Purification and biochemical characterization of the Epstein-Barr virus-determined nuclear antigen and an associated protein with a 53,000-dalton subunit.

0022-538X/80/09-0592/11$02.00/0 Vol. 35, No.

Purification and

Biochemical

Characterization

of the

Epstein-Barr Virus-Determined

Nuclear

Antigen

and

an

Associated

Protein

with

a

53,000-Dalton

Subunit

JANOSLUKA,'* HANSJORNVALL,2 ANDGEORGEKLEIN'

DepartmentsofTumorBiology'andChemistry ,2Karolinska Institutet, 104 01 Stockholm 60, Sweden

TheEpstein-Barrvirus-determined nuclear antigen (EBNA) was purified 700-fold toapparent

homogeneity

from Raji and Namalwa cellextracts bya

three-step

procedure

involving

heat treatment, DNA-cellulose

chromatography,

and

hydroxyapatite

chromatography.

Acid-fixed nuclear binding and complement

fixation were used to monitor antigenic specificity. Purified EBNA was also capable of

specifically

inhibiting

the

regular anticomplement

immunofluorescence

reaction for EBNA

against Raji

targetcells. The purified antigen hadamolecular weight of 170,000 to

200,000.

By

sodium

dodecyl

sulfate-polyacrylamide

gel

electrophoresis,

it

yielded

a

single

48,000-dalton (48K) monomer. An

EBNA-associated

protein

wasalso

purified

fromthesamecellextract.Ithadamolecular weight of about 200,000 and

yielded

asingle 53K protein band bysodiumdodecyl

sulfate-polyacrylamide

gel electrophoresis.

The same proteinwas alsofoundin

Epstein-Barr

virus-negative

B-cell

lymphoma

lines. The two types of protein werecharacterized

by

aminoacidcomposition and peptide mapping. The results showed that the 53K and 48K protein components have no long regions in common;this excludes thatthesmaller product arises by breakdown of the larger product. Residue distributionswere

different,

butan excessofhydrophilic residues wasfound in both

proteins,

suggestingacertain

overall

similarity

in properties. 53Kcomponents fromdifferentcelllines appearedtodiffer somewhat. Epstein-Barr virus-positive lines carry two 53K components, one of which may be a

slightly

modified 53K

product. Immunocomplexing

assayshowed that the 48K,

butnotthe

53K, protein

carries EBNAspecificity. In mixtures, the 53Kprotein

is

co-precipitated

with the48Kprotein. The data suggest thatEBNAmay

form

a

complex

with the53K

protein

within thecell.

Epstein-Barr virus

(EBV)-transformed

hu-mancells

regularly

express avirally

determined

nuclearantigen,EBNA (29). It is the only pres-ently known viral product that is

regularly

ex-pressed in

proliferating,

EBV DNA-carrying cells. All known viralgenome-positive

cells

ex-press the

antigen.

This includes virally trans-formed

lymphoblastoid

cell lines of normal ori-gin, Burkitt

lymphoma

cells in vitro andinvivo, and theepithelialtumorcells ofnasopharyngeal carcinoma.Thisfact,together with some

prelim-inary functional studies, has ledtothe sugges-tionthatEBNA maymediatethe

transforming

(immortalizing) effect of the virus, perhaps in analogy with the T-antigens of

smaller

onco-genic DNA viruses (G.

Klein,

J. Luka, and J. Zeuthen, Cold Spring Harbor Symp. Quant. Biol.,inpress).

EBNA has beendetectedby anticomplement immunofluorescence (29),

complement

fixation (14),and the acid-fixed nuclearbinding (AFNB) assay (27). It has been

partially

purified from

EBV-transformedlymphoid cells in several lab-oratories (3,18,21-23,27).

ForEBNA

purification,

ourgrouphas

previ-ously useda

four-step procedure

(21). The mo-lecular

weight

of the

antigen

was between 170,000

(170K)

and

230K,

as estimated

by

gel

filtration.EBNA isa

relatively

heat-stable pro-tein that binds to double-stranded DNA and

more

weakly

to

single-stranded

DNA.

Previ-ouslypurified productsgavetwosubunitbands by sodium dodecylsulfate

(SDS)-polyacrylam-ide gel electrophoresis. The molecular

weights

of themonomers

corresponded

to48K and53K. The sameproteinmoietieswereobtained when EBNA was isolated and

purified by

an immu-nocomplexing assay. In the latter

procedure,

EBNA-anti-EBNA immune

complexes

were separated from cell extracts

by

reacting

them withproteinA-Sepharoseand

subsequently

an-alyzing them by SDS-gel electrophoresis

(21).

