Evidence for an Adenovirus Type 2-Coded Early Glycoprotein

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0022-538X/78/0028-0314$02.00/0

Evidence for

an

Adenovirus

Type

2-Coded

Early

Glycoprotein

YUN-HUAJENG,WILLIAMS.M.WOLD,* AND MAURICEGREEN

Institutefor Molecular Virology, St.Louis University Schoolof Medicine, St. Louis, Missouri63110

Receivedfor publication28February 1978

Wehaveidentifiedanadenovirus type2(Ad2)-induced early glycopolypeptide

with an apparent molecularweightof 20,000 to 21,000 (20/21K), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 20/21K poly-peptide could belabeledin vivowith[3H]glucosamine.[35S]methionine- and

[3H]-glucosamine-labeled 20/21K polypeptides bound to concanavalin A-Sepharose

columns and were eluted with 0.2 M methyl-a-D-mannoside. The pulse-labeled

polypeptide appearedasasharp band withanapparentmolecular weight of 21K,

butafterachase it convertedtomultiplebands with an average molecular weight of 20K. Thisvariabilityinelectrophoretic mobility is consistent with glycosylation

or deglycosylation of the 20/21Kpolypeptide. Analysis of the pulse and

pulse-chase-labeled formsby using partial proteolysis indicated that the polypeptides

werehighlyrelatedchemically, butnotidentical. Most of the 20/21Kpolypeptide

islocalized in thecytoplasm fraction of infected cells lysed by Nonidet P-40. The 20/21K polypeptide and a 44K polypeptide, labeled with [35S]methionine or

[3H]glucosamineinAd2-infected humancells, were precipitated by a rat

antise-rumagainstanAd2-transformedratcell line (T2C4),butnotby antisera against

three other Ad2-transformed rat cell lines, or by serum from nonimmune rats. The partial proteolysis patterns of the 20/21K and the 44K polypeptides were

indistinguishable, indicating that the two polypeptides are highly related, and

suggestingthat the 44Kpolypeptide might beadimerof the 20/21K polypeptide.

The 20/21K polypeptide was also induced in Ad2-early infected monkey and hamstercells. These results imply that the 20/21K polypeptide is synthesized in infected human, monkey, and hamster cells, and in one but not all Ad2-transformedratcells.Thus, the 20/21K polypeptide is probably viral coded rather thancell coded and viral induced.

Polypeptides induced in cells during early

stages (i.e., before viral DNA replication) of infectionby human adenovirus type2(Ad2)are

of great interest becausetheymayregulateviral DNA replication, transcription, and cell

trans-formation (34). We have attempted to identify Ad2-induced early polypeptides by labeling in-fected and mock-inin-fected cells with [35S]methi-onine,subjectingtheproteinextracts to

electro-phoresis through sodium dodecyl

sulfate-poly-acrylamideslabgels(SDS-PAGE),and compar-ing the infected and mock-infectedpolypeptide bands seen in autoradiograms. We have also identified Ad2 early polypeptides by immuno-precipitation, using antisera against

Ad2-trans-formed rodent cells. Prominent early poly-peptide bands with the following approximate apparent molecularweights have been detected: 73,000 (73K), 53K, 21K, 19K, 15K, 11.5K, and 1lK (8, 10, 14). Additional minor polypeptide

bands of about 17.5K, 15.5K, 13.5K, 13K, 12K, 8.8K, and 8.3K can be observedin some gels, or

canbe immunoprecipitated by antisera against

certain lines ofAd2-transformed ratcells (Wold

and Green, unpublished data). Similar in

vivo-labeled polypeptides have been identified by

otherlaboratories (12,23). Similar

polypeptides

havealso been observedbycell-free translation

ofpolyribosomal RNA fromAd2-earlyinfected

cells andbyimmunoprecipitation, usingarabbit antiserumagainstearlyinfected HeLa cells(23).

Six polypeptides (72K, 44 to 50K, 19K, 15.5K,

15K, and

11K)

have been mapped by cell-free translation ofearlymRNA purified by hybridi-zation to restriction endonuclease DNA frag-ments(18).

