JOURNAL OF VIROLOGY, Sept. 1988, p. 3109-3119 Vol. 62, No. 9 0022-538X/88/093109-11$02.00/0 Copyright © 1988, American Society for Microbiology Expression and Complex Formation of Simian Virus 40 Large T and Mouse in Insect Cells DAVID, R. O'REILLY AND LOIS K. MILLER* Departments ofEntomology and Genetics, University of Georgia, Athens, Georgia 30602 Received 1 March 1988/Accepted 16 May 1988

Recombinant baculoviruses were constructed which express simian virus 40 large T antigen (SVT-Ag) or murine p53 to high levels in infected insect cells. Characterization of the expressed revealed that they display many properties of the corresponding mammalian-derived proteins. Both proteins are of wild-type size, localize to the nucleus, are recognized by several SVT-Ag- or p53-specific monoclonal antibodies, and are phosphorylated in this system. Complexes are formed between baculovirus-derived SVT-Ag and p53 after coinfection of insect cells with both recombinant viruses. After infection of insect cells with either virus individually, each can self-associate to form a variety of oligomeric species. Pulse-chase experiments indicated that both SVT-Ag and p53 are highly stable in insect cells, even in the absence of complex formation.

A variety of systems have been devel- able concerning the nature or site(s) of the oped recently with a view to achieving enhanced expression events involved (17, 34, 36). Baculovirus-derived human of eucaryotic proteins normally present at extremely low epidermal growth factor receptor displays an autophos- levels (44). A recently developed vector system with consid- phorylation activity like that of the wild-type protein (12). erable potential employs the baculovirus Autographa cali- We chose to study the expression of simian virus 40 fornica nuclear polyhedrosis virus (AcMNPV) to mediate (SV40) large T antigen (SVT-Ag) and murine p53, because expression of the cloned gene in insect cell cultures (25, 31, prior characterization of the numerous functions and post- 32). In this system, the gene is inserted in place of the translational modifications associated with these proteins AcMNPV polyhedrin gene, which is nonessential for viral has been extensive. SVT-Ag displays ATPase activity, replication in . Expression of the cloned gene is DNA-binding activity, and activity (4, 5, 43, 57). then under the control of the polyhedrin promoter, resulting The protein is essential for viral DNA replication (59) and in high levels of expression late in infection. may associate with the host cell DNA polymerase alpha (53). To date, a wide range of genes has been expressed by It can immortalize and transform primary and established using the baculovirus system and in many cases, biologically cell lines, and these properties may be related to its ability to activp proteins have been obtained (32a; reviewed in refer- bind and stabilize the host cell protein p53 (30, 38, reviewed ence 25). Characterization of the expressed proteins has in reference 37). p53 itself is an immortalizing (6, shown that insect cells can carry out at least some of the 20, 39) and is implicated in the passage of cells from Go to G1 posttranslational modifications which occur in mammalian (21). Both SVT-Ag and p53 are phosphoproteins, and the cells. Signal sequence cleavage has been demonstrated for nature and sites of phosphorylation are largely determined human alpha- and beta- and interleukin-2 and -3 (29, 45-49, 60). SVT-Ag is a particularly good model to study (27, 33, 54, 55), while the appropriate proteolytic cleavage of phosphorylation since there is much recent evidence linking human immunodeficiency virus and gag proteins and phosphorylation to the regulation of a number of its biolog- virus hemagglutinin is observed (16, 22, 26, 40). ical activities, especially viral DNA replication and DNA Polyomavirus large T antigen and the Drosophila Kruppel binding (11, 35, 48). protein display DNA-binding activity after synthesis in in- In this study, we describe the expression of SVT-Ag and sect cells (36, 42). Oligomerization, complex formation, or p53 by using the baculovirus expression system. The abun- both has been observed for baculovirus-expressed dantly expressed proteins are localized in the insect cell major antigen (7) and two influenza virus polymerase nucleus and adopt a structural conformation similar to that complex proteins (56). of their mammalian counterparts. Both proteins are phos- It is not yet clear whether all posttranslational modifica- phorylated and retain the ability to associate together after tions will be comparable in mammalian and insect cells. expression in insect cells. Glycosylation is one modification which appears to differ between mammalian and insect cells, but the significance of MATERIALS AND METHODS this difference is not yet known and several biologically Cells and viruses. Spodoptera frugiperda (fall armyworm) active have been by using already produced IPLB-SF21 cells (SF21 cells) (61) were maintained in TC-100 the baculovirus system. One of the posttranslational modi- medium (GIBCO Laboratories) supplemented with 10% fetal fications of is phosphorylation, because this key importance calf serum and 0.25% tryptose broth. Stocks of wild-type modification has been found to play a critical role in the (wt) virus, AcMNPV L-1 (24), were prepared and assayed as regulation of protein function in a variety of systems. It is described previously (31). Extracts of clone 6 rat cells (30), known that the c-myc, and Drosophila Kruppel protein, which express elevated levels of mouse p53, and AdS human T-cell leukemia virus type I p40" are phosphorylated SVR111-infected human 293 cells (10) containing SVT-Ag after expression in insect cells, but no information is avail- were provided by C. Prives. Construction of recombinant viruses. To generate recom- * Corresponding author. binant AcMNPVs expressing SVT-Ag and mouse p53, 3109 3110 O'REILLY AND MILLER J. VIROL.