In this paper, we show that the

previously

detectedmonomersrepresenttwodifferent

pro-592

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teins,only one of which carries thespecificityof thenuclearantigen. The smaller (48K) protein chainis theEBNA-specific subunit,whereasthe largerchain (53K)represents an EBNA-associ-ated protein. We also show that a similar or identical 53K protein subunit is present, but without EBNA, in

EBV-negative

human lym-phoma lines of B-cellorigin. It islikely that this protein isanalogoustothesupposedly transfor-mation-related protein composed of subunitsin the same size class (53K to 55K) that was re-cently detected in simian virus 40-, polyoma virus-, and adenovirus-transformed cells and even in anumber ofchemically and spontane-ouslytransformed cell systems (7, 17, 20).

Inaddition tothe purification procedure for theproteins with 48K and 53K components,we alsoreportcertainimmunological and structural studiesperformed with thetwoproteins.

MATERIALS AND METHODS Cells.Raji(9), Namalwa (12), and AW-Ramos (15) are EBNA-positive lymphoma lines that do not ex-press EBV antigens associated with the lytic cycle. Ramos (13) and BJAB (24) are EBV DNA- and EBNA-negativehumanB-celllymphomalines.

DHL-4and DHL-7areEBV-negativehistiocytic lymphoma lines of B-cell origin (8). 26CB-1 is a herpes virus papio-positive baboon lymphoblastoid line of B-cell origin.It carriesherpesvirus papioDNAand contains the associated nuclear antigen,HUPNA, cross-reac-tive withEBNA(26).Molt-4 (25) and 1301 (1, 31) are EBV-negative lines derived from acute lymphocytic leukemia ofthe T-cell type. All cell linesweregrown in RPMI1640medium(GIBCOLaboratories, Grand Island,N.Y.)supplementedwith 10% fetal calf serum,

100Uofpenicillinperml,and100

iLg

ofstreptomycin per mL Large amounts ofRaji cells were obtained from Pfizer Inc., New York, N.Y., through

arrange-ments made by the Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda, Md. LargeamountsofNamalwa cellsinfected with New-castledisease virus1to 2days before harvesting and freezingweregenerouslyprovided by C. B.Anfinsen, NationalInstitutes of Health, and N. B. Finter,

Well-comeResearchLaboratories,Beckenham, England. Sera. Three anti-EBNAantibody-positiveandtwo

anti-EBNAantibody-negativehuman sera were used for immunoprecipitation and for the monitoring of EBNApurification.Table1summarizes thepertinent serological informationforallreagents.Allserawere tested forthe anti-EBNA antibody subclass. Sera that contained anti-EBNA reactivity exclusively in the IgG3 subclass werenotused forimmunoprecipitation, since thisimmunoglobulin subclass does not bind to proteinA-Sepharose.

Other reagents. DNA-cellulose wasprepared by the method of Alberts and Herrick (2), employing native calf thymusDNA (Worthington Biochemicals Corp.,Freehold, N.J.) andcelluloseCF 11(Whatman, Inc., Clifton, N.J.). Hydroxyapatite (Bio-Gel HTP)

was from Bio-Rad Laboratories, Richmond, Calif. DEAE-Sephadex, Sephacryl S-200, and protein

A-Sepharose were from Pharmacia, Inc., Uppsala, Swe-den.A "C-labeledamino acid mixture was from the Radiochemical Centre, Amersham, England. Extra-cellularstaphylococcal protease I (V8) was from Miles Laboratories, Inc.,Elkhart, Ind.

Immunoprecipitation by protein A-Sepharose. Forimmunoprecipitation by proteinA-Sepharose, we slightly modified the earlier procedure (21). Cell ex-tracts(0.5ml) were preincubated with 10,lI of a 50% suspensionof proteinA-Sepharose for10minatroom temperature, and the protein A-Sepharose was dis-carded.The cellextractswerethenincubated with 20 ,ul of serum (2 hat roomtemperature, followed by 30 min at37°C), 20

pd

of theprotein A-Sepharose suspen-sionwasadded,and theincubationwascontinued for 20min at room temperature. ProteinA-Sepharose and bound immunecomplexeswererecovered by centrif-ugation. The pelletswerewashed six times in buffer containing 150 mM NaCl, 0.5M LiCl, 20mM Tris-hydrochloride(pH 8.0), and0.5mM phenylmethylsul-fonyl fluoride (PMSF). The bound complexes were

eluted with 60,Al ofSDS-gel electrophoresis sample buffer by heating to 100°C for 2 min. The eluted proteinswereanalyzed bySDS-gelelectrophoresis.

Gelelectrophoresisandfluorography.SDS-gel electrophoresis ina gradient of7 to 15% polyacryl-amidewasperformed by the method of Laemmli (16). Proteinsampleswerereduced and denatured before electrophoresisbyheatingat 100°C for2 mininthe presence of SDS and 2-mercaptoethanol. Coomassie brilliantblue R-250wasused forstaining, and fluorog-raphywasperformedby the method of Bonner and Laskey(4).

Urea-gelelectrophoresisina gradientof5 to 15% polyacrylamidewasperformedby the method of Pan-yim andChalkley(28). ForSDS-gel electrophoresisin

aseconddimension,thegelswere cutintolanes after staining, shaken for30min inSDSsample buffer,and

rerun.