Relatively little isknownabout thechemistry

of these polypeptides or their biological func-tions. The73Kpolypeptideisaphosphoprotein

(14, 17, 19,22) that is viral coded (9, 18, 30), that binds to single-stranded DNA (24, 27, 29), and

thatapparentlyfunctionsinviralDNA

replica-tion (28,31). This protein hasbeen purified to

homogeneity and partially characterized (19,

27). The53K (8, 16) and the 15K polypeptides

(8) are candidate transformation proteins be-causetheyare immunoprecipitated byantisera

against Ad2- or Ad5-transformed rodent cells.

314

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VOL. 28,1978

Genescoding polypeptides of 44 to 50K and 15K polypeptides have been mapped in the trans-forming region of the Ad2 genome (18). The 11K polypeptide is localized mainly in the nuclear fraction (3, 23; our unpublished data). The 73K, 21K, 15K, 11K, and possibly 8.3Kpolypeptides arepresent inasolublecomplex thatsynthesizes Ad2 DNA, suggesting a possible role for these in viral DNA replication (21).

Ishibashi and Maizel (13) reported that an Ad2-induced early 19K polypeptide could be labeled in vivo with [3H]glucosamine. In this report, we present evidence that our 21K

poly-peptide is an Ad2-coded earlyglycopolypeptide, in that it can be labeled with[3H]glucosamine,

it binds to concanavalin A (ConA)-Sepharose,

itsmobilitychangesinpulse-chase experiments,

it is immunoprecipitated by antisera to

Ad2-transformed cells (T2C4), and it issynthesized

in Ad2-infectedmonkey and hamster cells.

MATERIALS AND METHODS

Chemicals.

L-['S]methionine

(Met) (400 Ci/

mmol) and [3H]glucosamine (20 Ci/mmol) were purchased from New England NuclearCorp.;

1-/i-D-arabinofuranosylcytosine (ara-C), ConA-Sepharose, and methyl-a-D-mannoside were purchased from Sigma ChemicalCo.; acrylamidewaspurchased from Eastman Kodak Co.; Nonidet P-40 was purchased from Shell ChemicalCorp.; N,N-methlenebisacrylam-ide,N,N,N',N'-tetramethyladenediamine, and ammo-niumpersulfatewerepurchased from Bio-Rad Labo-ratories; andStaphylococcusaureusV8 proteasewas purchased from MilesLaboratories, Inc.

Cell culture and virus infection.Suspension

cul-turesof human KB cellsweregrown inEagle minimal

essential medium(MEM) containing5%horseserum. Cells at aconcentration of6x 106/mlwereinfected with100PFUof Ad2(strain 38-2) per cell in medium without horse serum. After 1 h ofadsorption, cells weresuspendedat aconcentrationof3.5x 105/mlin medium with 5% horseserum.Cyclohexamide (CH;25

,Lg/ml)wasaddedto someculturesat 1hpostinfection (p.i.). Ara-C (20,ug/ml) wasaddedat4hp.i. At 9 h p.i., cellswerewashed andsuspendedeither inwarm Met-free medium or medium with 10% the normal glucoseconcentration; both media contained5%horse

serumand20,ugof ara-C per ml.Cells,at a

concentra-tion of3.5 x 105/ml, werelabeled with[3S]Met (10

t,Ci/ml) or [3H]glucosamine (10

liCi/ml)

forvarious

times. At the end of the labeling period, cells were centrifuged and washed twice with cold

phosphate-bufferedsalinelackingCa2'and

Mg2e.

Mock-infected

cellsweresimilarly labeled,except that viruswas not added.

CV-1(monkey) cell monolayersweremaintained in 75-cm2 plastic flasks containing MEM with 5% calf serum,and in anatmosphere of 5%CO2.Nearly con-fluent monolayers were infected (or mock infected)

with Ad2 in MEM without serum. After 2 hof

adsorb-tion,MEMwith5% calf serum(withorwithout 25yg

of CH perml) wasadded to 25 ml, and incubation continued.Ara-C (20,ug/ml)wasaddedat 4hp.i. Cells

315

werelabeled from 9 to 20 hp.i. with [3S]Met in Met-free MEM containing 20

jig

of ara-C per ml.

