Expression and complex formation of simian virus 40 large T antigen and mouse p53 in insect cells.

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JOURNAL OF VIROLOGY, Sept. 1988,p. 3109-3119 0022-538X/88/093109-11$02.00/0

Expression and Complex Formation of Simian Virus

40 Large T

Antigen and Mouse p53 in Insect Cells

DAVID,R. O'REILLY ANDLOIS K. MILLER*

Departments ofEntomology and Genetics, UniversityofGeorgia, Athens, Georgia30602 Received1March1988/Accepted 16May 1988

Recombinantbaculoviruses wereconstructedwhich expresssimian virus40 largeT antigen (SVT-Ag)or

murinep53tohigh levels in infected insect cells. Characterization of the expressed proteins revealed that they displaymanyproperties of the corresponding mammalian-derived proteins. Both proteinsareof wild-type size,

localize to the nucleus, are recognized by several SVT-Ag- orp53-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 proteincanself-associatetoforma varietyof oligomeric species. Pulse-chase experiments

indicated that both SVT-Ag and p53arehighly stableininsectcells, evenin the absenceof complex formation.

A variety ofgene expression systems have been devel-oped recently withaviewtoachieving enhancedexpression of eucaryotic proteins normally present at extremely low levels(44). A recently developedvectorsystemwith consid-erable potential employs the baculovirus Autographa cali-fornica nuclear polyhedrosis virus (AcMNPV) to mediate expression of the clonedgenein insectcell cultures(25, 31, 32). In this system, the gene is inserted in place of the AcMNPV polyhedrin gene, which is nonessential for viral replication in cell culture. Expression of the cloned geneis then underthe control of thepolyhedrinpromoter,resulting inhigh levels of expression late in infection.

To date, a wide range ofgenes has been expressed by using the baculovirussystemand inmanycases,biologically

activp

proteins have been obtained (32a; reviewedin refer-ence 25). Characterization of the expressed proteins has shown that insect cells can carry out at least some of the posttranslational modifications which occur in mammalian cells. Signal sequence cleavage has been demonstrated for human alpha- and beta-interferon and interleukin-2 and -3 (27, 33, 54, 55), while the appropriateproteolytic cleavage of human immunodeficiency virus env and gag proteins and

influenza virus hemagglutinin is observed (16, 22, 26, 40). Polyomavirus large T antigen and the Drosophila Kruppel protein display DNA-binding activity after synthesis in in-sect cells (36, 42). Oligomerization, complex formation, or both has beenobserved forbaculovirus-expressed rotavirus majorcapsid antigen (7) andtwoinfluenzaviruspolymerase complex proteins (56).

It isnot yet clear whether allposttranslational modifica-tions will be comparable in mammalian and insect cells. Glycosylation is one modification which appears to differ betweenmammalian andinsectcells, but the significance of this difference is not yet known and several biologically active glycoproteins have already been produced by using the baculovirus system. One of the posttranslational modi-ficationsofkey importance isphosphorylation, because this modification has been found to play a critical role in the

regulation of protein function in a variety ofsystems. It is known that the Drosophila Kruppel protein, c-myc, and

humanT-cell leukemia virustypeIp40" arephosphorylated

after expression in insect cells, but noinformation is avail-*Correspondingauthor.

ableconcerning the natureorsite(s) of the phosphorylation

events involved (17, 34, 36). Baculovirus-derived human

epidermal growth factor receptor displays an autophos-phorylation activity like that of the wild-type protein (12).

We chose to study the expression of simian virus 40 (SV40) large T antigen(SVT-Ag) and murine p53, because prior characterization of the numerousfunctions and post-translational modifications associated with these proteins has been extensive. SVT-Ag displays ATPase activity, DNA-binding activity, and helicase activity (4, 5, 43, 57). Theprotein is essential for viral DNA replication (59) and mayassociate with thehost cell DNApolymerase alpha (53). It can immortalize and transform primary and established celllines, and these propertiesmaybe relatedtoitsabilityto bind and stabilize the host cellprotein p53 (30, 38,reviewed in reference37). p53 itself is animmortalizingoncogene (6, 20, 39) and isimplicatedinthepassageof cellsfrom

Go

toG1 (21). Both SVT-Ag and p53 are phosphoproteins, and the

nature and sites ofphosphorylation are largely determined

(29, 45-49, 60). SVT-Ag isaparticularly good modeltostudy phosphorylation since there is muchrecentevidence linking phosphorylationtothe regulation ofanumberof its biolog-ical activities, especially viral DNA replication and DNA binding (11, 35, 48).

Inthisstudy, we describe theexpression of SVT-Ag and

p53 by using the baculovirus expression system.The abun-dantly expressed proteins are localized in the insect cell

nucleus and adopt astructuralconformation similarto that oftheir mammalian counterparts. Both proteins are phos-phorylated and retain the ability to associate together after expression in insectcells.

MATERIALS AND METHODS

Cells and viruses. Spodoptera frugiperda(fall armyworm) IPLB-SF21 cells (SF21 cells)(61)weremaintained in TC-100 medium(GIBCO Laboratories) supplemented with10%fetal calf serum and 0.25% tryptose broth. Stocks ofwild-type (wt) virus, AcMNPV L-1 (24),wereprepared and assayedas

describedpreviously (31). Extractsof clone 6ratcells (30), which express elevated levels of mouse p53, and AdS

SVR111-infected human 293 cells (10) containing SVT-Ag were provided by C. Prives.

Construction ofrecombinant viruses. To generate

recom-binant AcMNPVs expressing SVT-Ag and mouse p53, 3109

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3110 O'REILLY AND MILLER

A

Bam HI EcoRI

1781 CIan B

3 ~~KpnI

ST-Ag

5190 pEV55SVT

(BgIU/StuI) 9.9 kb

p

Polyhedrin5'leoder SVT-Ag5leader

*(Bgl/StuD) I0

AAACCTATAAATAG ATC C CTAG G CTTTT

1t

(BgIll/Kpn

Pst I

Eco RI

XhoI pEV55p53 to

BgIll3 7.8kb

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

Polyhedrin5'leoder p53 5 leader

-0 BgIll Eco RI -AAACCTATAAATAGATCTCGAGAATTCCATCCTGG

XhoI

_-29y -105

FIG. 1. Structure of the transplacement plasmids pEV55SVTand pEV55p53 (notto scale). (A) InpEV55SVT, SV40 sequences from pSVT#5, including the cDNA copyof the SVT-Ag gene, areindicated by the hatched box. The arrow indicates the expected SVT-Ag transcript. SV40 nucleotide numbersaregiven. (B) ForpEV55p53,thestippled box represents thep53cDNAfrompSV53c.Again, thep53 transcript is indicated. In both plasmids, AcMNPV sequences which flank the polyhedrin genearepresented as openboxes(P5'and P3'). The pUC8 sequences are indicated byathin line. Selected restriction endonuclease sitesareshown.Sitesinparenthesesarethose present in the originalfragments whichweredestroyedduring the construction. ThesequenceofthepEV55-cDNAjunction is given below eachplasmid. Ineach case,the position of the fusion site relative to theAUG of the cloned gene is indicated.

cDNA copies of these genes were cloned first into the transplacementplasmidpEV55. Thestructureofthis vector hasbeen described previously(31,32).The cDNAencoding SVT-Agwasexcised from the plasmidpSVT#5(constructed byY.Gluzman) by digestion withStuI(nucleotide5191) and EcoRI (nucleotide 1780), yieldingafragment whichextends from 29 base pairs upstream of the ATG of SVT-Ag to approximately a kilobase downstream ofthe translational termination codon. This fragment was cloned into pEV55 whichhadbeen previously digestedwithBglII (filledinwith T4DNApolymerase) andEcoRI. Therecombinant plasmid obtained,pEV55SVT, is illustrated in Fig. 1A.

