Phosphorylation of the Budgerigar Fledgling Disease Virus Major Capsid Protein Vp1t

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 polyomavirus, 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. The supernatants were analyzed by SDS-PAGE on (Bio-Rad), pH 5-7 (Bio-Rad), pH 6-8 (Bio-Rad), and pH 12.5% polyacrylamide gels, stained with Coomassie blue, 3.5-10 (Pharmacia) ampholines. Figure lB shows the pres- and autoradiographed, as shown in lane 2 of Fig. 2A. These ence of at least five different isoelectric species of VP1, results demonstrate that three proteins from intact virus which have been designated A through E. From the which migrate identically to VP1, VP2, and VP3 (compare Coomassie blue staining reaction, there appear to be two Fig. 2A, lanes 1 and 2) and which are immunoprecipitated major isoelectric species, designated B and C, and three less with a monoclonal antibody against VP1 are phosphorylated. abundant species, designated A, D, and E. These five Previous studies showed that VP2 and VP3 are phosphory- species were consistently seen upon Coomassie blue staining lated to some extent in both murine polyomavirus (4, 32) and of two-dimensional gels. The fact that BFDV VP1 species B SV40 (32), but further information about the phosphoryla- anld C were predominant is consistent with observations tion of these minor capsid proteins has not been reported. reported for the murine polyomavirus VP1 species (4). Previous studies in our laboratory (2, 4) demonstrated that To obtain the approximate isoelectric points (pls) of these only the three most acidic isoelectric species of murine five isoelectric species, the chloramine T method (14) was polyomavirus VP1 (D, E, and F) are phosphorylated. Two- used to label purified BFDV with 1251 and 5 x 10~ cpm of ditnensional electrophoretic analysis and autoradiography virions was included in a companion tube gel, which was were performed on purified in vivo 32P-labeled BFDV to sliced into 2-mm segments after IEF. The locations of the determine which individual VP1 species are phosphorylated viral proteins were ascertained by counting the radioactivity and to investigate further the contention that VP2 and VP3 in each gel slice in a gamma counter, and the pH of each slice are also phosphorylated. Purified 1251-labeled BFDV was VOL. 66, 1992 NOTES 4553

A. B.

P- SLH-: e.1 C. Cr ... 0 -'J-- P-THHl- '- "