Cell fractionation. Infectedormock-infected cells labeled with [3S]Met were washed and then sus-pended in isotonic high pH buffer (33). All steps were at 0 to 4°C, and all buffers contained 1 to 2 mM phenylmethylsulfonyl fluoride. Cells were lysed by using 0.5% Nonidet P-40 for 5 min. Lysis was moni-tored by phase-contrast microscopy until 99% com-plete. Nuclei were pelleted by centrifugation at 200 x gfor3min.The cytoplasmic supernatant was obtained by centrifugation at 12,000xg for 20min to remove mitochondria. Isolated nuclei were resuspended in phosphate-buffered saline, and treated with 0.86% Tween 40 and 0.43% sodium deoxycholate to remove

outerand innernuclear membranes (11). The

nuclear-detergent suspension wasvigorously mixed in a Vortex mixer for 30 s, and the nuclei werecollected by cen-trifugation at 500 x g for 3 min. The pellet was resuspended in cold phosphate-buffered saline with the addition of 1% Triton X-100 and disrupted by sonic treatmentfor 10 min in a Raytheon sonic oscil-lator. The nucleoplasmsupernatant and nuclear pellet wereseparated bycentrifugation at 12,000xg for 20 min.Portions of subfractionated samples were precip-itated with 10% coldtrichloroacetic acid for analysis oflabeledpolypeptides.

ConA-Sepharosecolumnchromatography. Af-finity chromatography of glycoproteins on ConA-Sepharose columnswas carried out asdescribed by Stohlmanetal.(26),withthefollowing modifications. All stepswere at4°C. The column (1.5x 25cm) was firstequilibrated with10bed volumes of50 mM Tris-hydrochloride (pH 7.5), 50mM NaCl, 1 mM MnCl2,

and 2 mM phenylmethylsulfonyl fluoride (bufferA)

containing 0.2Mmethyl a-D-mannoside, followedby

buffer A containing 0.2% Nonidet P-40 and 0.2% so-dium deoxycholate (buffer A-detergent). The

cyto-plasmic protein fraction (no CH pretreatment) was diluted 25-fold with bufferA,andsodiumdeoxycholate

wasaddedto afinal concentration of 0.2%. Thesample wasdisrupted by sonic treatment(see above), centri-fuged at 12,000 x gfor20min, dialyzed extensively

against bufferA-detergent,and loadedontothe ConA-Sepharose column. The column was washed with bufferA-detergent untilnosignificantradioactive ma-terialwaseluted. The adsorbedproteinswereeluted withbufferAcontaining0.2Mmethyl-a-D-mannoside.

Thecolumnflow-through,wash,and

methyl-a-D-man-noside-elutedfractionswereconcentratedby

precipi-tation with cold 10%trichloroacetic acid andwashed

with acetone to removetrichloroacetic acid and

deter-gent.

Radioimmunoprecipitation ofproteins. T2C4

cells, fromanAd2-transformedratcell line(6),were

obtained from P. H. Gallimore. Antiserum was

pre-paredin ratsagainstextracts ofT2C4cells, and the

immunoglobulin G (IgG) fraction was purified (8).

Infected andmock-infected labeled cellextracts were

assayedforT2C4-specificpolypeptides bythe double-antibodyimmunoprecipitationprocedure,asdescribed earlier(8).Portionscontainingequalcountsper min-ute from infected and mock-infected samples were

incubated with T2C4ornonimmune rat IgG at4°C

for 18 h. Thengoatserumanti-ratIgGwasadded,and

thesamplewasincubated for2hat37°C.Precipitates

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werecollectedbycentrifugationfor90 sat23,000xg

in a Beckman 152microfuge,washed three times with

phosphate-bufferedsalinecontaining0.5M urea, 0.5%

Nonidet P-40, and 1% sodiumdeoxycholate,suspended

in anequalvolume ofgelelectrophoresis samplebuffer

without 2-mercapthoethanol, and analyzed by

SDS-PAGE.

SDS-PAGE and autoradiography. SDS-PAGE

was carried out as described elsewhere (14). Unless

statedotherwise,8to21%gradient acrylamide 10-cm

slab gels were used. Radiofluorography as described

by Bonner and Laskey (1) was used to detect

[1H]-labeledpolypeptides. The absorbance of bands in au-toradiogramswasmonitored byusingaJoyce-Loebel densitometer. The area under the absorbance tracing was calculated as the quantitative estimation of the intensityof bands.