To construct pEV55p53, a cDNA encoding murine p53 wasexcisedfromtheplasmidpSV53c(19)by digestionwith EcoRI andBglII (filled inwith T4 DNApolymerase). The resultant 1.33-kilobasefragment includes105basepairs of5' flanking sequence and extends 20 base pairs beyond the termination codon. Itwasinserted into pEV55thathad been previously digested with KpnI (bluntendedwithin T4 DNA polymerase) and EcoRI. The structure of the resultant plasmid, pEV55p53, is depicted inFig. 1B.

Toconfirm the structureofthepEV55-cDNAjunctionin both pEV55SVT andpEV55p53, the DNA sequence span-ning the junction was determined by double-stranded se-quencing of plasmid DNA, essentiallyasdescribedbyChen andSeeburg(3). The primerusedwas a17-mer correspond-ing to residues -26 to -42 in the polyhedrin leader se-quence.TheplasmidspEV55SVT andpEV55p53 containthe SVT-AG and p53 genes downstream from the AcMNPV polyhedrin promoter and flanked by polyhedrin 5' and 3' flanking sequences (Fig. 1).

Recombinant virusesexpressing these genes were gener-atedby replacement of thewtpolyhedringenein AcMNPV L-1 with the promoter-gene fusions from

pEV55SVT

and pEV55p53. To this end, 2 x

106

SF21 cells were cotrans-fected with 2 ,ug of viral DNA (isolated as described in reference 31) and 18 ,ug of eitherpEVS5SVTorpEV55p53, according to the procedure of Potter and Miller (41). At 5 days later, progeny virus were harvested and then re-plaqued. Plaquesgenerated byrecombinantviruswere iden-tified by visual screening for an occlusion-negative

pheno-type (31). The recombinant viruseswere subjectedtothree roundsofplaquepurification before large-scale virus stocks wereprepared. Viral DNA wasisolated, and the structures of the resultant viruses, vEV55SVT and vEV55p53, were verified by restriction enzyme analysis and Southern blot-ting.

Analysisof proteins synthesizedininfected cells.SF21 cells (106/35-mmPetridish)wereinfected withwtorrecombinant viruses at amultiplicity ofinfection (MOI) offrom 10 to50 (see figure legends). At the appropriate times postinfection (p.i.), the mediumwas removed and replaced with TC-100 lacking either methionine or phosphate. The cells were labeled 1 h later with 50,uCi of[35S]methionineor100,uCiof 32p; (New England Nuclear) in 0.5 ml of methionine- or phosphate-deficient medium. The lengths of the labeling periods are indicated in the individual figure legends. In certainexperiments,the [35S]methionine pulse labelingwas chased by incubation of the cells in TC-100 containing an excessofunlabeledmethionine. Thecellswererinsedthree times in cold phosphate-buffered saline (PBS; 8 mM Na2HPO4, 137 mMNaCl,0.5 mMMgCl2, 1.6 mMKH2PO4, 2.7 mMKCl [pH 8.0])andincubated in50 ,ul oflysisbuffer (1%NonidetP-40,150 mMNaCl,50 mMTrishydrochloride [pH 8.0]) containing 1 mM phenylmethylsulfonyl fluoride (Fluka), 40 ,uM pepstatin (Fluka), and 20 ,uM leupeptin (Fluka) for 30 min at 4°C. Lysates were stored at -80°C. Totalproteinspresentwerevisualizedbyelectrophoresisof portionsofthelysates through 10% sodiumdodecyl sulfate-polyacrylamide gels (SDS-PAGE; 23). Alternatively, sam-ples were immunoprecipitated with various antibodies (in tissue culturefluid)directedagainstSVT-Agorp53 (detailed in the text). Trial immunoprecipitation experiments were conducted to ensure the presence ofexcess

antibody.

Im-munoprecipitation experiments were carried out in NET buffer(140mMNaCl,5mMEDTA,0.05%NonidetP-40,50 mMTrishydrochloride [pH 8.0])containing 1mgofbovine serumalbumin perml,at4°Cfor 3to4h. Antigen-antibody complexes were collected by adsorptionto fixed Staphylo-coccus aureus (SigmaChemicalCo.) for 1h at4°C. Immu-noprecipitates were washed three times in NET buffer,

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BACULOVIRUS-DERIVED SVT-Ag AND p53 3111 eluted by being boiled for5min inthegel-loadingbuffer (23),

andanalyzed by SDS-PAGE.

Immunofluorescence studies. SF21 cells

(105)

were seeded onto glass coverslips (22 by 22 mm) and infected with the recombinantviruses at an MOI of 10. At the selected times p.i.,the cells were washed three times in cold PBS and fixed in 70% acetone-30% methanol at -20°C for 10 min. The fixativewas removed, and thecoverslips wereair driedand stored at -20°C. Before being stained, coverslips were incubated inPBS at room temperaturefor15min. ThePBS was removed by aspiration, and 50 ,ul of the appropriate dilution (determined empirically) of monoclonal antibody was placed on the coverslip. After incubation at 37°Cfor1 h in a moist environment, the coverslips were washed twice for 15 mineach in PBS at room temperature. The second antibody used was a fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulin antiserum (Sigma), and incubation conditions were as described above. After two further washes withPBS,thecoverslipsweremountedonto microscope slides and examined by using Nomarski orUV optics.

Sucrose gradient centrifugation. SF21 cells were infected with the recombinant viruses individually or together and lysedasdescribed above. Lysates(2 x 106 infected cellsper gradient) wereloadedonto5-ml lineargradients of5 to 20% sucroseinPBS andcentrifugedat55,000 x gfor16 hat

4°C.

Equal volume fractions (250 ,ul) were collected and immu-noprecipitated with PAb 419 orPAb 421.

RESULTS

Construction of recombinant viruses and analysisof SVT-Ag and mouse p53expression. cDNAsencoding SVT-Agand murine

p53

were cloned into the

transplacement

plasmid

pEV55asdescribed inMaterials andMethods.The resultant plasmidsaredepictedinFig. 1. In

pEV55SVT

(Fig. 1A),

the junction ofpolyhedrinandSVT-Agleader sequencesoccurs at position -29 relative to the

SVT-Ag

ATG,

while in

pEVSSpS3

(Fig. 1B),the

polyhedrin-p53

fusion isat

position

-105 relative tothe p53 ATG. The transplacement

plasmid

pEV55 provides the entire

polyhedrin promoter-leader

re-gion to drive gene

expression.