Bam HI Eco RI A 1781 CIan B (BgIll/Kpn I Pst I 3 ~~Kpn

ST-Ag Eco RI 5190 pEV55SVT Xho I to (BgIU/StuI) 9.9 kb pEV55p53 BgIll3 7.8kb

-'p~~~~~~~~~~~~~~~~~~~~~

p

Polyhedrin 5' leoder p53 5 leader Polyhedrin 5' leoder SVT-Ag 5 leader -0 BgIll Eco RI - * (Bgl /StuD) I0 AAACCTATAAATAGATCTCGAGAATTCCATCCTGG AAAC CTATAAATAG ATC C CTAG GCTTTT 1t XhoI _-29y -105 FIG. 1. Structure of the transplacement plasmids pEV55SVT and pEV55p53 (not to scale). (A) In pEV55SVT, SV40 sequences from pSVT#5, including the cDNA copy of the SVT-Ag gene, are indicated by the hatched box. The arrow indicates the expected SVT-Ag transcript. SV40 nucleotide numbers are given. (B) For pEV55p53, the stippled box represents the p53 cDNA from pSV53c. Again, the p53 transcript is indicated. In both plasmids, AcMNPV sequences which flank the polyhedrin gene are presented as open boxes (P5' and P3'). The pUC8 sequences are indicated by a thin line. Selected restriction endonuclease sites are shown. Sites in parentheses are those present in the original fragments which were destroyed during the construction. The sequence of the pEV55-cDNA junction is given below each plasmid. In each case, the position of the fusion site relative to the AUG of the cloned gene is indicated. cDNA copies of these genes were cloned first into the type (31). The recombinant viruses were subjected to three transplacement plasmid pEV55. The structure of this vector rounds of plaque purification before large-scale virus stocks has been described previously (31, 32). The cDNA encoding were prepared. Viral DNA was isolated, and the structures SVT-Ag was excised from the plasmid pSVT#5 (constructed of the resultant viruses, vEV55SVT and vEV55p53, were by Y. Gluzman) by digestion with StuI (nucleotide 5191) and verified by restriction enzyme analysis and Southern blot- EcoRI (nucleotide 1780), yielding a fragment which extends ting. from 29 base pairs upstream of the ATG of SVT-Ag to Analysis of proteins synthesized in infected cells. SF21 cells approximately a kilobase downstream of the translational (106/35-mm Petri dish) were infected with wt or recombinant termination codon. This fragment was cloned into pEV55 viruses at a multiplicity of infection (MOI) of from 10 to 50 which had been previously digested with BglII (filled in with (see figure legends). At the appropriate times postinfection T4 DNA polymerase) and EcoRI. The recombinant plasmid (p.i.), the medium was removed and replaced with TC-100 obtained, pEV55SVT, is illustrated in Fig. 1A. lacking either or phosphate. The cells were To construct pEV55p53, a cDNA encoding murine p53 labeled 1 h later with 50 ,uCi of [35S]methionine or 100 ,uCi of was excised from the plasmid pSV53c (19) by digestion with 32p; (New England Nuclear) in 0.5 ml of methionine- or EcoRI and BglII (filled in with T4 DNA polymerase). The phosphate-deficient medium. The lengths of the labeling resultant 1.33-kilobase fragment includes 105 base pairs of 5' periods are indicated in the individual figure legends. In flanking sequence and extends 20 base pairs beyond the certain experiments, the [35S]methionine pulse labeling was termination codon. It was inserted into pEV55 that had been chased by incubation of the cells in TC-100 containing an previously digested with KpnI (blunt ended within T4 DNA excess of unlabeled methionine. The cells were rinsed three polymerase) and EcoRI. The structure of the resultant plasmid, pEV55p53, is depicted in Fig. 1B. times in cold phosphate-buffered saline (PBS; 8 mM To confirm the structure of the pEV55-cDNA junction in Na2HPO4, 137 mM NaCl, 0.5 mM MgCl2, 1.6 mM KH2PO4, both pEV55SVT and pEV55p53, the DNA sequence span- 2.7 mM KCl [pH 8.0]) and incubated in 50 ,ul of lysis buffer ning the junction was determined by double-stranded se- (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris hydrochloride quencing of plasmid DNA, essentially as described by Chen [pH 8.0]) containing 1 mM phenylmethylsulfonyl fluoride and Seeburg (3). The primer used was a 17-mer correspond- (Fluka), 40 ,uM pepstatin (Fluka), and 20 ,uM leupeptin ing to residues -26 to -42 in the polyhedrin leader se- (Fluka) for 30 min at 4°C. Lysates were stored at -80°C. quence. The plasmids pEV55SVT and pEV55p53 contain the Total proteins present were visualized by electrophoresis of SVT-AG and p53 genes downstream from the AcMNPV portions of the lysates through 10% sodium dodecyl sulfate- polyhedrin promoter and flanked by polyhedrin 5' and 3' polyacrylamide gels (SDS-PAGE; 23). Alternatively, sam- flanking sequences (Fig. 1). ples were immunoprecipitated with various antibodies (in Recombinant viruses expressing these genes were gener- tissue culture fluid) directed against SVT-Ag or p53 (detailed ated by replacement of the wt polyhedrin gene in AcMNPV in the text). Trial immunoprecipitation experiments were L-1 with the promoter-gene fusions from pEV55SVT and conducted to ensure the presence of excess antibody. Im- pEV55p53. To this end, 2 x 106 SF21 cells were cotrans- munoprecipitation experiments were carried out in NET fected with 2 ,ug of viral DNA (isolated as described in buffer (140 mM NaCl, 5 mM EDTA, 0.05% Nonidet P-40, 50 reference 31) and 18 ,ug of either pEVS5SVT or pEV55p53, mM Tris hydrochloride [pH 8.0]) containing 1 mg of bovine according to the procedure of Potter and Miller (41). At 5 serum albumin per ml, at 4°C for 3 to 4 h. Antigen-antibody days later, progeny virus were harvested and then re- complexes were collected by adsorption to fixed Staphylo- plaqued. Plaques generated by recombinant virus were iden- coccus aureus (Sigma Chemical Co.) for 1 h at 4°C. Immu- tified by visual screening for an occlusion-negative pheno- noprecipitates were washed three times in NET buffer, VOL. 62, 1988 BACULOVIRUS-DERIVED SVT-Ag AND p53 3111 eluted by being boiled for 5 min in the gel-loading buffer (23), The kinetics of synthesis of these proteins were examined and analyzed by SDS-PAGE. by SDS-PAGE of [35S]methionine pulse-labeled proteins of Immunofluorescence studies. SF21 cells (105) were seeded infected cells (Fig. 2B). Synthesis of both the 94- and 53-kDa onto glass coverslips (22 by 22 mm) and infected with the proteins is detectable at 24 h p.i. and increases through 48 h recombinant viruses at an MOI of 10. At the selected times after infection. In both cases, the kinetics of synthesis are p.i., the cells were washed three times in cold PBS and fixed like those of polyhedrin, and a similar inhibition of host cell in 