Partial proteolysis. The relationship between

[;35S]Met-labeled 44K and 20/21K polypeptides

im-munopreciptatedby T2C4antiserumwasinvestigated, usingthe partial proteolysis procedure essentiallyas

describedby Cleveland andcolleagues (4).The

poly-peptidesimmunoprecipitated byT2C4 antiserum were

resolvedbySDS-PAGE, thegelsweredried, and the

fluorographs were developed. The 44K and 20/21K

bands were cut from thegel, andthe gel slices were

insertedinto wells (4 mm wide, 0.75 mm thick,

con-taining soaking buffer which consisted of: 0.125 M

Tris-hydrochloride [pH 6.8], 0.1% SDS, and 1 mm

EDTA) of a second slabgel,andhydratedbysoaking

for 30 min. The proteolysis gel consisted of a 4-cm

stacking gel of9% acrylamide (pH 6.8) and a 5-cm

runninggelof17% acrylamide (pH8.8). S. aureus V8

protease was diluted to 2 mg/ml in soaking buffer

containing10%glyceroland0.025%bromophenol blue,

and 10

Al

wasaddedtowellscontaining the44Kand

20/21Kgel slices. Electrophoresiswascarried out with

the gel apparatus connected to a circulating water

bath at 20°C. The sample was subjected to electro-phoresis (25 mA pergel) until the bromophenolblue

band had almost reached the bottom of thestacking

gel. The powerwas shut off for30 minto allow the

protease to digest the

[K5S]Met-labeled

polypeptides

that hadmigratedfrom thegel slices. Electrophoresis

wasthen continued until thedyereached the bottom

ofthe runninggel,and thegels were dried and fluo-rographed.

RESULTS

Identification of Ad2-induced early

poly-peptidesanddemonstration of

electropho-retic mobility changes in a 20/21K

poly-peptide in pulse-chase experiments. The

electropherographinFig. 1illustrates

[35S]Met-labeled Ad2-infected and mock-infected cell

polypeptides. Inthis and allotherexperiments,

ara-C (20

,ig/ml)

was added at 4 h p.i. to inhibit viral DNA replication. Therefore, cells were in early stagesofinfection. Lanes A to H in Fig. 1 showpolypeptides from cells incubated with CH before labeling, a procedure that enhances the synthesisofAd2-specific early polypeptides

rel-ative to hostpolypeptides (10). Six Ad2-induced

polypeptides areclearly visible: DBP (the single-stranded DNA binding protein of about 73K daltons), 20/21K, 19K, 15K, 11.5K, and 11K. Lanes A and B and lanes C and D show infected and mock-infected polypeptides labeled 9 to 10 and9 to 12 hp.i., respectively. In these lanes(A and C), the 20/21K polypeptide appeared as a fairly sharp band of about 21K daltons.

How-ever, when cells were labeled for long periods,

e.g., 9to 24 h p.i. (lanes E and F), or9to 12 h p.i. followed bya 12-h chase in complete MEM (lanes G and H), the intensity of the 21Kband decreased, and new bands ofroughly 20K

ap-peared. Lanes ItoL illustratepolypeptides from cells labeled without preincubation with CH (only DBP and the 20/21K bands are visible). Again, a distinct 21K band was apparent in cell

extractslabeled 9to 12hp.i. (lanesI andJ),but this band became reduced in intensity and seemed to increase in mobility in cells labeled for 9 to 24 hp.i. (lanes K and L). These results provide initial evidence that the 20/21K poly-peptide was modified after translation. Since glycosylation ofpolypeptides affects their SDS-PAGEmobility,weconcentratedonthe20/21K

polypeptide as acandidateglycopolypeptide.

Figure 2 illustrates the analysis ofthe pulse (30 min) 21K polypeptide and the pulse-chase (30 min followed by a 15-h chase) 19K to 20K forms of this polypeptide by the partial prote-olysisprocedure. Lane A shows the pulse-labeled 21K polypeptide before chromatography on

ConA-Sepharose, and lanes B toJ show

pulse-and pulse-chase-labeled forms (purified on

ConA-Sepharose). In this experiment, the

chasedpolypeptideformedabroad bandranging from apparentmolecularweightof 19Kto20K. LanesC, F,and I represent the 20Kregion,and lanesD,G, and J represent the 19Kregion.

Mostof thepartialproteolysis polypeptidesof

thepulseandpulse-chaseformsclearly coincide,

confirming that the 21K and 20K are highly

related.However,thepolypeptidesare not

iden-tical,because the chased forms, especially 19K,

contained at least one band (e.g., second from

bottom inlanes D and G) notfoundamongthe

proteolysis products of thepulse form. Note that in lanes A to G some of the polypeptide has

spontaneously"polymerized" to 44K. Thiswas

areproducible phenomenon.