The A of the

BglII

site in pEV55 corresponds to the A of the

polyhedrin

ATG in wt AcMNPV. pEV55 is therefore

expected

to

provide higher

levels of

expression

than vectors such as

pAc373

or

pEV51

which lack portions of the leader

region

(31, 54). The recombinant viruses vEVSSSVT and

vEVSSpS3

were con-structed by cotransfection of wt AcMNPV DNA with pEVSSSVT and pEVSSpS3

respectively,

to allow allelic replacement ofthe wtpolyhedrinsequenceswiththecloned gene.Recombinant viruseswereselected

by

screening

foran

occlusion-negative phenotype

since the

polyhedrin

gene is nolongerpresent.

Theexpression of

SVT-Ag

and

p53 by

these viruses was

examinedby SDS-PAGE

analysis

ofextracts derived from SF21 cells infected with wt or recombinant viruses for various times. The extensive accumulation of

polyhedrin

after infection with wt virus can be clearly seen in the Coomassie blue-stained

gel

presented in

Fig.

2A. After infection with

vEV55pS3,

highlevelsofa53-kilodalton

(kDa)

protein accumulate by 36 and 48 h

p.i.

This

protein

is not present in wt-infectedorinmock-infectedcells.

Similarly,

a novel 94-kDa protein accumulates by 36 and 48 h after infectionwith

vEVSSSVT,

although the steady-state levels of thisproteinaresomewhat lower than those of the 53-kDa

protein.

The kinetics of

synthesis

of these

proteins

wereexamined

by

SDS-PAGE of

[35S]methionine pulse-labeled

proteins

of infected cells

(Fig.

2B).

Synthesis

of boththe 94- and53-kDa

proteins

is detectable at24h

p.i.

andincreases

through

48 h after infection. In both cases, the kinetics of

synthesis

are

likethose of

polyhedrin,

andasimilarinhibition ofhost cell

protein

synthesis

is observedatlate times

p.i.

Toconfirmthatthe 94- and 53-kDa

proteins correspond

to

SVT-Ag

and

p53, respectively, immunoprecipitation

exper-imentswere

performed

withmonoclonal antibodies

specific

forthese

proteins.

Itcanbe seenin

Fig.

2C and D that the monoclonal

antibody

PAb

419,

whichis

specific

for

SVT-Ag

(15),

recognizes

the 94-kDa

polypeptide expressed

in vEVSSSVT-infected SF21 cells.

Similarly,

PAb

421,

an

anti-p53 antibody

(15),

specifically

immunoprecipitates

the 53-kDa

protein

from

vEV55-infected

cells

(Fig.

2C and

D).

Again,

nosuch

proteins

are

immunoprecipitated

fromwt-or

mock-infected

lysates.

Note that

polyhedrin

precipitates

spontaneously

in these

experiments

because it is

quite

insoluble in the

lysis

buffer used. As

before,

synthesis

of both

proteins

is detectable from 24 h after infection and lower levels of

SVT-Ag

areobserved than of

p53.

In these and certain

subsequent

experiments,

the amount of

sample

usedfrom

vEVSSpS3-infected

cellswasreduced becauseof the more efficient

expression

of this vector

(see

figure

legends).

Trial titration

experiments

were

previously

carried

outto ensure the presence ofexcess

antibody

inall

immu-noprecipitations.

Wenext carriedout

immunoprecipitations

ofhumancells infected with

Ad5SVR111,

arecombinant adenovirus which expresses

SVT-Ag,

or clone 6 rat

cells,

which express elevated levels of mouse

p53,

in order to compare the mammalian-derived

proteins

withthesame

proteins

synthe-sized in infected insect cells.

Figure

2E demonstratesthat

SVT-Ag

synthesized

in insect cells is

precisely

thesamesize

as

SVT-Ag

produced

inAdSSVR111 infected humancells.

Similarly,

theinsect-derived

p53

displays

anidentical

mobil-ity

to murine

p53

made in the cloned 6 cells. Note that in certain

experiments

theinsect-derived

SVT-Ag

is observed

as a doublet. This is also seen with

Ad5SVR111-infected

human cellsand mayreflect

postlysis degradation

which has been

frequently

observed for various

SVT-Ag

preparations

(58).

From the Coomassie blue-stained

gel

ofthe

samples

shownin

Fig. 2E,

we estimated thatthe recombinant bacu-loviruses

produced

from2-to10-fold more

SVT-Ag

and

p53

percellthantheir mammaliancounterparts. Onthebasis of

a

comparison

with Coomassie blue-stained

protein

stan-dards,

we estimate that

SVT-Ag

accumulated to

approxi-mately

25 to50

,ug/107 cells,

whereas

p53

levelsof60to150 ,ug/107cells are obtained(datanotshown).

Subcellular localization of

SVT-Ag

and

p53

ininsect cells. A series of immunofluorescence

experiments

were carriedout to examinethesubcellular localizationof

SVT-Ag

or

p53

in insectcellsatvarious times afterinfection with

vEV55SVT

or

vEV55p53,

respectively.

Inthe data

presented

in

Fig.

3,

SVT-Ag

expression

is

initially

observedat24 h

p.i.,

whereas low levels of

p53

are detectableat12 hafter infection. The moreextensiveaccumulation of

p53

is inagreementwith the

expression

studies described above. At 12 h

p.i.

when

expression

levelsare

lower,

both

proteins

are

predominantly

found in the nucleus. Later in

infection,

duetothe

high

level

expression

of these

proteins

andtothefact that the nucleus swells

considerably

afterbaculovirus

infection,

it is difficult

to establish whether there are

significant

amounts of these

proteins

in the

cytoplasm

as well. Thepatterns of accumu-VOL.62, 1988

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ti-FIG. 2. Proteinsynthesis in recombinantand wtAcMNPV-infected insect cells. SF21 cells were infected with vEV55SVT,vEV55p53, or wtAcMNPV at an MOIof 20. At 12, 24, 36, and 48 h p.i., cells were pulse labeled with[35S]methioninefor1h and thenlysed (50,ul/35-mm dish). (A and B)A20-and 3-,ul sample, respectively, of each lysate was analyzed directly by SDS-PAGE.ACoomassieblue-stained gel is shown inpanel A, andanautoradiograph is shown inpanelB.(C)A20-,ulsample of eachvEV55SVTlysate and 7

RI1

of eachvEV55p53lysate were immunoprecipitated with PAb 419 and PAb 421, respectively, before SDS-PAGE. The same respective volumes of wt-infected and mock-infected (mi) cells were analyzedascontrols. TheCoomassie blue-stained gel is shown. (D) The same as panel C except that 10,u1of thevEV55SVT lysates and 3

,u1

of eachvEV55p53 lysate were used for theimmunoprecipitation experiments. The gel was visualized by autoradiography. (E) Immunoprecipitates of vEV55SVT- or vEV55p53-infected lysates (48 h p.i.) were analyzed in parallel with immunoprecipitates of lysates ofAd5SVR111-infected293 cells or clone6 ratcells.Ineach case, the volume oflysate used for the mammalian samples representsthreetimes more cellsthan thequantity used for the insect cell samples. Mock-infected (mi) insect cells were included asnegative controls.Anautoradiographisshown. Themolecular sizemarkers (M)aregiven in kilodaltons, and the positions of SVT-Ag (T), p53,andpolyhedrin (PH) areindicated.

lation observed were unchanged after coinfection of SF21 cells with both viruses (data not shown).