70% acetone-30% methanol at -20°C for 10 min. The protein synthesis is observed at late times p.i. fixative was removed, and the coverslips were air dried and To confirm that the 94- and 53-kDa proteins correspond to stored at -20°C. Before being stained, coverslips were SVT-Ag and p53, respectively, immunoprecipitation exper- incubated in PBS at room temperature for 15 min. The PBS iments were performed with monoclonal antibodies specific was removed by aspiration, and 50 ,ul of the appropriate for these proteins. It can be seen in Fig. 2C and D that the dilution (determined empirically) of monoclonal antibody monoclonal antibody PAb 419, which is specific for SVT-Ag was placed on the coverslip. After incubation at 37°C for 1 h (15), recognizes the 94-kDa polypeptide expressed in in a moist environment, the coverslips were washed twice vEVSSSVT-infected SF21 cells. Similarly, PAb 421, an for 15 min each in PBS at room temperature. The second anti-p53 antibody (15), specifically immunoprecipitates the antibody used was a fluorescein isothiocyanate-conjugated 53-kDa protein from vEV55-infected cells (Fig. 2C and D). rabbit anti-mouse immunoglobulin antiserum (Sigma), and Again, no such proteins are immunoprecipitated from wt- or incubation conditions were as described above. After two mock-infected lysates. Note that polyhedrin precipitates further washes with PBS, the coverslips were mounted onto spontaneously in these experiments because it is quite microscope slides and examined by using Nomarski or UV insoluble in the lysis buffer used. As before, synthesis of optics. is detectable from 24 h after infection and Sucrose gradient centrifugation. SF21 cells were infected both proteins with the recombinant viruses individually or together and lower levels of SVT-Ag are observed than of p53. In these lysed as described above. Lysates (2 x 106 infected cells per and certain subsequent experiments, the amount of sample gradient) were loaded onto 5-ml linear gradients of 5 to 20% used from vEVSSpS3-infected cells was reduced because of sucrose in PBS and centrifuged at 55,000 x g for 16 h at 4°C. the more efficient expression of this vector (see figure Equal volume fractions (250 ,ul) were collected and immu- legends). Trial titration experiments were previously carried noprecipitated with PAb 419 or PAb 421. out to ensure the presence of excess antibody in all immu- noprecipitations. We next carried out immunoprecipitations of human cells RESULTS infected with Ad5SVR111, a recombinant adenovirus which Construction of recombinant viruses and analysis of SVT- expresses SVT-Ag, or clone 6 rat cells, which express Ag and mouse p53 expression. cDNAs encoding SVT-Ag and elevated levels of mouse p53, in order to compare the murine p53 were cloned into the transplacement plasmid mammalian-derived proteins with the same proteins synthe- pEV55 as described in Materials and Methods. The resultant sized in infected insect cells. Figure 2E demonstrates that plasmids are depicted in Fig. 1. In pEV55SVT (Fig. 1A), the SVT-Ag synthesized in insect cells is precisely the same size junction of polyhedrin and SVT-Ag leader sequences occurs as SVT-Ag produced in AdSSVR111 infected human cells. at position -29 relative to the SVT-Ag ATG, while in Similarly, the insect-derived p53 displays an identical mobil- pEVSSpS3 (Fig. 1B), the polyhedrin-p53 fusion is at position ity to murine p53 made in the cloned 6 cells. Note that in -105 relative to the p53 ATG. The transplacement plasmid certain experiments the insect-derived SVT-Ag is observed pEV55 provides the entire polyhedrin promoter-leader re- as a doublet. This is also seen with Ad5SVR111-infected gion to drive gene expression. The A of the BglII site in human cells and may reflect postlysis degradation which has pEV55 corresponds to the A of the polyhedrin ATG in wt been frequently observed for various SVT-Ag preparations AcMNPV. pEV55 is therefore expected to provide higher (58). From the Coomassie blue-stained gel of the samples levels of expression than vectors such as pAc373 or pEV51 shown in Fig. 2E, we estimated that the recombinant bacu- which lack portions of the leader region (31, 54). The loviruses produced from 2- to 10-fold more SVT-Ag and p53 recombinant viruses vEVSSSVT and vEVSSpS3 were con- per cell than their mammalian counterparts. On the basis of structed by cotransfection of wt AcMNPV DNA with a comparison with Coomassie blue-stained protein stan- pEVSSSVT and pEVSSpS3 respectively, to allow allelic dards, we estimate that SVT-Ag accumulated to approxi- replacement of the wt polyhedrin sequences with the cloned mately 25 to 50 ,ug/107 cells, whereas p53 levels of 60 to 150 gene. Recombinant viruses were selected by screening for an ,ug/107 cells are obtained (data not shown). occlusion-negative phenotype since the polyhedrin gene is Subcellular localization of SVT-Ag and p53 in insect cells. A no longer present. series of immunofluorescence experiments were carried out The expression of SVT-Ag and p53 by these viruses was to examine the subcellular localization of SVT-Ag or p53 in examined by SDS-PAGE analysis of extracts derived from insect cells at various times after infection with vEV55SVT SF21 cells infected with wt or recombinant viruses for or vEV55p53, respectively. In the data presented in Fig. 3, various times. The extensive accumulation of polyhedrin SVT-Ag expression is initially observed at 24 h p.i., whereas after infection with wt virus can be clearly seen in the low levels of p53 are detectable at 12 h after infection. The Coomassie blue-stained gel presented in Fig. 2A. After more extensive accumulation of p53 is in agreement with the infection with vEV55pS3, high levels of a 53-kilodalton (kDa) expression studies described above. At 12 h p.i. when protein accumulate by 36 and 48 h p.i. This protein is not expression levels are lower, both proteins are predominantly present in wt-infected or in mock-infected cells. Similarly, a found in the nucleus. Later in infection, due to the high level novel 94-kDa protein accumulates by 36 and 48 h after expression of these proteins and to the fact that the nucleus infection with vEVSSSVT, although the steady-state levels swells considerably after baculovirus infection, it is difficult of this protein are somewhat lower than those of the 53-kDa to establish whether there are significant amounts of these protein. proteins in the cytoplasm as well. The patterns of accumu- 3112 O'REILLY AND MILLER J. VIROL.