Elution ofpolypeptides from SDS-polyacryl-amide gels.The stainedpolypeptidebandswere cut

out,mincedtoabouta1-mmthickness,and extracted with 0.1% SDS-50 mM Tris-hydrochloride (pH 8.5) for24h. Thesupernatantwasremoved after centrif-ugation andfreeze-dried. The dry materialwas incu-batedwith ice-cold 10% trichloroacetic acidovernight

to remove SDS and Tris. After centrifugation, the supernatantwasdiscarded,and thepelletwaswashed withcoldacetone(-20°C)andcentrifuged.For amino acidanalysis,thepelletwassubjectedtoacid hydrol-ysis.

Peptidemappingafterenzymaticorchemical proteolytictreatment.Forenzymaticdigestions,the purifiedproteins(about20

ltg)

wereincubated with0.1

,.g

ofextracellularStaphylococcusaureusprotease for

5and 10 min.Incubationwasterminatedby trichlo-roacetic acidprecipitation. Thepelletswere washed withcoldacetone, solubilizedinSDS samplebuffer, andanalyzedbySDS-polyacrylamide gel electropho-resis.

For chemicalcleavages, the purified proteins (about

25

,g)

weredissolved in70

pi

of concentrated formic acid anddilutedto70% acid withwater.CNBr(50 mg)

wasadded, and thesampleswereleftat room

temper-ature for 24 h. The reagent and the solvent were

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http://jvi.asm.org/

594 LUKA, JORNVALL, AND KLEIN removed by repeated evaporations, and thepeptides

were analyzed by SDS-polyacrylamide gradient gel

electrophoresis.

Amino acid analysis. Sampleseluted from gels

werehydrolyzedundervacuumat1100Cfor 22 h in 6

MHCI containing0.5%phenol.Protein from the

col-umn-purified material was treated similarly, except

when hydrolysiscurveswereestablished,in whichcase

duplicate sampleswere analyzed afterhydrolysisat

about20, 48,and 72 h. Amino acidsweredetermined

onaBeckman121Manalyzer.

Determinationof the relative amount of 53K

protein chains present in different cells.

"IC-aminoacid-labeledproteins purifiedonDNA-cellulose

from different cells were analyzed by

fluorography-mediatedautoradiographyafterSDS-polyacrylamide gel electrophoresis. For the origin of the cells, see

Table3below.Thedevelopedfilmswereanalyzed by

densitometryfor the relativeamounts of 53Kprotein. High-mobilitygroupproteinswerepresentin thesame

relativeamounts inallcell extractsandwereused for

standardization.

RESULTS

Purification of EBNA and"mock EBNA." Inanearlier purification procedure, EBNAwas

solubilized from antigen-positive cells, chro-matographedonDNA-cellulose, and purified by a four-step procedure as described previously

(21).

In thepresentstudy, EBNAwaspurified

fur-ther andseparated from associated molecules by

athree-step procedure involving heattreatment

and at least two consecutive steps of column chromatography. Antigen activity was

moni-tored by AFNB and complement fixation. The flow scheme of thepurification and the deriva-tion ofthe variousprotein fractions areshown

inFig. 1. The resultsaresummarized in Table2. Theinitial extraction was performed in 0.15

M NaCl. Apreviously used higher salt

concen-tration, 1.7 M (21), increases the yieldslightly but is unfavorable since it also extracts addi-tionalDNA-binding proteins.

In the majority of the experiments, cell

ex-tracts were prepared from the Raji cell line;

Newcastle disease virus-infected Namalwacells wereused insomeexperiments. Since the

prop-erties of EBNA were the same from both cell

lines, the resultsarenotshownseparately. For EBNA purification, 25 g of frozen cell

pelletswassuspended in 100 ml of buffer

con-taining 150 mM NaCl, 20mM Tris-hydrochlo-ride (pH 8.0),1mMEDTA, and0.5mM PMSF. Thecells werethen centrifugedinaSorvall

SS-34 rotor at 18,000rpm for 60min at40C.The supernatant (fraction I) wasslowly addedto a

glass beaker inawaterbathat650C.After the entire solutionwasadded, the beakerwasleftat

650C for10min. The whole suspensionwasthen

rapidly cooled and centrifugedat15,000rpmfor

30min. Thesupernatantwasdialyzed againsta

buffer

containing

150 mM

NaCl,

20 mM Tris-acetate

(pH

6.5),

1 mM

2-mercaptoethanol,

0.5

mM

PMSF,

1 mM

EDTA,

and 10%

glycerol

(fraction

II).

A DNA-cellulose colunm

(2 by

45

cm)

con-taining double-stranded calf

thymus

DNA was washed

extensively

with the

dialysis

buffer. The

dialyzed

cell extract was then

applied

to the

column. After elution of most of the

proteins

with the

starting buffer,

thecolumnwaswashed with buffer

containing

200 mM NaCl

(Fig. 2).

