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 06510-8023Received 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-gpl6Oinwhich 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 T7promoter 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 beforelysis.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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Antibody
180-, _
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-0
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30
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60
120
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*-__ m_
80
-52.5
-1 2 3 4 5 6 7 8 9 1011 12 13 14
B.
E
BxE
co
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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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[image:3.612.142.464.70.514.2]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. Todeter-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 20min(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 ofblockade 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 wasimmunoprecipi-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.
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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
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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 thegpl20 molecules remained soluble and not associated with
CD4. Cells were then labeled with
[35S]methionine,
chasedfor 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 Hresis-tance withahalf time very similartothatof CD4expressed
+gpl60 a
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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 theER.
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[image:6.612.73.299.70.232.2]A.
Minutes 0
15
30 45 60 90 120 180C
CMCMCMCMCMCMCM
200
-gpl2O-
04t
92.5-
flfl69-1 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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[image:7.612.122.481.75.386.2]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 generaleffectson 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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