Epitope analysis of insect-derived SVT-Ag and p53. The data obtained in the experiments described above indicate that theinsect-derived SVT-Ag andp53eachdisplay at least one epitope known to be found on the corresponding wt proteins.Toinvestigate further thesimilarity of these recom-binantproteinstotheirmammalian counterparts, we exam-ined the ability of several other monoclonal antibodies to

recognizethem. In thisexperiment, infected SF21 cellswere pulselabeled with [35S]methionine48 hp.i.andlysedeither immediately or after a 3-h chase. The lysates were then immunoprecipitated with the appropriate antibodies and analyzed bySDS-PAGE. Theantibodies tested forSVT-Ag were PAb 416 and PAb 419, which recognize distinct epi-topestoward the N terminus ofSVT-Ag (15), andPAb 100, which recognizes a determinant present in the center ofa subsetofSVT-Agmolecules(13, 14, 50).The latterepitope, J. VIROL.

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BACULOVIRUS-DERIVED SVT-Ag AND p53 3113

vEV55SVT

vEV55p53

I

[image:5.612.56.564.71.619.2]

419

421

FIG. 3. Immunofluorescence localization of SVT-Ag and p53 in insect cells. SF21 cellsonglass coverslipswereinfected 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

immunofluorescenceanalysis. The primary antibodies usedwerePAb 419orPAb 421.Mock-infected cells (mi)wereprocessed in parallelas

controls. Thefixed and stained cellswere visualized by either UV (columns 1 and 3)orNomarski(columns 2 and 4) illumination.

which has been associated with theDNA-binding properties of SVT-Ag, is dependent on the molecule assuming an

appropriate tertiary structure,becauseantibody recognition isdestroyed by denaturation. The baculovirus-derived SVT-Ag displays all three epitopes, as shown inFig. 4A.

The antibodies used to analyze p53 were PAb 242, PAb 246, PAb 248, and PAb 421 (15, 63). The former three are

apparently specificformousep53while the PAb 421epitope is morehighlyconserved and is found onp53 molecules of several species (62). The PAb 242, PAb 246, and PAb 248

mi

12

24

36

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A

4}6

479 1QO

--Ps

.:.::.

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B

242 246 248 421

__

__b

_ __ -p53

mS P C MiC m Dr mi PC mi C mi P mi P C

FIG. 4. Epitope analysis ofinsect-derivedSVT-Ag(T)andp53.SF21cellswereinfectedwithvEVSSSVT(A) orvEVSSpS3(B) at anMOI of 20. At36 hp.i., the cellswere labeled with [35S]methioninefor 1 h.The cells wereeitherlysedimmediately (lanes P) orincubatedin completeTC-100 containinganexcessof cold methionine for3 hbeforelysis(lanesC). Then,10 Il of eachlysatewasimmunoprecipitated

withPAb 416, PAb 419, or PAb 100 (panel A) orwithPAb242, PAb 246, PAb 248, or PAb 421 (panel B).Mock-infected(mi)cell lysateswere

immunoprecipitated with each antibody as a control (lanes mi). Immunoprecipitated proteins were analyzed by SDS-PAGE, and the autoradiographs obtainedareshown.

epitopesarelocalizedtoward the N terminusofthemolecule (exons 1, 3, and 4, respectively), whereas the PAb 421 epitope isatthe extremeCterminus ofp53 (62). PAb 246 is of furtherinterest since it has been reported that the deter-minant recognizedbythisantibodyisstabilizedbycomplex formation with SVT-Ag. However, itcanbe seenfrom Fig. 4B thatthebaculovirus-produced p53 displays all four epi-topes, regardless of the presence of SVT-Ag. For both SVT-Agand p53, no significant difference was observed in theability ofanyantibodytorecognize these proteinsafter eitherapulseor apulse-chaselabeling (compare lanes P and C). This result indicates that all epitopes examined are present onboth recentlysynthesized andoldermolecules.

Complex formation between insect-derived SVT-Ag and p53. One of the most characteristic properties of SVT-Ag and p53 in mammalian systems is the ability to associate togethertoformatightcomplex(reviewedinreference37). Todetermine whetherthis associationtakes place in insect cells, we examined the sedimentation profiles of SVT-Ag andp53 through linear sucrosegradients. 35S-labeled lysates were prepared 48 h after infection of SF21 cells with vEV55SVT and vEV55p53 either individually or together, and thelysates werecentrifuged through sucrosegradients. After centrifugation, the gradients were fractionated and immunoprecipitated withPAb 419(anti-SVT-Ag)orPAb 421 (anti-p53). Figure 5A shows the sedimentation profile of SVT-Agextracted from SF21 cells infected withvEVSSSVT alone. While the protein sediments ratherheterogeneously, twomajor species can be distinguished (fractions 3 to 5 and fractions8 to10).This is similar to the sedimentation profile observedforSVT-Agextractedfrom SV40-infectedmonkey cells in which it has been proposed that the slower-sedi-menting form represents monomers and dimers, while the faster-sedimentingform corresponds to tetramers and higher oligomericforms (8). Thus, these data suggest that SVT-Ag synthesized in insect cells is also capable of forming a varietyofoligomeric forms.

Thesedimentationprofile of p53 extracted from SF21 cells infectedwith vEV55p53 aloneindicates that this protein also exists inavariety of oligomericforms (Fig. 5B). Again, two

species can be discerned(fractions 4 to 6 and fractions 14 to 17), although p53 appearsto sediment even more heteroge-neously than SVT-Ag.

After coinfection of SF21 cells with vEV55SVT and vEV55p53, immunoprecipitation of the gradient fractions with the anti-SVT-Ag antibody shows that much of the SVT-Agnow exists as heavy oligomeric forms which sedi-ment tothebottom ofthegradient(Fig. 5C, fractions 13to 17). p53 is nowfound tobecoprecipitated by the anti-SVT-Agantibody from the heavier fractions. These data indicate that complex formation has taken place between SVT-Ag and p53, and that the complexed forms of these proteins cosediment through the sucrosegradients,as seen in SV40-infected rodent cells (28).

Conversely, when the same gradient fractions are immu-noprecipitated with the anti-p53 antibody (Fig. 5D), p53 is found to sediment throughout the gradient and complexed SVT-Ag, which is coprecipitated with pS3 by the anti-p53 antibody, cosediments toward the bottom of the gradient.