This gives a fraction from which the

EBNA-associated

protein

with 53K subunits can be

purified (see

below). EBNA-containing

fractions

wereeluted with buffer

containing

1.0MNaCl. The EBNAeluatewas

pooled (fraction III)

and

dialyzed

against

10 mM

phosphate

buffer

(200

mM

NaCl,

10mM

phosphate [pH 7.5],

0.5mM

PMSF, 1 mM

2-mercaptoethanol).

After di-alysis, the solutionwas

centrifuged

at16,000rpm for30minat

4°C

to remove a

slight precipitate.

Thesupernatantwas

applied

to a

hydroxyapa-tite column

(0.9

by

10

cm)

prewashed

with the

phosphate

buffer. Elution was

by stepwise

in-creases in the

phosphate

concentration to 50

mM andto 140 mM

phosphate

buffer

(Fig. 3).

Thelast fraction

(fraction IV)

wasconcentrated

and

dialyzed against

150mMNaCl-20 mM

Tris-acetate (pH 6.5)-1mM EDTA-1mM

2-mercap-toethanol

and stored at

-70°C

inthe presence

of10%

glycerol.

This

procedure

ledto an

approximately

700-fold

purification

ofEBNA witha60%

yield.

The

purified

materialgavea

single

bandon

SDS-gel

electrophoresis (Fig.

1,

insert)

withamolecular

weight

corresponding

to 48K in relation to

marker

protein

subunits. This

preparation

gave

positive

AFNB and

complement

fixation

reac-tionsfor EBNA and inhibited the

anticomple-ment immunofluorescence assayfor

EBNA

on

Raji

cells.

TABLE 1. EBVantibody titers of the human serum reagents

Antibody titer

Donor Diagnosis of donor

against':

VCA EA EBNA

1 Burkitt's lymphoma 1,280 160 640

2 Burkitt'slymphoma 1,280 80 640

3 Burkitt'slymphoma 1,280 80 640

4 Normal 160 <2 320

5 Normal 160 <2 160

6 Normal <2 <2 <2

7 Normal <2 <2 <2

8 Normal <2 <2 <2

aAntibody titers were

determined

by

Werner

Henle

andGertrud Henle in the course of a previous collab-orativestudy. VCA, Virus capsid antigen; EA, early antigen.

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_, - = ~~~~~~~~~~~..Xt8

0 F,actlon yj

11Ililil---~ ~ ~ ~ ~~~.Pnnn5

Heat trea,ment

Fraction TI

DNA-

cellulosej|

I ... ..

~

EBNA

a 1:

Br4A

actlo 1

Hydroxyapat.te

- Fraction IV

IDEAE-Sephadex I

CrtK

I

EBNA

HydroxyoppaIte

53 KC+53Ku (RaJI) 53Kc (R3jmos)

53Kc

53Ku

FIG. 1. Purificationscheme.

TABLE 2. Purification of EBNA from25gof Rajicells

(U)Sp

act(U!

Purifica-Fraction Vol(ml) Protein(mg) AFNB

(Um)

tion fac- Yield (%) g) tor

I. Cell extract 120 310 1,200 4 (1) (100)

II. Heat-treatedcellextract 105 53 1,000 19 5 83

III. DNA-cellulose 26 0.64 700 1,100 270 58

IV. Hydroxyapatite 5 0.21 580 2,800 700 48

aReciprocal antigen titer endpoint for positive AFNB reaction.

Itwas

occasionally

necessarytoadda

DEAE-Sephadexstep toremove some low-molecular-weight contaminants, possibly degradation products. FractionIVwasthendialyzed against 150 mM NaCl-20 mM Tris-hydrochloride (pH

7.4)-i

mMEDTA-1mM2-mercaptoethanol-0.5

mM PMSF and bound to DEAE-Sephadex. EBNA was eluted between 200 and 300 mM NaCl.

Mock EBNAwas preparedfrom Ramos and BJAB cells in thesameway as theRaji prepa-ration up to and including the DNA-cellulose purificationstep (Fig. 1).

Purification of different 53K proteins. The0.2 M NaCl eluatefromthe DNA-cellulose colunm contained about 80% of the total 53K protein. This eluate was pooled and dialyzed against10 mMphosphatebuffer(200mMNaCl, 10 mM phosphate [pH 7.5], 0.5mM PMSF, 1 mM 2-mercaptoethanol). After dialysis, the

so-lutionwas appliedto ahydroxyapatite column (0.9 by10cm) prewashedwithphosphatebuffer. The materialwaseluted with50mMphosphate. Thisfractionwasconcentrated,dialyzed against Tris-acetate buffer (pH 6.5), and stored at -70°C. It gaveonebandbySDS-gel

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[image:4.510.81.426.70.491.2]

596

LUKA,

JORNVALL,

AND KLEIN

0.2M 1.0 M

I

I

10 20 30 40 50 60 70 80 90 100 110

[image:5.510.114.405.76.238.2]

FRAC TION NUMBER

FIG. 2. Chromatography ofheat-treatedRajicellextractonDNA-cellulose.Theextractwasappliedtothe column inabuffer containing0.15 MNaCl. Adsorbedproteinswereelutedstepwise byincreasing the NaCl concentration to0.2 M and thento1M.Theproteincontentofthefractionswasmeasuredby optical density at280nm(A280).EBNAactivitywasmonitoredbythe AFNBassay

(-0.8.

