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CD4

Is

Retained

in

the

Endoplasmic Reticulum by the

Human

Immunodeficiency Virus

Type 1

Glycoprotein

Precursor

BRUCE CRISE, LINDA BUONOCORE, AND JOHN K. ROSE*

Departments of Pathology and Cell Biology, Yale University School of Medicine, 310 Cedar Street, New

Haven,

Connecticut 06510-8023

Received 12 June1990/Accepted 14August 1990

We analyzed coexpression of the humanimmunodeficiency virustype 1glycoproteinprecursor,gpl60, and

its cellularreceptorCD4 in HeLa cellstodeterminewhether thetwomoleculescaninteract priortotransport tothe cell surface. Results of studies employing coprecipitation, analysis of oligosaccharide processing, and

immunocytochemistryshowed that newly synthesized CD4 andgpl60 formacomplex priortotransportfrom

the endoplasmic reticulum(ER). CD4 expressed by itselfwas transported efficiently from the ERtothe cell surface, but the complex of CD4 and gpl60 wasretained in the ER. This retention of CD4 within the ER is

probablya consequenceof the veryinefficienttransportofgpl60 itself (R. L. Willey, J. S. Bonifacino, B. J. Potts, M. A. Martin, and R. D. Klausner, Proc. Natl. Acad. Sci. USA 85:9580-9584, 1988). Retention of CD4 inthe ER by gp160maypartially explain the down regulation ofCD4 in human immunodeficiency virustype 1-infected T cells. Inhibition of CD4 transport appears to be a consequence of the interaction of two membrane-bound molecules, becauseacomplex of CD4 andgpl20 (the soluble extracellular domain of gp160)

wastransported rapidly and efficiently from the ER.

The envelope glycoprotein of the human

immunodefi-ciency virus type 1 (HIV-1) (2, 10) initiates infection by

binding to the cellular surface glycoprotein CD4 and

medi-atingthefusion of viralandcellular membranes (22, 23, 33).

Following HIV-1 infection, there is a progressive loss of

CD4-positive lymphocytes that severely compromises the

host immune system and leads to the development of the

acquired immune deficiency syndrome (AIDS).

CD4 is a cell surface glycoprotein found mainly on

T-helper cells and monocytes (30, 38). Infection of T cells

with HIV-1leadstoareduction in the cell surface expression

of CD4 (4, 11, 14). CD4 down regulation may be caused by

more than one mechanism. Reduced levels ofCD4 mRNA

andprotein aswell as reduced cell surface expression have

been reported previously (11, 42). Recently, expression of the HIV-1glycoprotein alone, inthe absence of other HIV-1

proteins, has been showntoreduce the level of CD4

expres-siononthe cell surface(13). Intracellularbindingof CD4by

gp160was suggestedasapossible mechanism ofCD4 down

regulation (11, 13, 36).

The surface glycoprotein of HIV-1 is synthesized as a

heavily glycosylated precursor (gp160) that is inefficiently

transported to the cell surface. Infact, the majority of the

protein is retained in the endoplasmic reticulum (ER) or

degraded inlysosomes. Only 5 to 15% of the total protein

synthesized is cleaved to form the mature, heterodimeric

gpl20/41 moleculeandexpressedonthecell surface(41). In

otherstudies,wehavefound thatCD4,incontrast togpl60,

istransported rapidlyandefficientlyfrom the ERthroughthe

Golgi apparatusto the cell surface. We therefore undertook

the work reported here to determine whether CD4 might

interact with gpl60 in the exocytic pathway and whether CD4 transport was blocked or retarded through interaction

with thepoorly transported gp160 molecule. Wereportthat

aspecificinteraction of CD4 andgpl60doesoccurin the ER

and that CD4boundto gp160isblocked in transportto the

*Correspondingauthor.

cellsurface. Otherconcurrentstudiesinthislaboratory have established that a soluble form of CD4bearing a signal for retention in the ER can interact with the HIIV glycoprotein

and block its exit from the ER (3a). Possible roles for the interaction between CD4 andgp160in HIVpathogenesisare

discussed.

MATERIALS ANDMETHODS

Constructionofplasmids.Theconstructionoftheplasmids

encoding gpl60, gp120, and CD4 under the control ofthe

bacteriophage T7 promoter (pBS-gp160, pBS-gp120, and

pBS-CD4, respectively) has been described previously (3a,

34). A plasmid encoding vesicularstomatitis virus Gprotein

under T7 promoter control (pBSG) was constructed by

excising the entire coding region ofpSVGL3 (31) by using

the restriction endonucleases XhoI and BamHI. This

frag-ment was isolated and cloned into Bluescript SK+

(Strata-gene, SanDiego, Calif.).