I:4b?S- 4e' 3EfI.-RICQUI. i4

t-, j.J , ......

Ir-, w- ., %,:

*1

> .,i

& ,; $z. .,

.. ^ < -. b .....

i #. " t _W,*WS ..,. -. .. El ..... %, E Z .... ,=\ __,,,1 ""_ii_W.:8!-s.t"h;' 9 , ti- X _ *. Z s ffi S - _ S | #s _ _ Si ..>wds i X F-."...... __....:...... > o ..:."" } 4F '* F-F .-- £.i. .,s: ,., .,,.|u...... *:: * ! *....:: S -E 1t*- .:...... S.F i.;

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s w tF :^

FIG. 2. Protein synthesis in recombinant and wt AcMNPV-infected insect cells. SF21 cells were infected with vEV55SVT, vEV55p53, or wt AcMNPV at an MOI of 20. At 12, 24, 36, and 48 h p.i., cells were pulse labeled with [35S]methionine for 1 h and then lysed (50 ,ul/35-mm dish). (A and B) A 20- and 3-,ul sample, respectively, of each lysate was analyzed directly by SDS-PAGE. A Coomassie blue-stained gel is shown in panel A, and an autoradiograph is shown in panel B. (C) A 20-,ul sample of each vEV55SVT lysate and 7 RI1 of each vEV55p53 lysate were immunoprecipitated with PAb 419 and PAb 421, respectively, before SDS-PAGE. The same respective volumes of wt-infected and mock-infected.*(mi)::*cellsggwere.:.ganalyzed....g<:as controls.. The Coomassie blue-stained gel is shown. (D) The same as panel C except that 10 ,u1 of the vEV55SVT lysates and 3 ,u1 of each vEV55p53 lysate were used for the immunoprecipitation experiments. The gel was visualized by autoradiography. (E) *Immunoprecipitates.:;0p..::of vEV55SVT- or vEV55p53-infected lysates (48 h p.i.) were analyzed in parallel with immunoprecipitates oflysates of Ad5SVR111-infected 293 cells or clone 6 rat cells. In each case, the volume of lysate used for the mammalian samples represents three times more cells than the quantity used for the insect cell samples. Mock-infected (mi) insect cells were included as negative controls. An autoradiograph is shown. The molecular size markers (M) are given in kilodaltons, and the positions of SVT-Ag (T), p53, and polyhedrin (PH) are indicated.