0 0.6.

OD cv

0.4-0.2.

50mM 140mM

I I

ti

lI

l

II|

I

-r-~~~~~l

J;~~~~~~~~~~~~~~~~~~

400 mM

10 20 30 40 S0 60 70 80

FRACTION NUMBER

FIG. 3. Chromatography of partially purified

EBNA on hydroxyapatite. The adsorbed proteins were eluted stepwise by increasing the phosphate

concentration ofthe buffer. Symbols as in Fig. 2.

A280,Optical densityat280nm.

resis (Fig. 1, insert),correspondingtoasubunit

molecularweight of about 53K.

The 53K proteinwas alsopresent in Ramos and BJAB cell extracts (Fig. 1, insert). It was

therefore purifiedfrom bothEBV-positive and -negative cells.

Incubation of the "C-aminoacid-labeled 53K proteins with EBNA-positive or -negative sera

and protein A-Sepharose failed to precipitate

any 53K protein. In the presence of the 48K

protein,partofthe53Kproteinwasprecipitated

by anti-EBNAantibody-positivesera,however.

The53K preparation from Rajicells was

nega-tiveinAFNBandcomplement fixation reactions for EBNA and didnotinhibit the anticomple-ment immunofluorescence assayfor EBNA on

Rajicells.

By gelelectrophoresis in acidicurea,the 53K

protein of the EBNA-negative lines gave only oneband, whereas the 53Kprotein derived from

EBNA-positive lines was resolved into two

bands (Fig. 1, insert). The single 53K protein

subunit of the EBV-negative lines has

electro-phoretic and chromatographic properties in

common with one type of53K chain from the

EBV-positive lines and is called

53Kc

for com-mon. The other component is found only in EBV-positivecellsand iscalled 53Kg for unique.

The two forms of 53K can be separated on a

DEAE-Sephadex column. The 53Kcform does notbindto DEAE-Sephadex in thepresenceof

150 mM NaCl,whereasthe

53K.

formbindsto thegelasshowninFig. 1, andcanbeelutedwith

250 mM NaCl.

Similarpurification of "C-amino acid-labeled cell extracts from B- and T-cells showed that 53K proteins are present in all permanent

B-cell-derived lines tested, in different amounts, butnotinT-celllines(1301andMolt-4), periph-eralB-cells, ormitogen-stimulated B-cells

(Ta-ble3).

Immunoprecipitation of EBNA. Immuno-precipitated material derived from the two EBNA-positive lines, RajiandNamalwa, by ex-posure to anti-EBNA-positive sera brought

downtwoprotein bands,detectedby fluorogra-phy, with molecular weights of about 48K and 53K(Fig. 4).Nosuchbandsweredetected when thesameextractswereexposedto anti-EBNA-negative sera orwhen EBNAantibodywas

re-actedwith extracts from EBNA-negativelines. Purified 53K proteins (a mixture of 53Kc and 53Kg) were notprecipitated byany of thesera

used,whereas thepurified48Kproteinwas

pre-cipitated by EBNA-positivesera.

Size of native EBNA and 53K protein. Partially purified EBNA wasderived from the

DNA-cellulose column step. It contained both the53K and48Kproteins.Itwas analyzedon a

Sephacryl S-200 column in 150 mM NaCl-20 I.2 .

0.8 .

0 0 N 0.6.

0.4. 0.2.

00

'-

I-ao

50 Z

41

.20

J. VIROL.

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TABLE 3. Presence andrelative amountofthe 53Kcomponent indifferent cell types

Cell Origin EBNA Relativeamtof53K'

NormalB-lymphocytes - Undetectable

ProteinA-stimulated B- - Undetectable

lymphocytes

Raji AfricanBurkitt'slymphoma + 100

Namalwa African Burkitt'slymphoma + 100

Ramos AmericanBurkitt'slymphoma - 60

AW-Ramos In vitro EBV-converted Ramos + 100

BJAB Burkitt'slymphoma-like lymphoma - 50

DHL-4 B-cell-derivedhistiocytic lymphoma - Low

DHL-7 B-cell-derivedhistiocyticlymphoma - Low

26CB-1 Herpesviruspapio-carrying baboon + 90

B-cell line

Molt4 T-cell-derivedacutelymphoidleu- - Undetectable

kemia line

1301 T-cell-derivedacutelymphoidleu- - Undetectable

kemia line InrelationtoRaji

cells,

which is100.

mMTris-acetate(pH 6.5)-i mM EDTA-0.5 mM PMSF-1 mM 2-mercaptoethanol in parallel withpurifiedEBNA and53Kproteins.The

par-tially purified EBNA gave a major peak at a

position correspondingtoamolecularweight of

about 200K. Thepurified EBNA and the puri-fied 53K protein gave similar results butwith additional protein peaks, representing

com-pounds with lower molecular weights (Fig. 5). The difference in patterns between the

sepa-ratedproteins and the mixture could have

sev-eralexplanations. One possibility is that the 48K protein is stabilized by complexing with the 53K protein, whereas when isolated in separated forms, both components may dissociate more

extensively.