Stability of baculovirus-produced SVT-Ag and p53. The results described above demonstrate that baculovirus-pro-ducedSVT-Ag and pS3arecapable ofassociatingtogether to formahigh-molecular-weightcomplex in insect cells. One of the consequencesattributed tocomplex formation in mam-malian cells is the stabilization ofp53, which is otherwise turned over very rapidly (38). We undertook a series of pulse-chase experiments to determine the stability of SVT-Ag and p53 in SF21 cells both with and without complex formation. SF21 cells were infected with vEVSSSVT and vEVSSpS3 either separately or together. At 36 h p.i., the cellswerepulselabeled with[35S]methioninefor 30 min and thenchased with an excessof cold methionine for selected times. The autoradiograph presented in Fig. 6 shows that bothSVT-Agand pS3are highly stable ininsectcells, with nosignificantturnoverobservedevenaftera25-h chase. No significant differences were observed when the SF21 cells werecoinfected with both viruses. The fact that coprecipi-tation of SVT-Ag and p53 was observed even after the 30-min pulse suggests that complex formation takes place rapidly in this system.

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A

vEV55SVT

U 171615 14 1312 1110 9 8 7 6 5 4 3 2 l

B

vEV55p53

U 17161514131211 109 8 7 6 5 4 3 2 1

*lo - W -,A

-..w.ww..MPM--o

ES~nhlIPsE-W'

.

419

-

p53

421

vEV55SVT+ vEV55p53

U 171615141312 1 109 8 7 6 5 4 3 2 1

UINI--KIMUe'M

-1--ShAmkm

-D

vEV55SVT+ vEV55p53

U 17 16 15 1453 4112 10 9 8 7 6 5 4 3 2 1

p53- _ .:::::C-w.

419

-p53

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),orcoinfected with both viruses(MOIs,50 and10, respectively) (CandD)for 36 h.Theywerethenpulselabeled

with [35S]methionine for 1 h before lysis. Lysates were centrifuged through linear 5 to 20% sucrose gradients, and the fractions were

immunoprecipitatedwith PAb419orPAb 421. ImmunoprecipitateswereanalyzedbySDS-PAGEandautoradiographedasdescribedinthe

legendtoFig.4.Fractionsarenumbered1through17 from thetopof thegradient.LaneU,Analiquotof thelysatenotsubjectedtogradient

centrifugationandthenimmunoprecipitated inparallel.

vEV55SVT P 1 2.5 5 10 25

vEV55SVT vEV55p53

P 1 2.5 5 10 25

vEV55p53 P 1 2.5 5 10 25

vEV55SVT

vEV55p53

P 1 2.5 5 10 25

x

b

-T

_

-p53

.,1 \.. /

419 421

FIG. 6. StabilityofSVT-Ag(T)and p53 in insect cells. SF21 cellswerepulselabeled with[35S]methioninefor 30minafter infection for 36hwithvEV55SVT, vEVSSpS3,orboth(MOIsasin thelegendtoFig. 5).Labeledcellswereeitherlysedimmediatelyafterthepulse(lanes

P)orincubated inTC-100 withexcesscold methionine for1,2.5, 5, 10,or25 h.Lysateswerethenimmunoprecipitatedandanalyzedasbefore.

C

4mwwdw..l---- .Am.

VOL. 62,1988

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[image:7.612.56.564.75.357.2] [image:7.612.137.476.482.700.2]

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A

B

vEV55SVT

vEV55p53

wt

12 24 36 48 12 24 36 4812 24 3648 mi

-T

vEV55SVT vEV55p53 wt 12 6r4- 2

M 12 2436i4812243648}2 2436 48

205-

116- 97.4-66)

-

45-

29- 97.4-

66--p53

e sete: t; i .iP

1 ....'

Z7ii3IF

C

VEVS5SVT

vEV55p53 vEV55SVTt

vEV55p53

ml 12 24 3648mi 12 24 36 481224 36 4812 24 3648

45-

29-D

vEV55SVT 12 24 3648

i

l

.

-_L.

-:

-T

-p53

vEV55p53 vEV55SVT vEV55p53

12 24 36 48 12 2436 4812 2436 48

116-97.4-

:...:S

::: ::

:#.F.:7..w:**@.."a

i

..K.'.-''...'= '" - T

.:...

66-

45--p53

205- 97.4- 66-

45-

29-_."..._ ...--::o

419 421 419 421 419 421 419 421

FIG. 7. Phosphorylation of SVT-Ag and p53 in insect cells. SF21 cellswereinfected withvEV55SVT(MOI, 20),vEV55p53(MOI, 20),

or wtvirus(MOI, 20),orcoinfectedwithvEV55SVT andvEV55p53(MOIs,50and10,respectively). At12, 24, 36,or48hp.i.,thecellswere

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 CandD). (AandC)Coomassieblue-stained

gels;(B and D)Autoradiographs.Thesizesof the molecular markersaregivenin kilodaltons(lane M),and thepositionsofSVT-Ag(T),p53, and polyhedrin (PH)areindicated. mi,Mock infected.

-T

-p53

PhosphorylationofSVT-Agandp53ininsect cells.Another notable characteristic ofSVT-Ag and p53 is that they are both phosphorylated in mammalian cells. We investigated whether the proteins produced by our expression system were phosphorylated by labeling SF21 cells with

32Pi

at selected times after infection withvEV55SVT, vEV55p53,or wt virus. The labeled lysates were then analyzed by SDS-PAGE; autoradiography (Fig. 7B) shows clearly that both

SVT-Ag and p53 are phosphorylated in this system. In vEV55p53-infected cells, themajorphosphoprotein present in the infected cell late in infection is p53. Immunoprecipi-tation before SDS-PAGE confirmed the identities of the phosphoproteins (Fig. 7D). Comparison of the autoradio-graphs(Fig. 7B andD) with theCoomassieblue-stainedgels (Fig. 7A andC) suggests that thedegreeofphosphorylation is maximalat24hp.i.,droppingtwo- tofivefoldby48h after J. VIROL.

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infection.

Analysis

of

SVT-Ag

and

p53

extracted from SF21 cellscoinfected with both

vEV55SVT

and

vEV55p53

reveals no

major

differences in the

degree

of

phosphorylation

of either ofthese

proteins

after

complex

formation(Fig.7C and

D).

DISCUSSION

Inthis

study,

wedescribetheconstructionand

analysis

of recombinant baculovirus vectors

expressing SVT-Ag

and

mouse

p53.

The vectors

produce

levels of these

proteins

ranging

from 50to 150

,ug/107

cells. These levelsare excel-lent

compared

with those ofthe

presently

available mamma-liangene

expression

systems.

Recently, Jeang

etal.

(18)

describedtheconstructionofa recombinant baculovirus

containing

the

coding

sequences for

SV40

large

T and smallt

antigens.

However,

thisvector includedSV40

early-gene

splice

sites,

andtheauthorsreport that

only

small t

antigen

is

synthesized

in

significant

amounts. There is little or no detectable accumulation of

large

T

antigen.

The fact thatwe have

readily

obtained the

expression

of

large

T

antigen

from a cDNA clone further demonstrates the

importance

of

using

intronlessgenes with the baculovirus

expression

system, asdiscussed

previously

(32).

