Association of human immunodeficiency virus type 1 envelope glycoprotein with particles depends on interactions between the third variable and conserved regions of gp120.

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JOURNALOFVIROLOGY, June 1993,p. 3639-3643 0022-538X/93/063639-05$02.00/0

Association of

Human

Immunodeficiency

Virus

Type

1 Envelope Glycoprotein with Particles Depends

on

Interactions between the Third Variable and

Conserved

Regions of gpl20

RONALD L. WILLEY*ANDMALCOLM A. MARTIN

Laboratory of Molecular Microbiology, NationalInstituteof

Allergy

andInfectiousDiseases,

Bethesda,

Maryland

20892

Received 8January 1993/Accepted 2 March 1993

Many regions within theenvelope of humanimmunodeficiency virustype1 (HIV-1)that affect itsstructure

and function have been identified. We have

previously

reported that the interaction of the secondconserved (C2) and third variable (V3)regions ofgpl20influences the

ability

ofHIV-1toestablishaproductive infection

in susceptible cells. To better understand the basis for this interaction,wehave conducted structure-function analysesofenvelopeexpressedfrommolecularproviral clones ofHIV-1 containing defined mutations in C2 and

V3 that

individually

andin combination

differentially

affect envelope function. The substitution ofaglutamine

for an asparagine residue (Q-267) at a potential asparagine-linked glycosylation site in C2, which severely impairsvirus

infectivity,

reduces intracellular processing of gpl60intogpl20, the association ofgpl20with

virions, and the

ability

ofgpl20to bindtotheH1V-1cell surface receptorprotein, CD4. The change ofan argininetoanisoleucine codon inV3 (I-308),inthepresence of the Q-267 mutation,restoresvirus

infectivity

tonearwild-typelevelsby increasing theamountofgpl20 associated with virionsascompared with the Q-267

mutantbut doesnotcompensatefor theQ-267-induced processing defect. The I-308change in thecontextof

the

wild-type

HIV-1 hasnoaffectonprocessing, association,orCD4 binding. Theseresults indicate that the

impaired

infectivity

ofthe Q-267mutantvirus is duetoamarked reduction intheamountof viriongp120and suggest that theinteraction ofC2 and V3stabilizes the association of gp120withgp4l.

The envelope of human immunodeficiency virus type 1 (HIV-1) consists of two glycoprotein subunits, gp120 and gp4l (2, 29). Both proteins are derived from a 160-kDa

precursor polyprotein (gpl60) which is cleaved during its intracellular transport through the endoplasmic reticulum

and Golgi complex (7, 28, 30). The mature gp120and gp4l cleavage products associate noncovalently (17) in virions,

and together they mediate viral attachment and entry into

CD4+ cells. Duringthepast severalyears, mutational anal-yses have been used to identify functional domains within bothgpl20andgp4l (3, 6, 8, 9, 11-13, 15-18, 20, 24,31, 33, 34). The cumulative results from these studies indicate that manyregionswithin bothgpl20 andgp4l influence specific envelopefunctions. Forexample, mutationsintroduced into four conservedregionsofgp120 impairitsbindingtothe cell surface viralreceptormolecule, CD4 (6, 24).The abilityof

gp4ltopromotefusion of the viral and host cellmembranes is reduced not only bymutations located within its amino-terminal and transmembrane regions (3, 12, 16) but alsoby changes in the third variable (V3) region ofgp120 (3, 13).

These resultsarenotparticularly surprising,sinceextensive disulfide bonding within gp120 (19) and the presence of oligomeric forms for both gp120 and gp4l (10, 25, 27) position nonadjacent regionsof theenvelope glycoproteinin closeproximity in HIV-1particles.

We have previously reported that the second conserved domain (C2) ofgp120 is critical for HIV-1 infectivity; the substitution ofaglutamineforanasparagineresidue(Q-267)

at apotential asparagine-linked glycosylationsite within C2 severely impairedtheabilityof HIV-1toestablisha

produc-*Correspondingauthor.

tive infectioninCD4+ T cells(33, 34). During these studies, however, spontaneous revertant viruses that were able to replicateto nearwild-typelevels emerged. Molecular

clon-ing and sequence analyses of the revertantproviral DNAs revealed that amino acid changes within the Cl and V3 regions ofgpl20 functionally compensated for the original glycosylation site mutation(33, 34). Since thisQ-267 muta-tiondidnotimpairthe abilityof solublegp120tobind CD4 (34) and theV3regionhas been reportedtoaffect a

postad-sorption step of the infectious cycle (3, 13), we suggested

that the structural interaction of Cl, C2, and V3 could influence the fusion of viral and host cell membranes(33).