Expression, radiolabeling,andimmunoprecipitationof

pro-teins. HeLa cells(approximately5 x 105cellsper6-cmdish)

wereinfectedwitharecombinantvaccinia virusencodingT7

polymerase (vTF7-3) (9)atamultiplicityof 10to25 in 0.5ml

of Dulbecco modified Eagle medium (DMEM) without

se-rum for 30 min at 37°C. The inoculum was then removed,

and cellswere transfected with 5 ,ugofplasmidDNA in 1.5

mlofDMEM, lackingserum,byusingaliposome-mediated

procedure similartothatdescribed by Felgneret al. (7)but

employing dimethyldioctadecyl ammonium bromide as the

cationic lipid (J. Rose, L. Buonocore, and M. Whitt, sub-mitted forpublication). Transfected cellswereincubated for

3 to4 h,washedoncewithphosphate-buffered saline (PBS)

(10 mM NaH2PO4, 10 mM Na2HPO4, 150 mM NaCl [pH

7.4]), and labeled for the timesindicated(see legendstoFig. 1-3 and 6) with [35S]methionine in DMEM lacking methio-nine. Cells were then washed once with ice-cold PBS and furtherincubated in DMEM with 5% fetalbovineserumand excessmethionineat 37°Cforvarioustimes.

Prior to immunoprecipitation, cells were washed once

with PBS and lysed in 1 ml of a solution (lysis buffer)

5585

0022-538X/90/115585-09$02.00/0

Copyright © 1990, American Society for Microbiology

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EDTA, and 10 mMTris hydrochloride, pH 7.4. Nuclei were

removed by centrifugation at 10,000 x gfor1min. ForCD4

immunoprecipitations, sodium dodecyl sulfate (SDS) was addedto aconcentration of0.1%tothe lysateand 100 ,ul of

OKT4 hybridoma (American Type Culture Collection)

cul-ture supernatant was thenadded.After incubation at 4°C for

60 min, antibody-antigen complexes were precipitated with fixed Staphylococcus aureus (Calbiochem-Behring) and

washed threetimes. OKT4 recognizes an epitope other than

the gpl60-binding site (17) and is capable ofbinding CD4

complexed to gpl60. Immunoprecipitations of gpl60 and

gpl20 wereperformed inlysis buffer plus0.2%SDSby using

1 ,ulof sheep anti-gpl20 serum (AIDS Research and Reagent

Program reagent number 288). After incubation at 37°C for

30 min, antigen-antibody complexes were precipitated and

washed asdescribed above. Immunoprecipitates were

ana-lyzedon 10% polyacrylamidegels containing SDS (16), and the gelswerepreparedfor fluorography, dried, and exposed to preflashed X-ray film by the method of Bonner and Laskey (3). Endoglycosidase H (endo H [37]) treatments of immunoprecipitations were performed by the method of

Rose and Bergmann (31).

Indirect immunofluorescence. Localization of CD4 was

performedoninfectedcells thatweretransfectedwith either pBS-CD4(5

[.g)

or amixture of pBS-CD4 andpBS-gpl6Oin

which pBS-gpl6O DNA was in fivefold excess (0.8 and 4.2

p,g, respectively). This DNA ratio gave two to three times more gpl60 than CD4 expression as judged by metabolic labeling(data not shown). Three hours after the transfection, the transfection medium was removed and replaced with 2 ml ofDMEM containing 5% fetal bovine serum, and the resulting medium was incubated for an additional 3 h. Cycloheximide (SigmaChemical Co.) was then added to the

mediumataconcentration of100,ug/ml, and thecells were

incubated for an additional 2 h before being washed once

with PBS and fixed. The fixation procedure was modified

from a procedure described by McLean and Nakane (25). The fixativewasprepared bycombining6 mlofH20 with 6

ml of 100 mM Na2HPO4 (pH 7.8) containing 220 mg of

lysine. NaIO4 (30 mg) was added to this solution and

vortexed thoroughly before the addition of 3.66 ml of8%

paraformaldehyde plus 8 mM NaOH. Cells were incubated

in fixative for 2 h at room temperature, washed once with

PBS plus 10 mMglycine (PBS-G), and soaked overnightin

PBS-Gbefore being permeabilizedin 1% TritonX-100.Cells wereincubatedwithOKT4hybridoma supernatant or sheep anti-gpl20serum(diluted1:200inPBS-G) for30 minat room

temperature and washed inPBS-G, followed by incubation

withrhodamine-conjugatedgoatanti-mouse (Cappel) or flu-orescein-conjugated rabbit anti-sheep (Jackson

Immunore-searchLaboratories) antibodies. Cellswereobserved witha

NikonMicrophot-FXmicroscope by epifluorescence

illumi-nation with a 40x oil immersion objective. Photographs

were taken with Ilford XP1 film developed by C41

process-ing.

RESULTS

CD4 andgpl60formacomplex when expressed in thesame cell.Todetermineif aCD4-gpl6Ocomplex could be detected

when both proteins were expressed together, we used the

vaccinia-T7 hybrid expression system described by Fuerst et

al. (9). Plasmid DNAs encoding

gpl60

and CD4 under T7

promoter control were transfected into HeLa cells that had

first been infected with a recombinant vaccinia virus,

DNA Antibody

None

o

cx.