CNBrpeptide mapping and staphylococ-cal protease V8 peptide mapping of 53K and 48Kprotein subunits. Peptide mapping

was performed after partial digestion with S. aureusV8protease by theprocedure of Cleve-land et al. (5). The EBNA and 53K protein componentswere compared. The purified

pro-teinsweretreated withtheenzymefor5and 10 min. The peptide fragmentsobtainedwere

elec-trophoresed througha7to15%polyacrylamide gel (Fig. 6). Three distinct peptides were

ob-tainedfromthe53K component. The48K

pro-tein monomer generated only two peptides.

Theywere notidenticaltoanyof the peptides derived fromthe 53Kcomponent.

The two proteins were also analyzed after

CNBrtreatmentin asimilar manner on a 7to

15% SDS-polyacrylamide gel (Fig. 7). The 53K

monomer gave one major polypeptide with a

molecularweight of about 24K and several

mi-nor components. The 48K monomer gave two

majorpolypeptidesatabout20K and 12K, with

no detectable minorcomponents.Thefact that

bothpatterns

give

large CNBr fragments is com-patible with the low methioninecontent ofthe

proteins

(Table

4; seebelow).

The two different peptide mapping experi-mentsthus demonstrated that the 48Kand the 53Kcomponents haveno long regions in com-monand that the 48Kmonomerisnot a degra-dation

product

ofthe 53Kcomponent.

Amino acid analyses. (i)

53K.

protein from

Raji

cells. The total composition of the pureproteinwasdeterminedbylarge-scale (20-to

30-rIg

samples) analysis in duplicate after three

different

times of

hydrolysis

(21, 49, and 72 h). The values obtained after correction for destruction(serine and

threonine,

with5and 3% additions to the 21-h values,

respectively)

and slowrelease

(valine

and

isoleucine,

with20and 25% increases,

respectively,

between 21 and 72 h) areshown in Table4. These data show that

the

53Kg protein

of

Raji

cells is

highly polar,

with dicarboxylic residues and

glycine

particu-larly

common and with a very low content of

hydrophobic

residues

containing

branched or

aromatic side chains.

(ii)

Comparisons

of thetwo53K

proteins

from

Raji cells.

Samples

fromdifferent

prepa-rationswere

compared

on asmallscale

(1

to10

,g)

after

single

hydrolysis

times.

Raji

cell-de-rived

53K.,

53Kc,

and their mixtureswere

ana-lyzed.

The standard deviation ofthe different

samples was large (about 10%), as might be expected, due to the

small

amounts ofprotein, the unidentical

purification

methods

(column

chromatographies

or

SDS-gel

electrophoresis),

and the

single

hydrolysis

times.

Despite

this,

all compositions weresimilar and showedno con-sistent variation with the

purification

methodor the type of

protein

analyzed.

It may be con-cludedthat the

purification

of the 53K

proteins

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598

LUKA, JORNVALL, AND KLEIN

1 2 3 4 5 6 7 8 9 10

53k-

-48k -w m "

residues, the tyrosine/phenylalanine ratio was consistently smaller in the 53K protein chains from Raji cells than inthe 53K protein chains fromRamoscells, and thesame wastrue for the ratio of the branched-chain residues isoleucine andvaline.

It can be concluded from these comparisons

of

composition,

performed inanidentical

man-ner, thatall 53K proteins analyzedare similar, but that consistent strain variations affect closely related residues rather than theoverall patterns of the proteins. Single samples ofthe 53Kcomponentfrom Namalwa and AW-Ramos cell lines were also analyzed, giving closely re-latedresidue

patterns

(datanotshown).

EBNA. Table 4 shows the results of large-scale analysis (25 ,ug). It is clear that the overall composition of EBNA (48K) is similar to the proteins with 53K subunits. EBNA also has

6004

[image:7.510.62.246.68.392.2]

400 200.

FIG. 4. Identification of 14C-labeled EBNA and 53K protein by immunoprecipitation, SDS-polyacryl-amide slabgel electrophoresis, and subsequent fluo-rography. Raji, Ramos, and BJAB cells were labeled separately by growth in a 14C-amino acid mixture. Extracts were treated with anti-EBNA-positive or -negative sera asdescribedin the text. (Lanes 1, 2, and 3)Raji cellextracttreated with three different anti-EBNA-positive sera; (lanes 4, 5, and 6) Raji cell

extracttreated withanti-EBNA-negative sera; (lanes

7and 8)Ramos cell extract treated with anti-EBNA-positive and -negative sera; (lanes 9 and 10) BJAB cell extract treated with anti-EBNA-positive and -negativesera.

wasreproducibleand that the two 53Kprotein types fromRajicells havehighlysimilar, ifnot identical,aminoacid compositions (Table 4).