Cellularlocalization of the 20/21K

poly-peptide. To further analyze the 20/21K

poly-peptide, we established which cellular fraction contained themajority of thepolypeptide. Ad2-infected and mock-infected cells were labeled

with[35S]Metfrom9 to 12hp.i.,lysedwith0.5%

NonidetP-40,and thenfractionatedintonuclear

pellet, nuclearmembrane, nucleoplasm,and

cy-toplasm. The vast majority of 20/21K

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Ad2 EARLY GLYCOPROTEIN

317 A

B

C

D

E

F

G

H

II

Ias

I hi

t .*_

6.r 14

I

A_

'._

.d.o

_.,.I

"

I

In

&I

UW

I

J

K

L

4-l :.

on-__n:~~~~~~~

-_

w a"

,sn

*

* f

flC_

l so nM AV.b.

40 -o"P

20/21

K-

4 19K

15K-

-11.5K

IIK

w

_n

[image:4.500.54.451.66.573.2]

- _.

FIG. 1. Identification ofAd2-inducedearlypolypeptides with andwithout CHpretreatment, and

pulse-chaseexperiments demonstrating mobility changesina20/21Kpolypeptide.Ad2-infectedandmock-infected

cellswereprepared.CH(25pg/ml)wasaddedtosomeculturesat1hp.i. (lanesAtoH).Ara-C(20pg/ml)was

addedto allcultures at4 hp.i.. At 9hp.i., allcultureswere washed andresuspendedin warmMet-free

medium containing ara-C and then labeled with[3S]Met forvarious timeperiods. Proteinextracts were

preparedandwereanalyzedby SDS-PAGE. Anautoradiograph ofadried 17-cmgelis shown. (A) Infected,

labeled9 to 10 hp.i.; (B)mockinfected,labeled9 to 10 hp.i.; (C, I)infected,labeled9to12 hp.i.; (D, J)mock

infected,labeled9 to12 hp.i.; (E, K) infected,labeled 9to24hp.i.; (F, L)mockinfected,labeled9 to24hp.i.; (G)infected,labeled9 to12hp.i.,andchasedfor12h;(H)mockinfected,labeled 9to12 hp.i.,and chasedfor 12 h.

DBP

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FIG. 2. Partialproteolysis (S. aureus V8protease)ofpulsed21K andpulse-chased 35S-labeled19to 20K

forms of the 20/21Kpolypeptide. Infectedcells werepulse-labeled with[35S]Met for30minorfor30min followed bya15-hchase.Cytoplasmextractswerepreparedandwerepurified by chromatographyon ConA-Sepharosefollowed bySDS-PAGE. The 21Kpulseand20Kor19K chasepolypeptidebandswerecutfromthe gelandanalyzed by partial proteolysisasdescribed in thetext,using 0.5, 5,or50yg ofproteaseperlane. (A)

Pulse-labeled 21Kpolypeptide before chromatography on ConA-Sepharose; (B, E, H) pulse-labeled 21K

polypeptide purifiedonConA-Sepharose; (C, F, I)pulse-chase-labeled20Kafter ConA-Sepharose; (D, G, J) pulse-chaselabeled19Kpolypeptide after ConA-Sepharose.

peptide was present in the cytoplasm fraction

(Table 1).

Labeling of the 20/21K polypeptide with

[3H]glucosamine, and immunoprecipitation

with antiserum directed against an

Ad2-transformed rat cell line (T2C4). Infected

and mock-infected cellswerelabeled with [3H]-glucosamineto testwhether the labelwas incor-porated intothe20/21K polypeptide.Asshown

in Fig. 3, the only Ad2-specific polypeptide la-beledwith[3H]glucosamine had about thesame electrophoretic mobility as the 20/21K poly-peptide (lane B). Antiserum against T2C4cells

precipitated the

'H-labeled

20/21K

polypep-tides, aswell asa second polypeptide ofabout

44K(lane D).The 20/21Kand44Kpolypeptides

werenotprecipitatedfrom infected cellextracts

bynonimmuneratsera (laneE),orfrom mock-infected cellextractsby either T2C4 or

nonim-mune ratsera (lanesFandG). The T2C4 anti-serum also immunoprecipitated viral specific early[35S]Met-labeled polypeptidesof 53K, 44K,

20/21K, 19K, 18K, 15K, 14.5K, 13.5K, 12K, and 11.5K (Wold and Green, unpublished data).