CD4

0

't

cmJ

0a)

180

-110

-gpl60

0

00)C\

I

80

-65

-52.5

-+

CD4

0

QCD

_e$ -gPl160

ga -CD4

1 2 3 4 5 6 7 8

FIG. 1. Coexpression and coprecipitation of CD4 and gpl60. HeLa cells (5 x 105 cells per plate) infected with a recombinant vacciniavirus, vTF7-3, weretransfectedwith cDNAsencoding CD4 (pBS-CD4[5

jug])

orgpl60 (pBS-gpl60 [5 ,ug])or with both pBS-CD4andpBS-gpl60 (1.25arld3.75 ,ug,respectively)orleft untrans-fected. Cells were pulse-labeled for 30 min with 50 ,Ci of [35S]methioninein 0.5 mlofmethionine-free medium and incubated in chase mediumcontaining excess methioninefor 30 min before

lysis.Cell lysatesweredivided in half andimmunoprecipitated with

monoclonalantibody (OKT4)toCD4(odd-numbered lanes)orwith sheepanti-gpl20serum(even-numbered lanes), and immunoprecip-itateswereanalyzed by SDS-PAGE followedbyfluorography. The DNAs used to transfect the cells are indicated above the lanes. Positions ofmolecularweight markers(in thousands)areshownon

theleft.Thepositions of CD4andgpl60areindicatedontheright.

vTF7-3.This virus encodes bacteriophage T7 RNA

polymer-ase, which transcribes the cytoplasmic plasmid DNA and

yields a high level of protein expression. When plasmids encoding two different proteins were cotransfected, there

wasgreater than95% coexpression, as determinedby

dou-ble-labelimmunofluorescence(datanotshown).

HeLa cells expressing gpl60, CD4, or gpl60 and CD4

were metabolically labeled with [35S]methionine for30min

and then incubated in the presence of excess unlabeled

methionine for an additional 30 min. Cells were lysed in

detergent,and thelysatesweredivided in half and immuno-precipitated with antibodies to gpl20orCD4 (Fig. 1). The immunoprecipitates were analyzed by SDS-polyacrylamide gelelectrophoresis (PAGE).Theresults show thatCD4and

gpl60 were expressed and specifically precipitated by the

antibodies (Fig. 1, lanes 3 and 6, respectively). In cells

cotransfected with cDNAs encoding gpl60and CD4, both

proteinswereexpressed andwereprecipitatedas acomplex

either by antibody to CD4 or gpl20 (note that gpl60 was

present in the immunoprecipitate prepared with anti-CD4

antibodies and CD4 was present in the immunoprecipitate

prepared with anti-gpl20antibodies [lanes 7 and 8]).

Wealsoperformedanexperimenttodetermine if CD4 and gpl60 expressedin separate cultures would formacomplex

after detergent lysis. Two plates of cells, one expressing

CD4 and oneexpressinggp160, werelabeledasabove. The

cells were thenremoved from the dishes and mixed before

lysis and immunoprecipitation. No complex formation was

detected (data not shown), suggesting that the association

detected aftercoexpressionwas occurring during

coexpres-sion in thesame cell.

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Minutes

Antibody

180-, _

110

-0

10

30

45

60

120

.O..o C o

m) mV)0 V)C co c CD ::CD)

*-__ m_

80

-52.5

-1 2 3 4 5 6 7 8 9 1011 12 13 14

B.

E

BxE

co

E

0

Minutes

FIG. 2. Rate ofintracellularbinding of CD4 and gp160. (A) Seven plates of HeLa cells (5 x 105cellsperplate) infected with vTF7-3 and transfected with pBS-CD4 (1.25 Fg) and pBS-gpl60 (3.75 ,ug) DNAs were pulse-labeled with 50 p.Ci of [35S]methionine in 0.5 ml of methionine-free mediumfor 10 min and then incubated in chase medium for the times indicated. Cellswerelysedatthetimesindicated, and celllysatesweredivided in half andimmunoprecipitated with either OKT4 (even-numbered lanes)orsheepanti-gpl20serum(odd-numbered

lanes) and analyzed by SDS-PAGE followed by fluorography. Background bands (labeled VV) precipitated from vTF7-3 infected, but untransfected, HeLa cellsareindicated incontrol lanes 13 and14(120-min chase). These bandsarealsoseenin lanes9through12.Positions

ofmolecular weight markers (in thousands)are shownatthe left. (B) Thepercentage of maximal CD4and gp160 bindingovertimewas

quantitated from densitometry of the data shown in panel A. The ratios of CD4togp160(lI)in immunoprecipitations prepared with OKT4 antibodyor the ratios ofgp160to CD4 (*) in immunoprecipitations prepared with anti-gp120 serum are expressedas percentagesof the highest ratio observed.