Several lines of evidence indicate that the baculovirus-derived

proteins

are similar to their mammalian counter-parts. The

proteins

are of normal size

(Fig. 2E)

and are

transported

to the nucleus

(Fig. 3), indicating

that the nuclear transport

signals

of both

SVT-Ag

and

p53

are

recognized

ininsect cells. Both

proteins

also appearto

adopt

a native conformation since

they

both

display

several

epi-topescharacteristic ofthewt

proteins (Fig. 4). Interestingly,

the baculovirus-derived

p53

is

clearly recognized by

the monoclonal

antibody

PAb

246,

even in the absence of

SVT-Ag (Fig.

4B).

Yewdell et al.

(63) reported

that this

epitope

is

generally

unstable in the absence of

SVT-Ag.

However,

those authors do report that the PAb 246

epitope

is present on

p53

from at least one

spontaneously

trans-formedmousecelllinein theabsence of

SVT-Ag.

Thebasis ofthis

phenomenon

isnotknown.

Sucrose

gradient centrifugation

analyses

indicated that baculovirus-derived

SVT-Ag

and

p53

are

capable

of

self-associating

toforma

variety

of

oligomeric

forms

(Fig. 5).

At least for

SVT-Ag,

thispropertyis

important

because there is much evidence

showing

that different

oligomeric

forms of the

protein display

different

posttranslational

modifications and

biological

functions in mammalian cells

(reviewed

in reference

43).

Our

experiments

also show that

baculovirus-produced

SVT-Ag

and

p53

can associate

together

in insect cells to forma

rapidly sedimenting high-molecular-weight

complex.

The

precise

role

played by complex

formationin mammalian cellsisnotyet clear but it may have

profound

effectsonthe

ability

of

SVT-Ag

to support viral DNA

replication

and immortalize or transform cells

(1, 9, 30;

for a

review,

see reference

37).

It istherefore of interesttoobserve thatthis property is retained

by

the

proteins

in insect cells. Our results suggest that some

SVT-Ag

and

p53

remain

uncom-plexed

in this system since the total amount of

SVT-Ag

immunoprecipitated

from these cells

(Fig.

SC)

is greater than

theamount

coprecipitated

with

p53 (Fig. SD).

The converse

is alsotrue.Since

p53

appears tobe inexcessof

SVT-Ag

in coinfected

cells,

thereis

likely

tobeacertainamountof free

p53

inthis system. In

addition,

thereis evidenceto suggest that

only

a

subpopulation

of

SVT-Ag

is

capable

of

binding

p53 (63). However, it is also difficult torule out the

possi-bility that there is some dissociation of the complex postlysis.

We notice that the sedimentation profile of p53does not change significantly in the presence orabsence ofSVT-Ag. Since the sedimentation profile of uncomplexed p53 in mammalian cells has not been reported, we do not know whether this is ageneral phenomenon.

Another interesting observationconcerning complex for-mation in insect cells is that a pulse-chase analysis (Fig. 6) revealed thatcomplex formation is very rapid in these cells and that association is complete in less than 30min. This is in contrast to the situation in mammalian cells in which Carroll andGurney (2) reported that although p53 israpidly incorporatedinto thecomplex, SVT-Ag enters the complex moreslowly, requiring from 3 to6 h for maximum incorpo-ration. Those authors proposed that this phenomenon indi-cates that newly made SVT-Ag requires some posttransla-tional modification before it can complex p53. More recently, Schmeig and Simmons (51) have postulated that the kinetics ofcomplex formation depends on the ratio of SVT-Agtop53 in thecell line studied and that competition between newly synthesized and complexed SVT-Ag is the major determinant of the rate of entry ofSVT-Ag into the complex. The rapidrateofcomplex formation observed here would tend to support the latter hypothesis; in our system there isample p53which should allow prompt entry ofnewly synthesizedSVT-Ag into the complex.

Thepulse-chase experimentsalso revealed that both SVT-Agandp53arehighlystable ininsect cells. This is in striking contrasttowhat is observed in mammalian cells inwhich, in theabsence ofSVT-Ag, p53ishighlyunstable(38). Although we areunsureofthebasis for thisenhanced stability ofp53 in insect cells, it should be noted that in this system, p53 expressiontakesplace late ininfection, when the virus has already largely shut down host cell protein synthesis and disrupted hostcell metabolism.

Further evidence that thebaculovirus system can express authentic mammalianproteinswasprovidedby the observa-tion that both SVT-Ag and p53 are phosphorylated in this system.ThedatapresentedinFig. 7 suggestthat phosphor-ylation is maximalat24hp.i.anddeclinesthereafter. It will now beimportanttodetermine thetype(s)andsite(s) ofthe

phosphorylation

events

involved,

since boththe natureand degree of phosphorylation appear to be critical for the correct functioning of SVT-Ag and p53. Recent evidence suggeststhat

phosphorylation

of serineresidues down regu-latesthe

ability

of

SVT-Ag

to supportviral replication (11, 35). This may ormaynot be mediatedby decreased

origin-binding

activity (11, 35, 52). It seems also that the appear-ance of higher oligomeric forms of SVT-Ag is coincident with greater phosphorylation ofthe protein as it ages (8). Furthermore, Samadetal. (45)reportthatatleasta compo-nent ofp53 phosphorylation is dependent on SVT-Ag. We believe that these proteins therefore provide a valuable model systemtoestablish whether insect cellscan

phosphor-ylation proteins

in a mannerqualitatively and quantitatively similartomammalian cells.

In summary, we have successfully used the baculovirus expressionsystemtodirect theefficientsynthesisofSVT-Ag andmurinep53ininsectcells. Theseproteinsareidenticalto thecorrespondingmammalianproducts byallcriteria exam-ined, and the levels ofexpression obtained compare favor-ablywith those of mammalianexpression systemspresently available. Recentlyobtained evidence indicates that baculo-virus-derived SVT-Ag is functional in an in vitro

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origin-dependent replication system (C. Prives, personal communication). These facts, coupled with the ease ofuse and inherent safety of the baculovirus system (32), should makethese vectors aconvenient source ofSVT-Agandp53 for in vitro biochemical studies. We anticipate that the furtheranalysisof these recombinantproteins and in partic-ular a more detailed characterization of their state of phos-phorylationwillallow us to better evaluate thepotential and limitations of thebaculovirus expression system.

ACKNOWLEDGMENTS

We thankCarol Prives for criticalreadingofthemanuscript,for providing lysatesof clone6-andAd5SVR111-infectedcells, and for pSVT#5and monoclonal antibodies PAb 416, PAb419, PAb421,

andPAb 100. We aregratefultoMarcusFechheimer forhelp with

theimmunofluorescenceexperiments. WealsothankEvelyne May and Jean-Claude Erhart for providing pSV53c and monoclonal antibodies PAb242, PAb 246, and PAb 248.

This workwas supported inpartby Public Health Service grant A123719 from the National Institute of Allergy and Infectious Diseases.