The 20/21K and 44K polypeptides precipi-tated by the T2C4 antiserum were assayed by

the partial proteolysis procedure (4) to test whethertheywerechemicallyrelated.

[35S]Met-labeled polypeptides were assayed instead of

TABLE 1. Distributionofradioactivityand the

20/21Kpolypeptideinsubcellularfractions

Mock-in-_fected Infected_cells ₂₀₂₁20/21K_totl 20/21K

/21tta

Fraction cells (%total cptoa (%total (%ctotal area)h CPM),

cpm), cpm),

Whole cells 100.0 100.0 1.5 100.0 Nuclear membrane 2.1 1.9 0.6 0.8 Nuclearpellet 8.9 9.5 1.0 6.3

Nucleoplasm 1.4 1.5 0.2 0.2

Cytoplasm 86.2 85.6 1.6 91.9

'Total ¢'S countsper minute in the infected whole-cell preparationwas278x10',and in themock-infected prepara-tionwas280x10.

Infected and mock-infected proteins were resolved by SDS-PAGE, andautoradiogramsweredeveloped.The auto-radiogramswerescannedbyusingaJoyce-Loebel densitom-eterto obtain the totalarea represented byeach fraction. These values indicate the percentage of the totalscan area

represented bythe 20/21K polypeptide,minuscontribution frommock-infectedpolypeptides.

'Calculationsarebasedon avalue of 100% for the esti-mated20/21Kcountsper minute in thewhole-cellfraction.

["H]glucosamine-labeled

polypeptides, because larger amounts of radioactivity could be ob-tained.

[35S]Met-labeled

polypeptides

immuno-precipitated by the T2C4 antiserum were

re-solved by SDS-PAGE, and fluorograms were

developed. The 20/21K and 44K bandswerecut

eV -w so

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Ad2 EARLY GLYCOPROTEIN 319

A

B

C

D

E

F

G

DBP

I

20/

21 K

15K

11.5K

I

IK

FIG. 3. Identification of polypeptides labeled in vivo with [3H]glucosamine andimmunoprecipitated by antiserumagainst 72C4 cells. Ad2-infected and mock-infected cells were labeled with[3H]glucosamine.Total

cellextractswereprepared and immunoprecipitated by using T2C4 IgG and nonimmune ratIgG. Equal

volumesof each precipitate were analyzed by SDS-PAGE. (A) Marker polypeptides:[3S]Met-labeled,early infectedpolypeptidesprepared by the CH enhancement procedure. (B-G),[3H]glucosamine-labeledextracts. (B)Infected, before immunoprecipitation; (C) mock infected, before immunoprecipitation; (D) infected extract

versusT2C4IgG; (E) infected extract versus nonimmune rat IgG; (F) mock-infected extract versus72C4IgG;

(G)mock-infectedextract versusnonimmune rat IgG.

from the driedgelandanalyzed bypartial

pro-teolysis as described above. The partial

prote-olysis slab gel is shown in Fig. 4. The same

polypeptide bands were generated by protease

digestion of the 20/21K and44K polypeptides,

indicating that they are highly related

chemi-cally. Proteolysis ofthe 20/21K and 44K

poly-peptidesby usingotherproteaseconcentrations

also indicated thattheyarehighly related.

Binding of the 20/21K polypeptide to

ConA-Sepharose and its elution by

methyl-a-D-mannoside. To obtain further evidence

that the 20/21Kpolypeptide isa

glycopolypep-tide, 35S-labeled proteinextracts weresubjected

toaffinity chromatographyoncolumns of

ConA-Sepharose. Onlycertain carbohydrate moieties

(a-D-glucopyranosides, and

a-N-acetyl-D-glucos-aminides) bindstronglytoConA(20), so that if

the20/21Kpolypeptide contains thesesugars it

should bind to the ConA-Sepharose column.

Infected and mock-infected cells were labeled

with

[35S]Met

for15h, and

cell

cytoplasmswere

preparedand thenloadedonto

ConA-Sepharose

columns. The columns were washed and then

eluted with 0.2 M

methyl-a-D-mannoside.