Rate ofgpl60andCD4 association. Todetermine therate

ofassociation betweengpl60andCD4,HeLa cells

express-ing both CD4 and gpl60 were pulse-labeled with [35S] methionine and incubated in the presence of unlabeled

methionine for various times(Fig. 2A). Detergent lysates of

the cells were divided into twoequal parts, immunoprecip-itated with antibodies to gpl20 or CD4, and analyzed by

SDS-PAGE (Fig. 2A). Immediately after the pulse (time

zero), there was no association ofnewly synthesized CD4

andgpl60 asjudged by lack ofcoprecipitation (lanes 1 and

2). By30min,someassociationwasevident(lanes5 and6).

Quantitation of the autoradiogram (Fig. 2B) showed that

maximalassociationoccurred after about 60minand that the half time for binding was approximately 30 to 40 min.

Precipitationoftwolabeled vaccinia virusproteinsoccurred

at later time points (Fig. 2A, bands marked VV). These

proteins are not derived fromCD4 orgp160, becausethey

werepresentwhen thecellswerenottransfectedwith DNAs

encodinggpl60 orCD4 (lanes 13 and14).

Our data are consistent with an earlier study indicating

A.

- >-VV 0. doo

.-W qmmm -M laom

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became competent to bind a soluble form of CD4 in vitro,

withahalf time of about 30 min (8), indicating that folding of

gp120 is required before binding of CD4. The long lag before binding observed here (Fig. 2) is probably also due to a requirementfor folding prior to binding.

Coexpression of gpl60 blocks oligosaccharide processing on CD4. Association of CD4 and gp160 with a half time of 30

min suggested that the association might be occurring early

in theexocytic pathway. To obtain information on where the

association was occurring and to analyze the effect of gp160 expressiononCD4 transport, we examined the processing of

the N-linked oligosaccharides on CD4 in the presence or

absence ofgpl60 expression. CD4 has two N-linked glycans,

oneof which acquires resistance to the enzyme endo H. The

high-mannose oligosaccharides added in the ER become resistant to cleavage by endo H after the sequential action of N-acetylglucosamine transferase and mannosidase II (15).

TheN-acetylglucosamine transferase is located in the medial

Golgi compartment, suggesting that this is the site where

endo Hresistance is acquired (6, 15).

Transfected HeLa cells expressing CD4 alone or CD4 and gpl60 were pulse-labeled with [35S]methionine and then incubated with excess unlabeled methionine for various times. CD4 was then immunoprecipitated from cell lysates.

Halfof each immunoprecipitate was digested with endo H,

and the remainderwas left untreated (Fig. 3). The

oligosac-charideson CD4expressed alone became endo H resistant,

with a halftime of approximately 45 min (Fig. 3A). It is

evident thatonly one of the two N-linked oligosaccharides

onCD4 became endo Hresistant,because the resistant band

migrated halfway between the fully cleaved and uncleaved material.

Coexpression ofCD4 and gp160 resulted in a dramatic inhibition ofoligosaccharide processing on CD4 (Fig. 3B). Also, theassociation ofCD4 withgp160isevident from the coprecipitation occurringat later times. Quantitation of the

results in Fig. 3A and 3B is shown in Fig. 3C. Only about

15% of the CD4 expressed with gpl60 obtained endo H

resistanceevenafter 3 h. It isalso evident that the oligosac-charides ofgp160 (which precipitated with CD4) remained

sensitive toendo H treatment (Fig. 3B). The shift in gp160

mobilityto an apparentmolecular mass of 90 kilodaltons is

consistent with removal ofall oftheoligosaccharides (29).

Evenwhen gp160wasexpressed alone, we did not observe processing of its oligosaccharides (data not shown). This

resultis consistent withan earlier report that only 5 to15%

of

gp160

istransported (41).

Specffic

inhibition of CD4 processing bygpl60. To

deter-mine if theeffectofgp160onCD4processing was specific or

due to a general inhibition of exocytosis by gp160, we

expressed anunrelatedprotein, the surface glycoprotein of

vesicular stomatitis virus (VSV) G protein, in the presence

and absenceofgp160 (Fig.4). Cells were pulse-labeled with

[35S]methionine

and incubated in the presence of unlabeled methioninefor various times. The oligosaccharides of VSV G protein expressed alone in HeLa cells became endo H resistant, with a half time of about 30 min. This rate was unchanged in the presence of gp160. We have noted that VSVGproteinprocessing in HeLa cells is somewhat slower than in other cells, where the half times are typically 15 to 20

min(28, 40). Immune precipitation withanti-gp120

antibod-ies and anti-VSV antibodantibod-ies indicated that gp160 was

ex-pressed at levels equivalent to VSV G protein in this

experiment

(datanot shown). We conclude that the effect of

blockade of exocytosis by gpl60.