lation observed were unchanged after coinfection of SF21 recognize them. In this experiment, infected SF21 cells were cells with both viruses (data not shown). pulse labeled with [35S]methionine 48 h p.i. and lysed either Epitope analysis of insect-derived SVT-Ag and p53. The immediately or after a 3-h chase. The lysates were then data obtained in the experiments described above indicate immunoprecipitated with the appropriate antibodies and that the insect-derived SVT-Ag and p53 each display at least analyzed by SDS-PAGE. The antibodies tested for SVT-Ag one epitope known to be found on the corresponding wt were PAb 416 and PAb 419, which recognize distinct epi- proteins. To investigate further the similarity of these recom- topes toward the N terminus of SVT-Ag (15), and PAb 100, binant proteins to their mammalian counterparts, we exam- which recognizes a determinant present in the center of a ined the ability of several other monoclonal antibodies to subset of SVT-Ag molecules (13, 14, 50). The latter epitope, VOL. 62, 1988 BACULOVIRUS-DERIVED SVT-Ag AND p53 3113 vEV55SVT vEV55p53

mi

12

I 24

36

419 421 FIG. 3. Immunofluorescence localization of SVT-Ag and p53 in insect cells. SF21 cells on glass coverslips were infected with vEV55SVT or vEV55p53 at an MOI of 10. Infections were allowed to proceed for 12, 24, or 36 h before the cells were fixed and processed for immunofluorescence analysis. The primary antibodies used were PAb 419 or PAb 421. Mock-infected cells (mi) were processed in parallel as controls. The fixed and stained cells were visualized by either UV (columns 1 and 3) or Nomarski (columns 2 and 4) illumination.

which has been associated with the DNA-binding properties The antibodies used to analyze p53 were PAb 242, PAb of SVT-Ag, is dependent on the molecule assuming an 246, PAb 248, and PAb 421 (15, 63). The former three are appropriate tertiary structure, because antibody recognition apparently specific for mouse p53 while the PAb 421 epitope is destroyed by denaturation. The baculovirus-derived SVT- is more highly conserved and is found on p53 molecules of Ag displays all three epitopes, as shown in Fig. 4A. several species (62). The PAb 242, PAb 246, and PAb 248 3114 O'REILLY AND MILLER J. VIROL.

A B 4}6 479 242 246 248 421 --Ps 1QO

.: .: :.

*: ::: ::: Yi...... :: ...... :.

.. .: ...... - __ _ __ p53 :. :: __b .:......

.. ::: .. :.:: :. *...;0..B!iS.:S-''...... *:.:.::.....::::.::...... Si.::: ...... :: mS P C MiC m Dr mi PC mi C mi P mi P C FIG. 4. Epitope analysis of insect-derived SVT-Ag (T) and p53. SF21 cells were infected with vEVSSSVT (A) or vEVSSpS3 (B) at an MOI of 20. At 36 h p.i., the cells were labeled with [35S]methionine for 1 h. The cells were either lysed immediately (lanes P) or incubated in complete TC-100 containing an excess of cold methionine for 3 h before lysis (lanes C). Then, 10 Il of each lysate was immunoprecipitated with PAb 416, PAb 419, or PAb 100 (panel A) or with PAb 242, PAb 246, PAb 248, or PAb 421 (panel B). Mock-infected (mi) cell lysates were immunoprecipitated with each antibody as a control (lanes mi). Immunoprecipitated proteins were analyzed by SDS-PAGE, and the autoradiographs obtained are shown. epitopes are localized toward the N terminus of the molecule species can be discerned (fractions 4 to 6 and fractions 14 to ( 1, 3, and 4, respectively), whereas the PAb 421 17), although p53 appears to sediment even more heteroge- epitope is at the extreme C terminus of p53 (62). PAb 246 is neously than SVT-Ag. of further interest since it has been reported that the deter- After coinfection of SF21 cells with vEV55SVT and minant recognized by this antibody is stabilized by complex vEV55p53, immunoprecipitation of the gradient fractions formation with SVT-Ag. However, it can be seen from Fig. with the anti-SVT-Ag antibody shows that much of the 4B that the baculovirus-produced p53 displays all four epi- SVT-Ag now exists as heavy oligomeric forms which sedi- topes, regardless of the presence of SVT-Ag. For both ment to the bottom of the gradient (Fig. 5C, fractions 13 to SVT-Ag and p53, no significant difference was observed in 17). p53 is now found to be coprecipitated by the anti-SVT- the ability of any antibody to recognize these proteins after Ag antibody from the heavier fractions. These data indicate either a pulse or a pulse-chase labeling (compare lanes P and that complex formation has taken place between SVT-Ag C). This result indicates that all epitopes examined are and p53, and that the complexed forms of these proteins present on both recently synthesized and older molecules. cosediment through the sucrose gradients, as seen in SV40- Complex formation between insect-derived SVT-Ag and infected rodent cells (28). p53. One of the most characteristic properties of SVT-Ag Conversely, when the same gradient fractions are immu- and p53 in mammalian systems is the ability to associate noprecipitated with the anti-p53 antibody (Fig. 5D), p53 is together to form a tight complex (reviewed in reference 37). found to sediment throughout the gradient and complexed To determine whether this association takes place in insect SVT-Ag, which is coprecipitated with pS3 by the anti-p53 cells, we examined the sedimentation profiles of SVT-Ag antibody, cosediments toward the bottom of the gradient. and p53 through linear sucrose gradients. 35S-labeled lysates Stability of baculovirus-produced SVT-Ag and p53. The were prepared 48 h after infection of SF21 cells with results described above demonstrate that baculovirus-pro- vEV55SVT and vEV55p53 either individually or together, duced SVT-Ag and pS3 are capable of associating together to and the lysates were centrifuged through sucrose gradients. form a high-molecular-weight complex in insect cells. One of After centrifugation, the gradients were fractionated and the consequences attributed to complex formation in mam- immunoprecipitated with PAb 419 (anti-SVT-Ag) or PAb 421 malian cells is the stabilization of p53, which is otherwise (anti-p53). Figure 5A shows the sedimentation profile of turned over very rapidly (38). We undertook a series of SVT-Ag extracted from SF21 cells infected with vEVSSSVT pulse-chase experiments to determine the stability of SVT- alone. While the protein sediments rather heterogeneously, Ag and p53 in SF21 cells both with and without complex two major species can be distinguished (fractions 3 to 5 and formation. SF21 cells were infected with vEVSSSVT and fractions 8 to 10). This is similar to the sedimentation profile vEVSSpS3 either separately or together. At 36 h p.i., the observed for SVT-Ag extracted from SV40-infected monkey cells were pulse labeled with [35S]methionine for 30 min and cells in which it has been proposed that the slower-sedi- then chased with an excess of cold methionine for selected menting form represents monomers and dimers, while the times. The autoradiograph presented in Fig. 6 shows that faster-sedimenting form corresponds to tetramers and higher both SVT-Ag and pS3 are highly stable in insect cells, with oligomeric forms (8). Thus, these data suggest that SVT-Ag no significant turnover observed even after a 25-h chase. No synthesized in insect cells is also capable of forming a significant differences were observed when the SF21 cells variety of oligomeric forms. were coinfected with both viruses. The fact that coprecipi- The sedimentation profile of p53 extracted from SF21 cells tation of SVT-Ag and p53 was observed even after the infected with vEV55p53 alone indicates that this protein also 30-min pulse suggests that complex formation takes place exists in a variety of oligomeric forms (Fig. 5B). Again, two rapidly in this system. VOL. 62, 1988 BACULOVIRUS-DERIVED SVT-Ag AND p53 3115