LITERATURE CITED

1. Braithwaite,A.W.,H.-W.Sturzbecher,C.Addison,C.Palmer, K. Rudge, and J. R. Jenkins. 1987. Mouse p53 inhibits SV40 origin-dependent DNA replication. Nature (London) 329:458-460.

2. Carroll, R., and E. G. Gurney. 1982. Time-dependent

matura-tionof the simian virus40largeTantigen-p53 complexstudied by usingmonoclonal antibodies.J. Virol. 44:565-573.

3. Chen,E.Y., andP. H. Seeburg. 1985. Supercoil sequencing:a

fast andsimple method forsequencing plasmidDNA. DNA 4: 165-170.

4. Clark, R.,M.Tevethia,and R.Tjian.1984.TheATPaseactivity ofSV40large Tantigen, p. 363-368. InG.Vande Woude, A. Levine, W. Topp, and J. Watson (ed.), Cancer cells, vol. 2. ColdSpring HarborLaboratory,ColdSpring Harbor, N.Y. 5. Dean, F. B., P. Bullock, Y. Murakami, C. R. Wobbe, L.

Weissbach,andJ.Hurwitz.1987. Simianvirus 40 DNA replica-tion: SV40largeTantigenunwindsDNAcontainingtheSV40 originofreplication. Proc. Natl. Acad. Sci. USA84:16-20. 6. Eliyahu, D., A. Raz, P. Gruss, D. Givol, and M.Oren. 1984.

Participationofp53cellulartumorantigenintransformation of normalembryoniccells. Nature(London)312:646-649. 7. Estes, M. K., S. E. Crawford, M.E. Penaranda, B. L.Petrie,

J. W. Burns,W.-K. Chan,B.Ericson,G. E.Smith, and M. D. Summers. 1987. Synthesisandimmunogenicityof therotavirus major capsid antigen usingabaculovirusexpressionsystem. J. Virol.61:1488-1494.

8. Fanning, E., B. Nowak, and C. Burger. 1981. Detection and characterization ofmultiple forms of simian virus 40 large T

antigen.J. Virol. 37:92-102.

9. Gannon,J.V.,and D. P. Lane.1987.p53and DNApolymerase alphacompeteforbindingtoSV40Tantigen.Nature(London) 329:456-458.

10. Gluzman, Y., H. Reichl, and D. Solnick. 1982. Helper-free

adenovirus type 5 vectors, p. 187-192. In Y. Gluzman, (ed.),

Eucaryoticviralvectors.ColdSpringHarborLaboratory,Cold Spring Harbor,N.Y.

11. Grasser, F. A., K. Mann, and G. Walter. 1987. Removal of serinephosphatesfrom simian virus40largeTantigenincreases itsabilitytostimulateDNAreplicationinvitro buthasnoeffect

onATPaseorDNAbinding.J. Virol. 61:3373-3380.

12. Greenfield, C., G.Patel,S. Clark, N.Jones,andM. D.

Water-field. 1988. Expressionof thehumanEGFreceptor withligand

stimulatable kinase activity in insect cellsusing abaculovirus

vector.EMBO J. 7:139-146.

13. Gurney, E. G., R. Harrison, andJ. Fenno. 1980. Monoclonal antibodies against simian virus 40 T antigens: evidence for distinctsubclasses oflargeTantigenandforsimilaritiesamong non-viralTantigens. J. Virol.34:752-763.

14. Gurney,E.G.,S.Tamowski,and W. Deppert.1986. Antigenic bindingsites ofmonoclonal antibodies specificforsimian virus 40largeTantigen.J. Virol.57:1168-1172.

15. Harlow, E.,L. V.Crawford,D. C.Pim,and N. M. Williamson.

1981. Monoclonal antibodiesspecificfor simian virus 40tumor

antigens.J. Virol. 39:861-869.

16. Hu,S.-L.,S.G.Kosowski, and K.F.Schaaf. 1987.Expression ofenvelopeglycoproteinsof humanimmunodeficiencyvirusby

aninsect virus vector. J. Virol.61:3617-3620.

17. Jeang, K.-T., C.-Z. Giam, M. Nerenberg, and G. Khoury. 1987.

Abundant synthesisof functional human T-cell leukemia virus

type I p40x protein ineucaryotic cells by usingabaculovirus expressionvector.J. Virol. 61:708-713.

18. Jeang, K.-T., M. Holmgren-Konig, and G. Khoury. 1987. A

baculovirusvector can expressintron-containing genes.J.

Vi-rol. 61:1761-1764.

19. Jenkins,J.,K.Rudge,P.Chumakov,andG. Currie. 1985.The

cellularoncogene p53can beactivatedby mutagenesis.Nature

(London) 317:816-818.

20. Jenkins, J., K. Rudge, and G. Currie. 1984. Cellular

immortal-ization by acDNA clone encoding the

transformation-associ-ated phosphoproteinp53. Nature(London)312:651-654.

21. Kaczmarek, L., M. Oren, and R. Baserga. 1986. Cooperation

between the p53 proteintumorantigen and platelet-poorplasma

in theinduction of cellular DNAsynthesis. Exp. Cell Res. 162:

268-272.

22. Kuroda, K., C. Hauser, R. Rott, H.-D. Klenk, and W. Doerfler.

1986.Expressionof the influenza virushaemagglutininin insect

cells byabaculovirusvectors. EMBOJ. 5:1359-1365.

23. Laemmli, U. K. 1970. Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature (London)

227:680-685.

24. Lee, H. H., and L. K. Miller. 1978. Isolation of genotypic

variants ofAutographa californica nuclear polyhedrosisvirus.

J. Virol. 27:754-767.

25. Lucknow, V. A., and M. D. Summers. 1988. Trends in the

development of baculovirus expression vectors. Bio-Tech-nology 6:47-55.

26. Madisen, L., B. Travis, S.-L. Hu, and A. F. Purchio. 1987.

Expression of the humanimmunodeficiency virus gag gene in

insect cells. Virology158:248-250.

27. Maeda,S.,T.Kawai,M.Obinata,H.Fujiwara,T.Horiuchi, Y. Saeki, Y. Sato, and M. Furusawa. 1985. Production of human alpha-interferoninsilkwormusingabaculovirusvector.Nature (London) 315:592-594.

28. McCormick, F., and E. Harlow. 1980. Association of a murine

53,000-dalton phosphoprotein with simian virus 40 large T antigenintransformed cells. J.Virol. 34:213-224.

29. Meek, D. W., and W. Eckhart. 1988.Phosphorylationof p53in

normal and simian virus 40-transformed NIH 3T3 cells. Mol.

Cell. Biol. 8:461-465.

30. Michalovitz, D.,D.Eliyahu,and M.Oren. 1986. Overproduction

ofprotein p53 contributes to simian virus 40-mediated

transfor-mation. Mol. Cell. Biol. 6:3531-3536.

31. Miller, D. W., P. Safer, and L. K. Miller. 1986. An insect

baculovirus host-vector system for high level expression of

foreigngenes, p. 277-298. In J. K. Setlow and A. Hollander, (ed.), Genetic engineering, vol. 8. Plenum Publishing Corp.,

NewYork.