CD4 bound togpl60is retained in the ER. The results of

the metabolic labeling experiments followed by endo H

treatment indicated that the CD4bound togpl60 contained

unprocessed oligosaccharides (Fig. 3). This lack of CD4

processing could be explained in two ways. CD4 bound to

gpl60 could have been retained at a sitepriortothemedial

Golgi compartment or, alternatively, gpl60 binding to CD4

might have had a more direct effect on oligosaccharide

processing, such as inhibiting the access of processing

enzymes to the CD4 oligosaccharides. To decide between

these alternatives, we used indirect immunofluorescence

microscopy toexamine the localization of CD4 synthesized

in the presence or absenceof gp160.

HeLa cells expressing CD4, gp160, or CD4 and gpl60

were treated with cycloheximide for 2 h and fixed with a

modified paraformaldehyde fixative (25) to maintain the

epitope recognized by the OKT4 antibody. Cycloheximide

treatment blocks protein synthesis without disrupting

pro-tein transport through the exocytic pathway (12). This

treat-mentallowedsufficient time for CD4 molecules made in the

absenceofgpl60 to reach the cell surface. After fixation, the

cells were made permeable with detergent (to allow entry of

antibody) andincubated with OKT4 antibody followed by a

rhodamine-conjugated second antibody. Figure 5A shows

that CD4 expressed alone reached the plasma membrane

(notethat theoutline of cell is labeled). When cells were not

permeabilized, only the surface outline was seen. In perme-abilized cells, CD4 was also seen in a punctate pattern which

may represent endocytic vesicles (27). When CD4 was

expressed with gpl60, CD4 was not detectable at the cell

surfaceand was seen only after permeabilization (Fig. 5C). CD4 appeared in a reticular pattern throughout the

cyto-plasmand in the nuclear membrane (ring stain around the

nucleus). This pattern is typical of proteins localized in the

ER (26, 31).Localization of gp160 expressed alone (Fig.5B)

gave a similar pattern of fluorescence, indicating that much

ofthe gp160 remains in the ER during the cycloheximide

treatment. This localization is consistent with the lack of

oligosaccharide processing on gpl60. The localization of

CD4 in cellsexpressing gpl60 and the lack of

oligosaccha-ride processing on CD4 bound to gp160 indicates that CD4 bound to gp160 is blocked from exiting the ER.

CD4-gpl2O complexes are not retained in the ER. During

transportof gpl60 to the cell surface, the molecule is cleaved

to form two noncovalently associated subunits, gpl20 and

gp41 (1). gp4l is derived from the C terminus of gpl60 and

contains the membrane-spanning domain, while gpl20 com-prises most of the extracellular portion and contains the binding site for CD4 (4, 24). We had observed that gpl20 expressed alone (from a mutated clone encoding only the

soluble gpl20portion ofgpl60) was also transported

ineffi-ciently from the ER (3a). Therefore, we wanted to determine if gpl2O-CD4 complexes would form in the ER and be transported or retained.

Wefirst expressedgpl20alone and examined itssecretion

(Fig. 6A). HeLa cells expressinggpl20 were pulse-labeled

with

[35S]methionine,

and then gpl20 was

immunoprecipi-tatedfrom the cell lysatesor medium at various times after

the addition of unlabeled methionine. A small amount of

gpl20wasdetectable in the medium after 1 h, and about 15%

was secreted after 3 h. We were unable to detect any endo

H-resistant oligosaccharides on the intracellular gp120 at any

time(Fig. 6A, lane 19). In contrast, allgpl20in themedium

contained some resistant oligosaccharides (indicating Golgi

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A.

Minutes

CD4(+CHO)-m CD4(-CHO)

-Minutes

0 10 20 30 45 60 90 120 180

- - _ _ _ _- DIVV

1 3m4 me l am

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

0 10 20 30 45 60 90 120 180

No

_m

_o m_ -gpl60

(+CHO)

- _ gpl60

(-CHO)

CD4(+CHO)

--CD4(-CHO)

-_

--p

Dv-

se

-6-_am_0_ p _ -- __

-,.

-_m go On _0f _ _

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

C.

10

0

co

80 60 40

20

0

30 60 90 120 180

Mirules

FIG. 3. Oligosaccharide processing onCD4 expressedin the presence and absence ofgpl60. Nine plates ofHeLa cells (infectedand transfected as described in the legend to Fig. 2) expressing CD4 (A) or CD4 and gpl60 (B) were labeled for 10 min with 50 ,uCi of

[35S]methionine in 0.5 mlof methionine-free medium and thenincubated in chase mediumcontainingexcessunlabeled methionine forthe

times indicated. Immunoprecipitates of cell lysates were prepared with OKT4 antibody, divided in half, and digested with endo H (even-numbered lanes)orleftundigested (odd-numbered lanes). Sampleswereanalyzed by SDS-PAGEandautoradiographed (AandB).The positions of CD4andgp160witholigosaccharides (+CHO)and afteroligosaccharide cleavage(-CHO)areindicated. Thetwobackground bandsalso seen innontransfectedcells(Fig. 2)appear here andarelabeledVV.The fraction of CD4 with endoH-resistantoligosaccharides

ateachtimepointwasquantifiedby scanningdensitometryof afluorographed gel (C). processing), since it ran as a broad band after endo H

digestion at a position between the undigested and fully

digested material (lane 17). Thelack ofprocessed

oligosac-charides on cell-associated gpl20 suggeststhat secretion is

veryrapid once the protein reaches the medial Golgi com-partment.