A B vEV55SVT vEV55p53 U 171615 14 1312 1110 9 8 7 6 5 4 3 2 l U 17161514131211 10 9 8 7 6 5 4 3 2 1

*lo - W -, A - ..w.ww..MPM--o ES~nhlIPsE-W' - . p53

419 421

C D vEV55SVT + vEV55p53 vEV55SVT + vEV55p53 U 171615141312 1 109 8 7 6 5 4 3 2 1 U 17 16 15 1453 4112 10 9 8 7 6 5 4 3 2 1

UINI--KIMUe'M-1--ShAmkm-

-w .Am. p53- _ .:::::C . 4mwwdw..l---- -p53

419 421 FIG. 5. Sucrose gradient analysis of insect-derived SVT-Ag (T) and p53. SF21 cells were infected with vEV55SVT (MOI, 50) (A), vEV55p53 (MOI, 10) (B), or coinfected with both viruses (MOIs, 50 and 10, respectively) (C and D) for 36 h. They were then pulse labeled with [35S]methionine for 1 h before lysis. Lysates were centrifuged through linear 5 to 20% sucrose gradients, and the fractions were immunoprecipitated with PAb 419 or PAb 421. Immunoprecipitates were analyzed by SDS-PAGE and autoradiographed as described in the legend to Fig. 4. Fractions are numbered 1 through 17 from the top of the gradient. Lane U, An aliquot of the lysate not subjected to gradient centrifugation and then immunoprecipitated in parallel.

vEV55SVT vEV55SVT vEV55SVT vEV55p53 vEV55p53 vEV55p53

P 1 2.5 5 10 25 P 1 2.5 5 10 25 P 1 2.5 5 10 25 P 1 2.5 5 10 25

x -T

_ -p53

.,1 \.. / 419 421 FIG. 6. Stability of SVT-Ag (T) and p53 in insect cells. SF21 cells were pulse labeled with [35S]methionineb for 30 min after infection for 36 h with vEV55SVT, vEVSSpS3, or both (MOIs as in the legend to Fig. 5). Labeled cells were either lysed immediately after the pulse (lanes P) or incubated in TC-100 with excess cold methionine for 1, 2.5, 5, 10, or 25 h. Lysates were then immunoprecipitated and analyzed as before. 3116 O'REILLY AND MILLER J. VIROL.

A B vEV55SVT vEV55p53 wt vEV55SVT vEV55p53 wt 12 6r4- 2 12 24 36 48 12 24 36 48 12 24 3648 mi M 12 24 36i4812 2436 48}2 243648 i 205- l 116- 97.4- 97.4- -T -T .- - 66- 66) - - -p53 45- -p53

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C D VEVS5SVT vEV55p53 vEV55SVTt vEV55p53 vEV55SVT vEV55p53 vEV55SVT vEV55p53 ml 12 24 36 48 mi 12 24 36 48 12 24 36 48 12 24 36 48 12 24 3648 12 24 36 48 12 24 36 48 12 24 36 48 205- 116- : :: :: 97.4- 97.4- :#.F.:7..w:**@.."a ..K.'.-''... '= '" - T -T

66- 66- -p53 45- -p53 45- :...... :S 29- 29- i .:... _."...... _... --::o 419 421 419 421 419 421 419 421 FIG. 7. Phosphorylation of SVT-Ag and p53 in insect cells. SF21 cells were infected with vEV55SVT (MOI, 20), vEV55p53 (MOI, 20), or wt virus (MOI, 20), or coinfected with vEV55SVT and vEV55p53 (MOIs, 50 and 10, respectively). At 12, 24, 36, or 48 h p.i., the cells were labeled with 32p, for 1 h before lysis. Samples of the lysates were either analyzed directly by SDS-PAGE (panels A and B) or were immunoprecipitated with PAb 419 or PAb 421 as described in Materials and Methods (panels C and D). (A and C) Coomassie blue-stained gels; (B and D) Autoradiographs. The sizes of the molecular markers are given in kilodaltons (lane M), and the positions of SVT-Ag (T), p53, and polyhedrin (PH) are indicated. mi, Mock infected.