32. Miller,L. K.1987.Baculoviruses forforeigngeneexpressionin

insectcells,p.457-465.InR.Rodriguez,and D.Denhardt(ed.),

Vectors:asurveyofmolecularcloningvectorsandtheiruses.

Butterworth's, Stoneham, Mass.

32a.Miller, L. K. 1988. Baculovirusesas geneexpression vectors.

Annu. Rev. Microbiol.42:177-179.

33. Miyajima,A., J.Schreurs,K.Otsu,A.Kondo, K.-I.Arai,andS.

Maeda.1987. Useof thesilkworm,Bombyxmori, andaninsect

baculovirus vectorfor high-level expression and secretion of

biologicallyactivemouse interleukin-3.Gene 58:273-281. 34. Miyamoto, C., G. E. Smith, J. Farrell-Towt, R. Chizzonite,

M.D.Summers,and G.Ju. 1985. Productionof human c-myc protein in insect cells infected with abaculovirus expression

vector. Mol. Cell. Biol.5:2860-2865.

J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

(11)

35. Mohr, I. J., B. Stillman,and Y. Gluzman. 1987. Regulation of

SV40DNAreplication by phosphorylation ofTantigen.EMBO J.6:153-160.

36. Ollo, R., and T. Maniatis. 1987. Drosophila Kruppel gene

product produced in a baculovirus expression system is a nuclearphosphoprotein that binds DNA. Proc.Natl.Acad. Sci.

USA84:5700-5704.

37. O'Reilly,D. R.1986.p53andtransformationbySV40. Biol.Cell 57:187-196.

38. Oren, M., W. Maltzman, and A. J. Levine. 1981.

Post-transla-tional regulation of the54Kcellulartumorantigen innormaland transformed cells. Mol. Cell. Biol. 1:101-110.

39. Parada, L.,H.Land, R. Weinberg, D.Wolf, and V. Rotter. 1984.

Cooperation betweengeneencoding p53tumourantigen andras in cellulartransformation. Nature (London) 312:649-651.

40. Possee, R. D. 1986. Cell-surface expression of influenza virus

haemagglutinin in insectcellsusing abaculovirusvector.Virus Res. 5:43-59.

41. Potter, K. N., and L. K. Miller. 1980. Transfection of two

inverterbrate cell lines with DNA of Autographa californica nuclearpolyhedrosis virus. J. Invertebr. Pathol.36:431-432.

42. Rice, W. C., H.E. Lorimer, C. Prives, and L. K. Miller. 1987.

Expression of polyomaviruslarge Tantigenbyusinga baculo-virusvector. J.Virol.61:1712-1716.

43. Rigby, P., and D. Lane. 1983. The structure and function of

simian virus40largeTantigen.Adv. ViralOncol.3:31-57. 44. Rodriguez,R., and D. Denhardt (ed.). 1987.Vectors:asurveyof

molecularcloningvectorsand theiruses.Butterworth's,

Stone-ham,Mass.

45. Samad, A., C. Anderson, and R. Carroll. 1986. Mapping of phosphomonoester and apparent phosphodiester bonds ofthe

oncogene product p53 from simian virus 40-transformed 3T3

cells. Proc. Natl. Acad.Sci. USA 83:897-901.

46. Scheidtmann, K. H. 1986. Phosphorylation of SV40 large T

antigen: cytoplasmic and nuclear phosphorylation sites differin their metabolic stability. Virology 150:85-95.

47. Scheidtmann,K.H., B. Echle, and G. Walter.1982.Simian virus 40largeTantigen is phosphorylatedatmultiple sites clustered

in two separateregions. J. Virol.44:116-133.

48. Scheidtmann, K. H., M. Hardung, B. Echle, and G. Walter. 1984. DNA-bindingactivity of simian virus 40 largeTantigen correlates withadistinctphosphorylation state.J. Virol. 50:1-12.

49. Scheidtmann, K. H., A. Kaiser, A. Carbone, and G. Walter. 1981. Phosphorylation of threonine in theproline-rich

carboxy-terminalregionof simianvirus40 Tantigen.J.Virol.38:59-69.

50. Scheller, A.,L.Covey,B.Barnet, and C. Prives. 1982. A small

subclass of SV40Tantigen binds to theorigin ofreplication.

Cell 29:375-383.

51. Schmeig, F.,andD. T.Simmons. 1984.Intracellular location and

kinetics ofcomplexformation between simian virus 40 Tantigen

andcellular proteinp53. J. Virol. 52:350-355.

52. Simmons,D.T.,W.Chou,and K.Rodgers. 1986. Phosphoryla-tiondownregulatestheDNA-binding activityof simianvirus40 Tantigen.J. Virol.60:888-894.

53. Smale, S. T., and R. Tjian. 1986. T-antigen-DNA polymerase

alpha complex implicatedin simian virus 40 DNAreplication.

Mol.Cell. Biol. 6:4077-4087

54. Smith,G. E.,G.Ju,B. L.Ericson, J. Moschera,H.-W. Lahm,

R. Chizzonite, and M. D. Summers. 1985. Modification and

secretion of human interleukin-2 produced ininsect cells by a

baculovirusexpression vector. Proc. Natl. Acad. Sci. USA82:

8404-8408.

55. Smith,G. E., M. D. Summers,andM.J. Fraser. 1983. Produc-tion of human beta-interferon in insect cells infected with a

baculovirusexpressionvector. Mol. Cell. Biol. 3:2156-2165.

56. St.Angelo, C., G. E. Smith, M. D. Summers,and R. M.Krug.

1987. Two of the three influenza viral polymerase proteins

expressed using baculovirus vectorsformacomplex ininsect cells. J. Virol. 61:361-365.

57. Stahl, H., P. Droge, and R. Knippers. 1986. DNA helicase

activity ofSV40 largetumorantigen. EMBOJ. 5:1939-1944.

58. Tegtmeyer, P., K. Rundell,and J.K.Collins. 1977.Modification

ofsimian virus40proteinA. J. Virol.21:647-657.

59. Tooze, J. 1981. DNA tumor viruses, p. 125-296. InJ. Tooze

(ed.), Molecularbiology oftumorviruses, 2nd ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

60. VanRoy, F., L. Fransen, and W. Fiers. 1981. Phosphorylation

patterns oftumorantigens in cells lytically infected or

trans-formedby simian virus40. J. Virol. 40:28 44.

61. Vaughn, J. L., R. H. Goodwin, G. L. Thompkins, and P. McCawley. 1977. The establishment of two cell lines from the insectSpodopterafrugiperda (Lepidoptera; Noctuidae). In Vi-tro13:213-217.

62. Wade-Evans,A., andJ. Jenkins.1985. Preciseepitope mapping of themurine transformation associated proteinp53. EMBO J. 4:699-706.

63. Yewdell,J. W., J. V.Gannon,andD. P. Lane.1986.Monoclonal antibody analysis of p53 expression in normal and transformed cells. J. Virol. 59:444 452.

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