To determine whetherintracellularCD4-gpl2O complexes

formed in the cells and whetherthey were transported, we

coexpressed gpl20 at about a twofold molar excess over

CD4 in HeLa cells. Under theseconditions, nearlyall ofthe

CD4 associated with gpl20 and

approximately

half of the

gpl20 molecules remained soluble and not associated with

CD4. Cells were then labeled with

[35S]methionine,

chased

for varioustimes,andimmunoprecipitated withantibodyto

CD4. Half ofthe immunoprecipitate from each time point

was treated with endo H, and the remainder was left

untreated (Fig.6B). Analysisof the samples bySDS-PAGE

showed thatassociation ofgpl20with CD4 occurredwhile

theoligosaccharides on both proteins werestill sensitive to

cleavage byendoH, suggesting bindinginthe ER

(Fig.

6B,

lanes 5 to 8). The rate of association was similar to that

observed withgpl60 and CD4(Fig. 2).

The CD4 associated with gpl20

acquired

endo H

resis-tance withahalf time very similartothatof CD4expressed

+gpl60 a

0 -1- . I I . I T

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5

30 45 60

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FIG. 4. Processing of oligosaccharides on vesicular stomatitis virus(VSV) G protein expressed in the presence and absence of

gpl60. Five plates of HeLa cells (5 x 105cells perplate) infected

withvTF7-3 and transfectedwith pBS-G aloneorbothpBS-G and

pBS-gpl60werelabeled for 10min with 50,uCi of [35S]methionine in

0.5ml ofmethionine-free medium and incubated in chase medium containing excess unlabeled methionine for the times indicated.

Immunoprecipitates ofcell lysates were prepared from each time point withrabbit anti-VSVserumand incubated in thepresence or

absence of endo H. Thepercent of endo H-resistant oligosaccha-rides on G protein at each time point was quantitated from a

fluorographed gel andplotted. Symbols: *, -gpl60; E, +gpl60.

alone (-45 min), indicating that its transport was not

blockedorslowedby associationwithgpl20. Wealsonoted

thatthegp120boundtoCD4attained endo H resistance. The

fully endo H-sensitive form of gp120migrated slightly slower

thanfully glycosylated CD4 (7 and 8), whereas gpl20 with

endo H-resistantoligosaccharides migratedas abroad band,

presumably duetoheterogeneous processing ofits multiple

oligosaccharides (Fig. 6B, lanes8, 10, 12, 14, and 16). This

heterogeneity made it difficult to quantitate the amount of

endo H-resistantgp120 directly, butonthe basisof therate

ofdisappearanceof the endo H-sensitive band ofgpl20, we

estimated that the gpl20 associated with CD4 was being

processed at the same rate as CD4. This result would be

expected for two molecules passing through the exocytic

pathwayas acomplex. Thegpl20 associatedwith CD4 also

showed an increase in apparent molecular weight at later

timepoints, presumably duetothe additionofterminalsialic

acid on its oligosaccharides (lanes 9, 11, 13, and 15). By

carryingoutasecondround ofimmunoprecipitations onthe

samesamples with antibody to gp120,we werealso ableto

followtherateofprocessing and secretion of thegpl20that

was notassociated with CD4 (about 50% of the total). This

material appeared to be processed like gpl20 expressed alone. Thecell-associatedprotein showed no endo H resis-tance, while a small endo H-resistant fraction (10 to 15%)

was found in the medium after3 h (data notshown).

Theseresultsindicate that CD4transportis notslowed by association with gpl20. Instead, it appears thatgpl20-CD4

complexes are transported at the normal CD4 rate, much

fasterthan gp120 expressed by itself.

DISCUSSION

The results presented here show that specific binding of theHIV-1precursorglycoprotein (gpl60)totheCD4 recep-tor occurred within the ER when both proteins were

ex-pressedin thesamecell. Bindingwasobserved afterashort

(;lD4

anti-CD4

B.

antI9p120

C.

CD4

FIG. 5. Detectionof CD4andgpl60 byindirect immunofluores-cence. HeLacells(5x 105 cellsperplate)platedoncoverslipswere

infectedwith vTF7-3 andtransfectedwithpBS-CD4,pBS-gp160,or

amixture of bothplasmids(4.2and 0.8 ,ug,respectively).Cellswere

treatedwithcycloheximide(100,ug/ml)6hposttransfectionfor 2 h and subjected to fixation (see Materials and Methods). The cells were then permeabilized by treatment with 1% Triton X-100 and treated with either hybridoma supernatantcontaining OKT4 anti-body (A and C) orwith sheepanti-gp120 serum(B), followed by

treatmentwith fluorescent secondary antibodies (rhodamine-conju-gated anti-mouse orfluorescein-conjugated anti-sheepantibodies). Twotypicalexamplesareshown in eachcase.

lag period and occurred with a half time of about 30 min.