The

columnflow-through, wash,andeluted fractions

were analyzed by SDS-PAGE. Only the

man-noside-elutedinfected cell fraction

(lane

F)

con-tained detectable 20/21K polypeptide

(Fig.

5).

These results suggest that the 20/21K

poly-peptide contains sugar residues

specific

for

ConA.

The20/21K

polypeptide

is

synthesized

in

Ad2-infected

monkey

cells.The

immunopre-cipitation of both

[3H]glucosamine

and

[35S]-Met-labeled 20/21K

polypeptide by

the T2C4

antiserum suggests that the 21K

polypeptide

is viral coded rather than cell coded and viral induced. The20/21Kpolypeptide

(and

theDBP,

11K, 8.8K, 8.3K polypeptides) was

synthesized

inAd2-early infected

monkey

(CV-1)

cells

(Fig.

6). The 21K, DBP, and 11K were also

synthe-sized in Ad2-early infected hamster cells (not

shown). Theseresults

provide

further evidence

that the 21K, DBP, and 11K

polypeptides

are

viral coded.

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320 JENG, WOLD, AND GREEN

.- nP

'U

44

I

,*

iK I~~~~~~~~~~~~~~~~~~~~7

4KM

I

FIG. 4. Partialproteolysis (S.aureus V8protease) of the 44K and20/21K[35S]Met-labeledpolypeptides immunoprecipitatedbyT2C4 antiserum(A) 44K, (B) 21K. Bothpolypeptides were treated with 5 pLg of protease.

DISCUSSION

Our results

provide

strong evidence that the

Ad2-induced 20/21K

polypeptide

is a

glycopo-lypeptide. The

polypeptide

canbelabeled with

[3H]glucosamine,

and it binds to

ConA-Sepha-rose columns and is eluted with

methyl-a-D-mannoside. When labeled with

[35S]Met

for

short periods (30 min to 3 h), it appears as a

single, relatively sharp band with an apparent

molecular weight of about 21K. However, if

la-beled for long periods, or if pulse-labeled and

thenchased, the polypeptideappears as multiple

bands with apparent molecularweights of 20K

to21K. Partial proteolysisdataindicatethat the

pulse-labeled 21K and the pulse-chase-labeled

20K polypeptides are highly related, although

notidentical. Both pulse (30min)and pulse (30

min)-chase (12 h in complete MEM) forms of

[35S]-labeled

20/21K polypeptidebind to

ConA-Sepharose, and therefore apparently are

glyco-sylated. The multiple bands of the chased

20/21K polypeptide may indicate heterogeneity in sugar content, because glycosylation affects

(usually decreases) the SDS-PAGE mobility of

polypeptides.We cannotexclude the possibility

that there is another distinct polypeptide of

A

B

C

'.

-1

F

[image:7.500.90.192.77.295.2]

-~~~~~~~~.

FIG. 5. ConA-Sepharose column chromatography: identification of[3S]Met-labeled Ad2-infected and

mock-infected polypeptides in the flow-through, wash, and methyl-a-D-mannoside eluted fractions.

Ad2-infectedandmock-infectedcellswerelabeled with[3S]Met from9to24 hp.i. Thecytoplasm fractionwas

prepared andchromatographedonConA-Sepharose.Appropriate column fractionswerepooled and analyzed

bySDS-PAGE. (A)Markerpolypeptides: Ad2-infectedKBcells extracts labeled with [3SJMet by the CH

enhancementprocedure; (B) infected, flow-through fraction; (C)mock infected, flow-through fraction; (D)

infected,washfraction; (E)mockinfected, wash fraction; (F) infected, methyla-D-mannosideeluate; (G) mock

infected,methyl-a-D-mannosideeluate.

..)

R

... s_

1;lcx

.

2

)

-t

.5K-~

5K

I; ....

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Ad2 EARLY GLYCOPROTEIN 321

Ad2

Infecti

CV-1

Cell

A

B

C

ft It _ ir

t

I

;b

DBP(79K)-21K

--IlK

._Auh

tF~~~~~~~

i. (.- I

f.

4.0* k 1

a

_i

8.8K-8.3K

about 20K to 21K that cannot be detected in thesesingle-dimensiongels.