Phosphorylation of SVT-Ag and p53 in insect cells. Another SVT-Ag and p53 are phosphorylated in this system. In notable characteristic of SVT-Ag and p53 is that they are vEV55p53-infected cells, the major phosphoprotein present both phosphorylated in mammalian cells. We investigated in the infected cell late in infection is p53. Immunoprecipi- whether the proteins produced by our expression system tation before SDS-PAGE confirmed the identities of the were phosphorylated by labeling SF21 cells with 32Pi at phosphoproteins (Fig. 7D). Comparison of the autoradio- selected times after infection with vEV55SVT, vEV55p53,or graphs (Fig. 7B and D) with the Coomassie blue-stained gels wt virus. The labeled lysates were then analyzed by SDS- (Fig. 7A and C) suggests that the degree of phosphorylation PAGE; autoradiography (Fig. 7B) shows clearly that both is maximal at 24 h p.i., dropping two- to fivefold by 48 h after VOL. 62, 1988 BACULOVIRUS-DERIVED SVT-Ag AND p53 3117 infection. Analysis of SVT-Ag and p53 extracted from SF21 p53 (63). However, it is also difficult to rule out the possi- cells coinfected with both vEV55SVT and vEV55p53 reveals bility that there is some dissociation of the complex no major differences in the degree of phosphorylation of postlysis. either of these proteins after complex formation (Fig. 7C and We notice that the sedimentation profile of p53 does not D). change significantly in the presence or absence of SVT-Ag. Since the sedimentation profile of uncomplexed p53 in DISCUSSION mammalian cells has not been reported, we do not know whether this is a general phenomenon. In this study, we describe the construction and analysis of Another interesting observation concerning complex for- recombinant baculovirus vectors expressing SVT-Ag and mation in insect cells is that a pulse-chase analysis (Fig. 6) mouse p53. The vectors produce levels of these proteins revealed that complex formation is very rapid in these cells ranging from 50 to 150 ,ug/107 cells. These levels are excel- and that association is complete in less than 30 min. This is lent compared with those of the presently available mamma- in contrast to the situation in mammalian cells in which lian gene expression systems. Carroll and Gurney (2) reported that although p53 is rapidly Recently, Jeang et al. (18) described the construction of a incorporated into the complex, SVT-Ag enters the complex recombinant baculovirus containing the coding sequences more slowly, requiring from 3 to 6 h for maximum incorpo- for SV40 large T and small t . However, this vector ration. Those authors proposed that this phenomenon indi- included SV40 early-gene splice sites, and the authors report cates that newly made SVT-Ag requires some posttransla- that only small t antigen is synthesized in significant tional modification before it can complex p53. More amounts. There is little or no detectable accumulation of recently, Schmeig and Simmons (51) have postulated that large T antigen. The fact that we have readily obtained the the kinetics of complex formation depends on the ratio of expression of large T antigen from a cDNA clone further SVT-Ag to p53 in the cell line studied and that competition demonstrates the importance of using intronless genes with between newly synthesized and complexed SVT-Ag is the the baculovirus expression system, as discussed previously major determinant of the rate of entry of SVT-Ag into the (32). complex. The rapid rate of complex formation observed here Several lines of evidence indicate that the baculovirus- would tend to support the latter hypothesis; in our system derived proteins are similar to their mammalian counter- there is ample p53 which should allow prompt entry of newly parts. The proteins are of normal size (Fig. 2E) and are synthesized SVT-Ag into the complex. transported to the nucleus (Fig. 3), indicating that the The pulse-chase experiments also revealed that both SVT- nuclear transport signals of both SVT-Ag and p53 are Ag and p53 are highly stable in insect cells. This is in striking recognized in insect cells. Both proteins also appear to adopt contrast to what is observed in mammalian cells in which, in a native conformation since they both display several epi- the absence of SVT-Ag, p53 is highly unstable (38). Although topes characteristic of the wt proteins (Fig. 4). Interestingly, we are unsure of the basis for this enhanced stability of p53 the baculovirus-derived p53 is clearly recognized by the in insect cells, it should be noted that in this system, p53 monoclonal antibody PAb 246, even in the absence of expression takes place late in infection, when the virus has SVT-Ag (Fig. 4B). Yewdell et al. (63) reported that this already largely shut down host cell protein synthesis and epitope is generally unstable in the absence of SVT-Ag. disrupted host cell metabolism. However, those authors do report that the PAb 246 epitope Further evidence that the baculovirus system can express is present on p53 from at least one spontaneously trans- authentic mammalian proteins was provided by the observa- formed mouse cell line in the absence of SVT-Ag. The basis tion that both SVT-Ag and p53 are phosphorylated in this of this phenomenon is not known. system. The data presented in Fig. 7 suggest that phosphor- Sucrose gradient centrifugation analyses indicated that ylation is maximal at 24 h p.i. and declines thereafter. It will baculovirus-derived SVT-Ag and p53 are capable of self- now be important to determine the type(s) and site(s) of the associating to form a variety of oligomeric forms (Fig. 5). At phosphorylation events involved, since both the nature and least for SVT-Ag, this property is important because there is degree of phosphorylation appear to be critical for the much evidence showing that different oligomeric forms of correct functioning of SVT-Ag and p53. Recent evidence the protein display different posttranslational modifications suggests that phosphorylation of residues down regu- and biological functions in mammalian cells (reviewed in lates the ability of SVT-Ag to support viral replication (11, reference 43). 