(iii) Comparisons of the 53K proteins from differentstrains.The 53Kprotein chain of the Ramos cell line was also analyzed on a smallscale, as shown in Table 4. It was closely similar to the 53K chains of the Raji cell line. There were somedeviations, but they cannot be distinguished from experimental variations when considered

individually.

It ismore signifi-cant, however, that some of the ratios between closelyrelated residues were different in allRaji versus all Ramos samples. Thus, for aromatic

a-0

600.

4004

200.

800.

600-400.

200.

catalose alk

Vo phophatase cyt.C.

A

I

I

B

II

I

I

C

I

I

I

I

10 20 30 40 50 60 70

FRACTION NUMBER FIG. 5. Chromatography of48Kprotein (A), 53K

protein (B), andamixtureof53Kand48Kproteins (C)onSephacrylS-200 in abuffercontaining0.15M

NaCl.The column wasprecalibratedwithamixture

ofbluedextran,catalase, alkalinephosphatase,and

cytochromec.EBNAwaslocalizedby AFNB assay.

Theprotein concentrationwas asinFig.2.

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The purity ofthe proteinwasshownby SDS-polyacrylamide gel electrophoresis. It was ob-tained inanapproximately 55% yield aftera 700-foldpurification byathree-step procedure.The heat stability of EBNA was exploited for the initial extraction,followed bytwocolumn chro-matography steps. The cell extraction method was based on the fact that EBNA is rapidly dissociated from the nucleus in frozen-thawed cells alreadyat alow-salt concentration. This is in contrast tothe majority of the DNA-binding proteins that remain associated with thecellular DNA in thecellpellet.

Inagreementwith earlier results, the

biologi-cally

active form of EBNA had a molecular

weight of 170Kto200K. Itcanbe dissociatedto a lower-molecular-weight form in high salt, re-taining its antigenic specificity in both comple-mentfixation andAFNB assays (the latter

al-_ .._.

a

b o

d

e

f

FIG. 6. SDS-polyacrylamide gel electrophoresisof proteins before and aftertreatment with S. aureus

protease V8 (0.1

pg).

(a) 53K; (b) 53K, V8 protease treated(5min); (c)53K, V8 protease treated(10 min); (d)48K; (e)48K, V8 proteasetreated(5min);(/) 48K, V8 protease treated (10 min).

aboutaquarterofallresidues of the

dicarboxylic

(or

amidated)

typeandalowcontentof

highly

hydrophobic

residues. It is not rich in basic

residues. EBNA is

clearly

different from the 53K component,however. In

particular,

thevalues of glycine,

threonine,

and methionineareso dissim-ilarastoexclude that the48Kchainisa

break-down

product

of the 53K component. When

these andother valuesare

considered,

it iseven

unlikely

that these two types of

protein

chain

canhaveanylongsegmentsincommon,despite theoverall similaritiesin

polarity.

DISCUSSION

EBNA and its associated 53K proteins have been purified separately and analyzed for the first time. We could show that the EBNA com-ponent is a 48Kprotein. EBNAspecificity was shown by both complement fixation and the AFNBtest,involving the reconstitution of acid-extracted nuclei and chromosomes with the DNA-binding protein,

followed

by ordinary an-ticomplement immunofluorescence staining for EBNA.

[image:8.510.69.239.80.369.2]

a

b

c

d

FIG. 7. SDS-polyacrylamide gel electrophoresisof proteins before andafter treatment with CNBr. (a) 53K; (b)53K, CNBrtreated; (c)48K; (d) 48K, CNBr

treated.

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600 LUKA, JORNVALL, AND KLEIN

TABLE 4. Amino acidcompositionof 53K componentsand EBNA

Aminoacid composition" offollowing

pro-Amino tein:

acid

53K,

53KC 53K Ra- EBNAe

iRajib RajiC Mos ~BA

Asx 11.2 11.5 11.7 10.0

Thr 4.7 3.2 4.2 6.2

Ser 7.1 7.0 9.9 7.5

Glx 14.0 13.4 14.8 16.2

Pro 8.2 6.9 6.0 5.4

Gly 13.0 15.1 17.3 8.0

Ala 7.7 6.5 5.9 9.3

Val 5.2 5.2 3.2 5.7

Met 1.3 0.7 0.3 1.9

lie 2.4 1.9 2.6 3.0

Leu 4.7 5.0 5.0 6.7

Tyr 0.6 1.1 1.8 1.7

Phe 2.9 3.4 2.8 2.8

Lys 9.2 9.7 6.8 9.2

His 2.2 2.7 2.2 1.4

Arg 5.6 6.7 5.5 5.1

aValues areexpressed in moles percent, disregard-ingcysteine/cystine andtryptophan.