Thislagperiod probablyrepresentsthe timerequiredforone orbothproteinstofold and displaytheirrespective binding

sites. An earlier study of the time required for gp120 (the

portionofgp160 containing theCD4-binding site) tofold in

vivo and then bind CD4 invitro also determined the half time

tobe 30 min(8). Thefoldingofgpl20may therefore be the

rate-limiting step in the association. The topology of CD4 bindingtogpl60in theERisnotentirelyclear. Onenormally envisions the extracellular domains of CD4 and the HIV-1 glycoprotein interacting from separate membranes during

virus-cell or cell-cell interactions. Our results indicate that

theseextracellular(luminal)domainscanalsointeract within

the membranes of the ER. It is not clear if the interaction

occurs between molecules thatarenextto each otheror on opposite sides ofER cisternae.

Wealso observedaprofound inhibition of CD4 transport

tothecell surface when CD4wasexpressedin thepresence

of gp160. Greater than 95% of CD4 expressed alone was

transported efficiently from theERthroughthe Golgi

appa-ratus, whereitacquiredanendoH-resistantoligosaccharide

(half time, -45 min). In contrast, when expressed in the

presenceofexcessgp160, 85to90%of CD4 didnotacquire

any endo H-resistant oligosaccharides and was not

ex-pressed on thecell surface. Indirect immunofluoresence of CD4 expressed with excess gpl60 showed that CD4 was

retainedby

gpl60

inside thecells in apatterntypicalof the

ER.

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[image:6.612.73.299.70.232.2]
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A.

Minutes 0

15

30 45 60 90 120 180

C

CMCMCMCMCMCMCM

200

-gpl2O-

04

t

92.5-

flfl

69-1 2 3 4 5 6 7 8 9 10 1112131415

180

200 hA _C

92.5

- *

69-

it

16 17 18 19

Minutes 0 15 30 45 60 90 120 180

gpl

20(+CHO)-92.5- ]gpl2oEndo Hr

69-t

gpl20(-CHO)- -CH

CD4-( 4 4 4 _ -CD4 EndoHr

46

-1 2 3 4 5 6 7 8 910111213141516

FIG. 6. Transport and secretion of gpl20 expressed in thepresenceand absence of CD4. (A) Nine plates of HeLa cells (5 x 105 cellsper

plate) expressing gp120werepulse-labeled with [35S]methionine for10min and incubated in chase medium for the times indicated. Celllysates

orchase medium(C and M,respectively)wereimmunoprecipitated with anti-gpl20 antibodies. Immunoprecipitates of cellular and secreted

gpl20 (after incubation for 3 h in chase medium)weretreated with endo H (lanes 17 and 19)orleft untreated(lanes 16 and 18). Positions of

molecularweight markers (in thousands)areindicated. (B) Eight plates of HeLa cells (5 x 105 cellsperplate)wereinfected with vTF7-3 and transfected with bothgp120 and CD4 (1.2 ,ug of pBS-gpl20 and 3.8 ,ug of pBS-CD4perplate). These cellsexpressing gpl20 and CD4were

pulse-labeled with [35S]methionine for 10minand incubated in chase medium for the times indicated.Immunoprecipitateswerepreparedwith

OKT4and incubated in thepresence(even-numbered lanes)orabsence(odd-numbered lanes) of endo H. Endo H-resistantformsofCD4and

gpl20 (gpl20 Endo Hr and CD4 Endo Hr)areindicated. The positions of gp120 with oligosaccharides (+CHO) and without oligosaccharides (-CHO)areshown. Allsamples (A and B)wereanalyzed by SDS-PAGE followed by fluorography.

Theblockade of CD4transport observed hereappears to

result from CD4 associationwith thevery slowly and

inef-ficiently transportedgpl60 molecule (41). Inagreementwith

thisearlier studyongpl60transport (41), wefind that most

(90to95%)ofgpl60 synthesized isnotdelivered tothe cell

surface but isdegraded insidethe celloraccumulates in the

ER (unpublished results). An inhibition of surface

expres-sion ofCD4as aconsequenceof HIV-1 infection(4, 11, 14,

36)or ofexpressionofgpl60alone (13)has been observed

previously. In the latter study, the surface expression of

CD4 was reduced after expression in gpl60 was initiated. The resultspresentedhere indicatethat the inhibition of CD4 transportfrom the ERbygpl60canbeasgreatas10-fold. In

fact, CD4thatescapes thegpl60blockade maybedoing so

in cells thatarenotcoexpressinggpl60inexcess overCD4.

