses, is divided into early and late phosphorylated. coding regions. The early region codes for two proteins, For purification of BFDV, primary cultures of chicken which have been designated the large T and small T antigens embryo cells were prepared as described previously (29). by analogy with other papovaviruses. However, the large T Infected cultures were maintained at 39°C in serum-free antigen of BFDV is considerably smaller than the large T Dulbecco's modified Eagle's medium (DMEM). BFDV was antigen of either SV40 or polyomavirus. Another distinction purified from infected-cell lysates as described previously is that an open reading frame for middle T antigen found in for polyomavirus (28). The CsCl gradients used in the rodent polyomaviruses is not present in the BFDV genome. purification were prepared by the method of Brunck and The late region of the genome codes for three structural Leick (7) as described in greater detail elsewhere (6). Prior to proteins, VP1, VP2, and VP3, with apparent molecular CsCl purification, sucrose-pelleted virus was treated with a masses of 42, 39, and 29 kDa, respectively. As with SV40 1:1 mixture of phosphate-buffered saline (PBS; 10 mM and mouse polyomavirus, VP3 appears to be identical to the sodium phosphate [pH 7.2], 150 mM NaCl) and RIPA buffer carboxy terminus of VP2. Recently, a sequence in the (20 mM morpholinepropanesulfonic acid [MOPS, pH 7.0], carboxy terminus of VP2 has been shown to confer nuclear 150 mM NaCl, 1% [wt/vol] desoxycholate, 1% [vol/vol] localization on this protein (34). A comparison of the struc- Nonidet P-40, 0.1% sodium dodecyl sulfate [SDS], 2 mM tural proteins between this avian polyomavirus and its EDTA) containing 10 ,ug each of the protease inhibitors aprotinin, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L- phenylalanine chloromethyl ketone, and phenylmethylsulfo- * Corresponding author. nyl fluoride per ml. The lower band, containing intact BFDV t Contribution 92-439-J from the Kansas Agricultural Experiment virions, was collected from the CsCl gradients and used for Station, Kansas State University, Manhattan, KS 66506. all subsequent analyses. 4551 4552 NOTES J. VIROL. A. TABLE 1. Characteristics of BFDV virion VP1 isoelectric species Species Apparent pla 3P incorporated' 66- B. A 6.45 B 6.30 :-;: A B C D E vvv C 6.15 45I -VP,, - D 6.05 + E 5.85 + 245- 29- 4444! -VP3 a Apparent pls were determined by incubating a gel slice containing the respective species in 1 ml of distilled water and measuring the pH at room temperature. b Determined by autoradiography of in vivo 32P-labeled BFDV virions 214- electrophoresed on an O'Farrell (30) two-dimensional gel. -, nonphosphory- lated species; +, phosphorylated species. FIG. 1. Electrophoretic analysis of BFDV structural proteins. (A) The three BFDV structural proteins and host-contributed his- was then measured in distilled water. The pIs of the five tones were resolved by SDS-PAGE (12.5% polyacrylamide) (21) and isoelectric species of VP1 ranged from 6.45 to 5.85, as shown visualized by Coomassie blue staining. Sizes (in kilodaltons) were 1. The minor were also shown to obtained Dalton Mark VII-L molecular size' in Table capsid proteins by electrophoresing within this range shown in so estima- markers (Sigma) in an adjacent lane, and the BFDV structural have pIs (as Fig. 2B), proteins and host-contributed histones are indicated. (B) The five tion of their pIs was made by comparing their positions in the isoelectric species of BFDV VP1 (designated A [most basic] to E IEF dimension with the positions of the VP1 species. [most acidic]) were resolved by IEF in the first dimension and To study protein phosphorylation, BFDV was labeled in SDS-PAGE in the second dimension and visualized by Coomassie vivo with 3 Pi by removing the serum-free DMEM from blue staining. chicken embryo cells at 18 h postinfection and replacing the medium with serum-free Eagle's medium containing 20% of the normal phosphate concentration and supplemented with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (21) approximately 3.6 x 108 cpm of 32p; (carrier free; ICN was performed on purified BFDV virions with a 12.5% Radiochemicals, Irvine, Calif.) per 100-mm2 dish. Infection acrylamide gel and a 0.33% bisacrylamide cross-linker. The was allowed to proceed until the cultures showed cytopathic three BFDV structural proteins as well as the host-contrib- effect, at which time the cells were harvested and the virus uted histones are shown in Fig. 1A. The VPlIVP2NVP3 ratio was purified as described above. appears to be quite similar to that found in the murine For analysis of phosphorylation by SDS-PAGE, 2.5 x 104 polyomavirus and SV40 (40), with the maj'or capsid protein cpm of purified intact 32P-labeled virions was first immuno- VP1 constituting at least 75% of the total protein of all three precipitated with anti-BFDV VP1 monoclonal antibody viruses. In order to determine whether isoelectric heteroge- 4F10F4 (13) to eliminate possible contaminating cellular neity exists in the BFDV major capsid protein, as it does in proteins. Four volumes of RIPA buffer were added to the murine polyomavirus and SV40, two-dimensional electro- mixture, which was then rocked at 4°C for 2 h, after which 50 phoresis wa's performed on purified virus by the method of pl of a 20% suspension of Staphylococcus aureus Pansorbin O'Farrell,, essentially as described previously (2, 4, 12, 30). cells (Calbiochem, San Diego, Calif.) in RIPA buffer was Prior to electrophoresis, 20 p.g of BFDV virions was dis- added. The mixture was rocked at 4°C for an additional 45 rupted in 2% SDS-5% 2-mercaptoethanol for 2 min at 1000C min, and the particulate material was pelleted by centrifuga- and acetone precipitated. The viral pellets were then resus- tion and washed three times in RIPA buffer, twice in PBS, pended in a 25-pAl solution composed of 9.5 M urea, 5% and once in distilled water. The final pellet was resuspended 2-mercaptoethanol, 2% Nonidet P-40, and 2% ampholines. in SDS-PAGE sample buffer (21), boiled for 2 min to elute The ampholine mixture used for isoelectric focusing (IEF) the antigen-antibody complexes from the cells, and recentri- consisted of equal volumes of pH 2.5-5 (Pharmacia), pH 4-6 fuged.
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