VP2 14- VP3 P-T1 YR- FIG. 2. (A) SDS-PAGE analysis of 32P-labeled BFDV virions. Lane 1, BFDV structural proteins separated by SDS-PAGE (12.5% polyacrylamide) and stained with Coomassie blue. Lane 2, autoradiography of purified in vivo 32P-labeled and immunoprecipitated BFDV structural proteins separated by SDS-PAGE. Positions of size markers are indicated (in kilodaltons). (B) AutoradiograVhy of purified BFDV proteins which were labeled in vitro with 12 I (105 cpm), dissociated, and separated by IEF in the first dimension and 1 2 SDS-PAGE in the second dimension as described in the text. (C) Autoradiography of purified BFDV proteins labeled in vivo with 32p FIG. 3. Phosphoamino acid analysis of BFDV VP1. Approxi- (105 cpm), immunoprecipitated, dissociated, and separated by IEF mately 2.5 X 104 cpm of 32P-labeled BFDV was used for phos- in the first dimension and SDS-PAGE in the second dimension. phoamino acid analysis. Lane 1, Phosphoamino acid standards (1 jig each of phosphotyrosine [P-TYR], phosphothreonine [P-THR], and phosphoserine [P-SER]) visualized following electrophoresis by spraying the plate with 0.2% ninhydrin in acetone and heating briefly also subjected to two-dimensional gel analysis (as described to allow color development. Lane 2, 32P-labeled phosphoamino above) and autoradiography to illustrate the migration pat- acids of BFDV VP1 visualized by autoradiography of the thin-layer terns of all BFDV structural proteins as well as to serve as a plate. reference autoradiogram for identifying the BFDV phosphoproteins, as will be discussed later. An agarose plug containing molecular weight markers (Sigma, St. Louis, seems to merge with the basic side of species D represented Mo.) was electrophoresed adjacent to the tube gel in order to an additional form of VP1 which was phosphorylated. How- facilitate estimation of protein molecular weights. Autoradi- ever, this signal was not observed consistently. ography of the two-dimensional gel of iodinated virions (Fig. Studies by Tan and Sokol (38, 39) revealed that phospho- 2B) showed that in addition to the five VP1 protein species, serine is the only phosphoamino acid present in SV40 VP1. two other proteins were present. By using the molecular size Anders and Consigli (2) found phosphorylation of both markers as a guide, it was demonstrated that one of these threonine and serine residues of VP1 from murine polyoma- proteins had a molecular mass of approximately 39 kDa, virus, with phosphothreonine accounting for approximately while the other showed a molecular mass of 29 kDa. These 70% of the polyomavirus VP1 phosphoamino acids and molecular masses are in agreement with the predicted mo- phosphoserine accounting for the other 30%. In order to lecular masses of VP2 and VP3, respectively. In addition, determine which amino acid(s) in BFDV VP1 serves as the both proteins were observed in two-dimensional gels when a phosphate acceptor, we performed phosphoamino acid anal- monoclonal antibody made against VP1 was used to immu- ysis on in vivo 32P-labeled VP1. BFDV structural proteins noprecipitate intact BFDV in RIPA buffer prior to dissocia- were resolved by SDS-PAGE and electrophoretically trans- tion (data not shown). An additional minor protein which ferred to an Immobilon-P membrane (Millipore) according to migrated at about 40 kDa was detected near the basic end of the manufacturer's instructions. The region of the membrane some gels, but this protein was not coprecipitated by the containing VP1 was visualized by autoradiography and ex- monoclonal antibody and may represent a copurifying cellu- cised. Acid hydrolysis of VP1 was performed as described lar protein. We therefore believe that the more acidic 39-kDa previously (19), and thin-layer electrophoresis was carried protein, which has a pl (about 6.3) nearly equivalent to that out at pH 3.5 as described by Cooper et al. (8). Figure 3, lane of VP1 species B, is VP2 and that the 29-kDa protein, which 1, shows the thin-layer electrophoretic separation and nin- has a pl of about 6.45, is VP3. hydrin staining of the phosphoamino acid standards. The For determination of which species of BFDV VP1 are relative positions of three discrete spots, representing phos- phosphorylated, 32P-labeled virions were first immunopre- phoserine, phosphothreonine, and phosphotyrosine, were cipitated with anti-BFDV VP1 monoclonal antibody 4F10F4 equivalent to their expected positions under these buffer (as described above), and two-dimensional electrophoresis conditions (8). Lane 2 shows the autoradiogram of labeled and autoradiography were performed (Fig. 2C). BFDV phos- phosphoamino acids found in the 32P-labeled BFDV VP1. phoproteins were identified by alignment of this autoradio- Phosphoserine was the only phosphoamino acid found in gram with that of the 125I-labeled virions (Fig. 2B). From this BFDV VP1. Overexposure of the autoradiogram failed to alignment, it appears that only the two most acidic species reveal the presence of either 32P-labeled phosphothreonine (species D and E) of VP1 as well as VP2 and VP3 were or phosphotyrosine. phosphorylated. It is possible that the faint signal which Bolen et al. (4) were able to ascribe several different 4554 NOTES J. VIROL. functions to VP1 of the mouse polyomavirus. These include tions, but it is interesting that the species of the murine a structural role, a role in the virus's ability to adsorb to and polyomavirus involved in these interactions were all phos- agglutinate guinea pig erythrocytes, a role in virus attach- phorylated. The presence of at least five isoelectric species ment to cellular receptors, resulting in productive infection, of BFDV VP1, two of which are phosphorylated, suggests and an unknown role suggested by the binding of VP1 to the that charge effects produced by phosphorylation may be virion DNA-protein core. At least six isoelectric species of superimposed on charge effects generated by other, as yet the mouse polyomavirus major capsid protein VP1 have unidentified, modifications. been identified (2, 4, 15, 24, 27, 32), and all of them appear to The function of phosphorylation of the minor capsid contain the identical amino acid sequence (2). In the murine proteins in the BFDV life cycle also remains to be eluci- polyomavirus studies by Bolen et al. (4), VP1 species A was dated. Further characterization of these phosphoproteins the only species associated with the DNA-protein core, should provide avenues for speculation as to the role of their whereas species D and F appeared to be involved in agglu- phosphorylation in the life cycles of BFDV and the other tination of guinea pig erythrocytes and species E was re- papovaviruses and will be the focus of a separate study. quired for virus adsorption to specific cellular receptors. In Here we report the existence of five isoelectric species of addition, structural roles appear to be served by species B the major capsid protein of an avian polyomavirus, BFDV, through F. Modifications which result in murine polyomavi- and we identify phosphorylation as a particular modification rus VP1 species of different pIs include phosphorylation of which contributes to differences in pIs. Posttranslational serine and threonine (1, 2, 4, 15, 24, 32), acetylation (4), modifications of proteins may be a means by which certain sulfuration of tyrosine (25), and hydroxylation of proline viruses can maximize the genetic economy of a genome (26). having a limited coding capacity. In this scenario, single Within the papovaviruses, posttranslational modifications translation products are differentially modified to carry out are not limited to the mouse polyomavirus. Two-dimensional separate and specific functions. The discovery of this phe- analysis of SV40 VP1 demonstrated the presence of several nomenon in BFDV, coupled with knowledge of its occur- different isoelectric forms (31), which are attributed to rence in murine polyomavirus and SV40, may demonstrate posttranslational modifications. Whether specific functions how papovaviruses have circumvented the limitations im- can be similarly assigned to the five isoelectric species of posed on them by their small genome size. BFDV VP1 remains to be shown. The current finding that VP1 of BFDV exists as multiple isoelectric species lends further support to the contention that posttranslational mod- This investigation was supported by Public Health Service grant ification of the major capsid protein is a general property of CA07319 from the National Cancer Institute, by NASA NAGW no. papovaviruses. A posttranslational modification of BFDV 1197 and 2328, and by the Wesley Foundation of Wichita, Kans. VP2 as well as of VP2 of SV40 and murine polyomavirus, J.I.H. is also a predoctoral fellow under National Cancer Institute namely, myristoylation, has also been reported (20, 36, training grant CA09418. 37). We thank Daryl Riley, Kevin Mapes, and LaDonna Grenz for The functional significance of BFDV VP1 phosphorylation technical assistance. remains elusive. 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