Our results also strongly suggest that the

20/21K

polypeptide

is coded

by

an

early

Ad2

gene, and is not a cellcodedpolypeptideinduced

by

Ad2. Both

[3H]glucosamine

and

[35S]Met-labeled 20/21K polypeptide were immunopre-cipitated by rat antiserum against T2C4 cells. T2C4 is a line ofAd2-transformed rat cells (6) that contains all four of the Ad2 early gene

blocks(7) andsynthesizes RNA derived fromall

four blocks (5). Thepolypeptidewas not precip-itatedby nonimmune rat serum, orbyrat

anti-sera against three other Ad2-transformed rat

cells (notshown). Therefore, itisprobable that

the Ad2 sequences present in T2C4 cells (but not in the other 3 Ad2-transformed rat lines) synthesize the 20/21K polypeptide. A 20/21K

polypeptide was also induced in Ad2-early

in-fectedmonkey and hamster cells. These results suggest that the 20/21K polypeptide is viral coded, because it isimprobable (but not

impos-sible) that a cell-coded 20/21K polypeptide

would be synthesized by Ad2-infected human,

monkey, andhamstercells,andbyonebut not

allAd2-transformed rat cells.

We are currently attempting to purify the

20/21K polypeptideforfurther chemical,

phys-ical,

and

biological

characterization.The

protein

seemstoexist in

multiple

charge

forms,

because

it is present in fractions eluted from

ion-ex-change

resins

by

salt concentrations of from 10

mM to 0.4 M.Also,it seems to exist in

multiple-size forms, as reflected by its behavior on gel

filtration columns. An exampleof this is shown in Fig. 3, where the 20/21K polypeptide

spon-taneously polymerizedto44K,the 44K

sponta-neouslyconverted to21K,andboth the 44K and

the20/21K polypeptide polymerizedtospecies

ofhighmolecularweight. Thistype of apparent size andchargeheterogeneityistypicalofmany

glycoproteins.We have notattemptedtoreduce

7* and alkylate the 20/21K

polypeptide

to

deter-minewhethersulfhydralgroups areinvolvedin

the polymerization of thepolypeptide.

Chin and Maizel (2) reported that a

35S-la-beled

polypeptide

(E2)was acomponent of the

plasma membrane. E2, apparently, is the same

polypeptide that Ishibashi and Maizel (13)

re-portedtobelabeledwith[3H]glucosamine. The

20/21Kpolypeptide probably correspondstoE2, because we have seen no indication that any otherearly proteinisglycosylated. Ourfinding

I

M M

CH

CH CH

CH+

FIG. 6. [35S]Met-labeledpolypeptides induced in Ad2-early infected monkey (CV-1) cells, with and without CH(25pg/ml)pretreatment. (A)Infected, no

CH;(B)infected,withCH; (C) mock infected, no CH; (D) mockinfected, with CH.

VOL. 28,1978

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JENG, AND GREEN

that the 20/21K polypeptide is located mainly in the cytoplasm of Nonidet-P40-lysed cells would be consistent with thepossibility that this polypeptide is a membrane component.

How-ever,wehavealso found the20/21Kpolypeptide in a complex purified from lysed nuclei that synthesizes Ad2 DNA (21). The 20/21K poly-peptide can also be immunoprecipitated by

T2C4 antiserum from the nucleoplasm of cells. Therefore, anuclear role forthispolypeptide is notexcluded.

Glycoproteinsarewidelydistributed innature

and serve a variety of structural, lubricating, enzymatic, hormonal, and plasma membrane-as-sociatedfunctions (15, 25,32). The finding that

anAd2-earlygeneproduct isaglycoprotein is of

interest,especially if it is foundtoserve a regu-latory function. Studies on the 20/21K poly-peptide should be ofinterestnotonly regarding Ad2 replication, but also the general role of glycosylation in protein function.

ACKNOWLEDGMENTS

We thank H. Thorntonforassistance in cell culture andin

preparation ofthe T2C4antiserum,and C.Devine for

tech-nical assistance.WearegratefultoP.H.Gallimoreforagift ofthe T2C4cells.

Thisworkwassupported byPublic Health Servicegrants Al01725-19from the National InstituteofAllergyand

Infec-tious Diseases and CA 21824-01 from the National Cancer

Institute, and by contract NOI CP 43359 from the Virus

Cancer Program within the National Cancer Institute.

W.S.M.W. waspartially supported byafellowshipfrom the

Medical ResearchCouncil of Canada.M.G. is therecipientof

aResearchCareer Award(5K06AI 04739)fromthe National

InstituteofAllergyand InfectiousDiseases.

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