'Duplicate analyses at three times of hydrolysis,

with correctedvalues for threonine,serine, valine, and isoleucine (see text).

c Average of sixanalysesonthree different prepa-rations(singlesamplesof1to 10

t.g

each). Standard deviations between differentpreparations,±0to2for individual values(average,1.1).

d Average of threeanalyseson twodifferent prepa-rations. Standard deviationsbetween different prepa-rations,±0.5to2for individual values (average, 1.0).

'Average of fiveanalyseson alarge scale (25,ug)of samples from two different preparations. Standard deviationsbetween differentpreparations, +0.1to 2.5

forindividual values (average, 1.1).

waystestedinthepresenceofaprotein carrier suchasbovineserumalbuminorovalbumin).

The

purified

EBNA hasasingle subunit,48K

in size. This EBNA monomer could also be isolated byimmunoaffinity chromatographyon protein A-Sepharose together with a second component,

approximately

53K in size. Both of these subunitswerealso noticed in thecourseof theprevious purification method,but the pres-entlycharacterized 48KEBNAcomponent was firstinterpretedas adegradation product ofthe 53K component, which was then thought to represent EBNA (21). In the present work, we showed that the 53Kproteinchain can be sep-arated from the 48K protein chain. Only the latter has the

antigenic

specificityof EBNA.

Therelationshipsamongthevarious forms of the 53K protein chains, on the one hand, and between these and EBNA, on the other hand, werestudied byanalyzing theamino acid com-position and by peptide mapping with CNBr

andstaphylococcal extracellularprotease. Both thecomposition (Table4) and thepeptide pat-terns (Fig. 6 and 7) show that EBNA and 53K are different protein chains without extensive common structures.This is also compatible with thedifferent origins ofthe two proteins. In all probability, EBNA is determined by the viral genome, whereas the 53K protein chains are clearly ofcellular origin, as shown by the pres-enceof

53Kc

inEBV-negative cell lines.

Comparisons between EBNA and the 53K protein revealed some superficial similarities: bothare polar, arepoor inhighlyhydrophobic residues, and have no excess of basic residues. Of course, amino acid analyses alone do not permit extensive

conclusions,

but it may be noted that the compositions are different from those typical for globular enzymes (6) and, in relation to DNA binding, that they are also different from those typical for most histones (11) and someother chromatin structural pro-teins (11),butperhaps less different from

com-positions

of

T-antigens

(10, 30) and adenoviral

DNA-binding protein (19). It must be empha-sized, however, thatthese overall similaritiesof residue distributions donotaffect the conclusion that EBNA and the 53K

protein

are different protein chains.

A53Kprotein subunitwas also foundin two EBV-negative

lymphoma

lines (BJAB and Ra-mos) butnot intwo acutelymphoid leukemia-derived T-celllines (1301 and Molt-4) and nor-malormitogen-stimulated B-cells. Peptide map-ping showed no differences among the various 53K protein chains. The amino acid composi-tionsalsosuggestthat the 53Kcomponentsfrom two different cell lines (Ramos and Raji) have only small differences in residues with related properties,asin thecaseof straindifferences of otherproteins (32). On the other hand,wecould detectnodifferencesincompositionsorin pep-tide patterns between the two forms of 53K components inRaji cells. It ispossible that the

unique

53K form inRaji,

53K.,

wasderived from

thecommon

53KW form,

perhaps bysomeminor modificationresulting from viral transformation. Immunoaffinity chromatography on protein A-Sepharose showed that the 53Kprotein was precipitated only in the presence of the 48K protein.This indicates that EBNA ismainlyor exclusivelypresent in the form ofacomplexwith the 53K protein. This conclusion is also sup-ported by the native sizes of the isolated and mixedproteins asrevealedby the

comparisons

ofSephacrylchromatographies.

The complexing of 48K and 53K protein chains isparticularly interesting in view of the fact that LaneandCrawford describedasimilar

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complexing

phenomenon between simian virus 40 large T-antigen and a 53K component in simian virus

40-transformed cells

(17). Linzeret al.reportedthepresence ofa 53Kcomponentin adenovirus-transformed cells complexing with adenovirus T-antigen (20). They also found a similar 53K protein in mouse teratocarcinoma cells and, at amuch lower content, in normal thymus tissue. 53K components were also re-ported in chemically induced mouse sarcomas (7).

These findings suggest that proteins in the 53Ksize class may be relatedtotransformation. Moreover,inDNAvirus-transformed

cells, they

are

complexed

with

T-antigen,

and as we now

have shown, in EBV-transformedcells, theyare complexed with EBNA. The roleplayed by this complex in the transformation process is now openfor study with pure andpartly character-izedcomponents.

ACKNOWLEDGMENTS

This research was supported in part by Public Health

ServicecontractNO1CP 33316 from the Division of Cancer Cause and Prevention, National Cancer Institute, and by grants from the Swedish CancerSociety (projects 107 and 620).

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