35). This may or may not be mediated by decreased origin- Our experiments also show that baculovirus-produced binding activity (11, 35, 52). It seems also that the appear- SVT-Ag and p53 can associate together in insect cells to ance of higher oligomeric forms of SVT-Ag is coincident form a rapidly sedimenting high-molecular-weight complex. with greater phosphorylation of the protein as it ages (8). The precise role played by complex formation in mammalian Furthermore, Samad et al. (45) report that at least a compo- cells is not yet clear but it may have profound effects on the nent of p53 phosphorylation is dependent on SVT-Ag. We ability of SVT-Ag to support viral DNA replication and believe that these proteins therefore provide a valuable immortalize or transform cells (1, 9, 30; for a review, see model system to establish whether insect cells can phosphor- reference 37). It is therefore of interest to observe that this ylation proteins in a manner qualitatively and quantitatively property is retained by the proteins in insect cells. Our similar to mammalian cells. results suggest that some SVT-Ag and p53 remain uncom- In summary, we have successfully used the baculovirus plexed in this system since the total amount of SVT-Ag expression system to direct the efficient synthesis of SVT-Ag immunoprecipitated from these cells (Fig. SC) is greater than and murine p53 in insect cells. These proteins are identical to the amount coprecipitated with p53 (Fig. SD). The converse the corresponding mammalian products by all criteria exam- is also true. Since p53 appears to be in excess of SVT-Ag in ined, and the levels of expression obtained compare favor- coinfected cells, there is likely to be a certain amount of free ably with those of mammalian expression systems presently p53 in this system. In addition, there is evidence to suggest available. Recently obtained evidence indicates that baculo- that only a subpopulation of SVT-Ag is capable of binding virus-derived SVT-Ag is functional in an in vitro SV40- 3118 O'REILLY AND MILLER J. VIROL. origin-dependent replication system (C. Prives, personal 14. Gurney, E. G., S. Tamowski, and W. Deppert. 1986. Antigenic communication). These facts, coupled with the ease of use binding sites of monoclonal antibodies specific for simian virus and inherent safety of the baculovirus system (32), should 40 large T antigen. J. Virol. 57:1168-1172. make these vectors a convenient source of SVT-Ag and p53 15. Harlow, E., L. V. Crawford, D. C. Pim, and N. M. Williamson. 1981. Monoclonal antibodies specific for simian virus 40 tumor for in vitro biochemical studies. We anticipate that the antigens. J. Virol. 39:861-869. further analysis of these recombinant proteins and in partic- 16. Hu, S.-L., S. G. Kosowski, and K. F. Schaaf. 1987. Expression ular a more detailed characterization of their state of phos- of envelope glycoproteins of human immunodeficiency virus by phorylation will allow us to better evaluate the potential and an insect virus vector. J. Virol. 61:3617-3620. limitations of the baculovirus expression system. 17. Jeang, K.-T., C.-Z. Giam, M. Nerenberg, and G. Khoury. 1987. Abundant synthesis of functional human T-cell leukemia virus ACKNOWLEDGMENTS type I p40x protein in eucaryotic cells by using a baculovirus expression vector. J. Virol. 61:708-713. We thank Carol Prives for critical reading of the manuscript, for 18. Jeang, K.-T., M. Holmgren-Konig, and G. Khoury. 1987. A providing lysates of clone 6- and Ad5SVR111-infected cells, and for baculovirus vector can express intron-containing genes. J. Vi- pSVT#5 and monoclonal antibodies PAb 416, PAb 419, PAb 421, rol. 61:1761-1764. and PAb 100. We are grateful to Marcus Fechheimer for help with 19. Jenkins, J., K. Rudge, P. Chumakov, and G. Currie. 1985. The the immunofluorescence experiments. We also thank Evelyne May cellular oncogene p53 can be activated by mutagenesis. Nature and Jean-Claude Erhart for providing pSV53c and monoclonal (London) 317:816-818. antibodies PAb 242, PAb 246, and PAb 248. 20. Jenkins, J., K. Rudge, and G. Currie. 1984. Cellular immortal- This work was supported in part by Public Health Service grant ization by a cDNA clone encoding the transformation-associ- A123719 from the National Institute of Allergy and Infectious ated phosphoprotein p53. Nature (London) 312:651-654. Diseases. 21. Kaczmarek, L., M. Oren, and R. Baserga. 1986. Cooperation between the p53 protein tumor antigen and platelet-poor plasma LITERATURE CITED in the induction of cellular DNA synthesis. Exp. Cell Res. 162: 1. Braithwaite, A. W., H.-W. Sturzbecher, C. Addison, C. Palmer, 268-272. K. Rudge, and J. R. Jenkins. 1987. Mouse p53 inhibits SV40 22. Kuroda, K., C. Hauser, R. Rott, H.-D. Klenk, and W. 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