DuringanHIV-1 infection, iftherate ofsynthesisofgpl60

exceeds that ofCD4,theexcessgpl60mayeffectivelyblock transportofnewly synthesizedCD4tothecell surface. This mechanism of downregulationof the receptormightprevent

rebinding of virustoinfected cells.

Thedisappearance ofCD4+ Tlymphocytes isahallmark

of AIDS. Several mechanismsthroughwhich CD4+ T

lym-phocytes are depleted have been proposed. Formation of

T-cell syncytia leading to cell death (19, 20, 35) or direct

killingof infectedoruninfected cells(withshedgpl20bound

toCD4) bythe host immune system(21)mayberesponsible

forthe disappearance ofCD4-positive cells. A blockade of

CD4transportbygpl60 might partially explaindecreases in

CD4+ T lymphocytes without cell killing by HIV-1. Also,

depletion of CD4 from the surface of lymphocytes would

result in loss of adhesion(5)and signaling (32, 39)functions of CD4 and probably interfere with the normal immune

response.

In addition to studying the interaction of CD4 with the HIV-1 glycoproteinprecursorgpl60, wealso examined the interaction of CD4 withgpl20 expressed byitself. We found that this solubleportionof thegpl60molecule wassecreted

fromcells, asexpectedfrom earlier studies(18). The

secre-tion of gpl20, like the transport of gpl60, was slow and

inefficient, with only about 15% being released from cells

after 3 h. Like gpl60, soluble gpl20 was also able to bind CD4 in the ER when the two proteins were coexpressed.

Interestingly, bindingof the solublegpl20moleculetoCD4

inthe ER did notinhibitCD4transportatall. Judgingfrom

the rate ofcarbohydrate processing on bothmolecules,the

complex of CD4and gpl20 (which contained about half of

B.

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GolgiapparatusasfastasCD4expressedbyitself(half time,

-45min). Thus,gpl20 transportwasgreatly acceleratedby

virtue of association with the more rapidly transported,

membrane-bound CD4 molecule.

An important unanswered question iswhy is CD4

trans-port blocked by association with membrane-associated

gpl60 butnotbyassociation with solublegpl20.Theanswer

does notappear to be differences in rates orefficiencies of

gpl20 andgpl60 transport, since both molecules are trans-portedwithsimilarinefficiency. Possiblythecomplexoftwo

membrane proteins, gpl60 and CD4, forms cross-bridges

within the ER cisternae. Such complexesmight notbe able

to be incorporated into transport vesicles budding fromthe ERormighteveninhibitvesicle formation.Itisunlikelythat

association of CD4 with the solublegpl20would cross-link

the ER, and gpl20 would be expected to be transported

whilecomplexedtothe membrane-bound CD4atthenormal

CD4rate.

We considered thepossibility thatgpl60-CD4 complexes

arecapable ofinhibitingallproteintransportfrom the ERby interfering with the normal ER structure. Such complexes

could thenbedirectly responsiblefor T-cellkilling.Wehave

not, however, detected any effect ofgpl60-CD4 complexes

in the ERontransportofathirdprotein,the VSV Gprotein,

fromthe ER. We are now investigatingthe possibility that

higher

levels ofgpl60-CD4 complexes would have general

effectson proteintransportfrom the ER.

It should be noted that the mechanism by which gpl60

retains CD4 in theERisprobably unrelatedto theretention

of the transported fraction gpl20 or gpl60 by the

sCD4-KDELconstruct thatwe described recently (3a).This

solu-ble CD4 molecule was designed with a specific signal for

retention of soluble proteinsin the ER(26).

ACKNOWLEDGMENTS

We thank D. Brown, L. Chong, A. Shaw, M. Whitt, and P. Zagouras for helpful suggestions andcomments onthemanuscript. This workwassupported by Public Health ServicegrantA1-24345 from the National Institutes of Health. The AIDS Research and ReferenceReagent Program oftheNationalInstituteofAllergyand Infectious Diseases providedtheanti-gpl20serum(Michael Phelan, contributor) used in thesestudies.

ADDENDUM IN PROOF

Conclusions very similar to those presented here were

reachedindependently byM.A. Jabbar and D. P. Nayak,J.

Virol., in press.

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Figure

FIG.1.fected.vaccinia(pBS-CD4CD4inHeLalysis.monoclonaltheitatessheepDNAsPositions[35S]methionine chase Coexpression and coprecipitation of CD4 and gpl60
FIG. 2.oftransfectedantibodyuntransfected,lanes)quantitatedhighestcellmethionine-free molecular Rate of intracellular binding of CD4 and gp160
FIG. 3.atpositionsbandstimes(even-numberedtransfected[35S]methionine each Oligosaccharide processing on CD4 expressed in the presence and absence of gpl60
FIG. 5.andacence.treatedweretreatedgatedbodytreatmentinfectedTwo mixture Detection of CD4 and gpl60 by indirect immunofluores- HeLa cells (5 x 105 cells per plate) plated on cover slips were with vTF7-3 and transfected with pBS-CD4, pBS-gp160, or of both p
+2

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