To better understand the interaction of the C2 and V3

regions, we examined envelope structure and function by usingthewild-type proteinandQ-267,

Q-267/I-308,

andI-308 envelope mutants of HIV-1 (Fig. 1). Immunoblot analyses

had previously indicated that gp120 production is not af-fectedbytheQ-267substitution(34).We have also observed that theI-308change doesnotaffectprocessing and release ofgpl20 (data not shown). Since the

Q-267/I-308

revertant had not been examined in this regard (33), quantitative pulse-chase analyses of envelope processing were carried out toevaluate theamountofgp120 producedandreleased from cells overtime. ThepNL4-3, Q-267, and

Q-267/I-308

proviral plasmidsweretransfected into HeLa cellsby using

calcium phosphate-precipitated DNA (31). At 18 to 20 h posttransfection,the cellswerepulse-labeledfor 30minwith [35S]methionine in methionine-free RPMI 1640 medium and then chased for 2, 4, and 6 h in complete RPMI medium

lacking radioactive label as previously described (31). Cell

pellets and the corresponding cell-free supernatants were

collectedimmediatelyafter thepulseand at each chasetime 3639

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3640 NOTES

gpl20

V5 gp4l 856aa

0 v1/v2 V3 V4 v 5a

C C2cc4ci

IC

1 l+

N/H \ COOH

L L L N G B L N N T R x 8 I

...Q . . ...I ..

267 308

FIG. 1. Schematic representation of the HIV-1 gpl60envelope glycoprotein indicatingconserved(CltoC5)and variable(Vi toV5) regionswithingpl20.Boxesspecifyamino aciddifferences between thewild-typeandmutantproviralclones. aa,aminoacids.

point and then subjectedto detergent lysis (31). Thegpl60

andgpl20 envelope proteinswereimmunoprecipitatedwith

apolyclonalrabbit antiserum directed againstgpl20,

resus-pendedinsamplebuffer(2%sodium dodecylsulfate[SDS], 1%2-mercaptoethanol, 1%glycerol,65 mM

Tris-hydrochlo-ride [pH6.8]), separatedon8% SDS-acrylamide-AcrylAide gels, and visualized by fluorography (31). The amount of labeled gpl60 and gpl2O recovered from the cells and mediumatthe various timepointswasquantitatedbyusing aFujixBas 2000Bio-Image Analyzer (Fuji).

The results of the pulse-chase analysis (Fig. 2) indicated that although comparable amountsofgpl60wereproduced

followingthepulse-labeling,theamountofprocessedgpl20

presentafter 2 hof chasewasreducedinboth theQ-267-and

Q-267/I-308-transfected cells (36 and 38%, respectively) compared with cells transfected with pNL4-3 (wild type).

Thisfindingis consistent withourpreviousobservation that

amutationaffectingtheadjacent266 codonimpairs process-ing by preventprocess-ingtransportofgpl60outof theendoplasmic

reticulum (31). Themigration ofboth the gpl60and gpl20 mutant proteins in the Q-267- and Q-267/I-308-transfected

cells was slightly faster than that of the corresponding

pNL4-3wild-type species (Fig. 2), suggestingthattheQ-267

mutation eliminatedanN-linkedglycosylationsite. Itshould be noted that although the I-308 revertantchange restores infectivity (Table 1), this substitution failed to compensate for the Q-267-induced processing defect. Thus, reduced

gpl20 production alone in the Q-267 mutant does not

ac-countfor thenoninfectiousphenotypeof thisvirus(Table 1).

The diminished gpl60 processing in the Q-267-transfected

cells did not cause a reduction in the amount of gpl20

released into the medium. Infact, proportionately moreof theQ-267cell-associatedgpl20wassecretedcomparedwith the wild-type envelope, since identical amounts of both

gpl2Oswerepresentin themediumby6 h ofchase(Fig.2).

Thiswasnotobserved for cellstransfected withthe replica-tion-competent Q-267/I-308 mutant; somewhat less gpl20

wasreleasedduring thesametime period.

Since a larger proportion of the gpl20 produced in the

Q-267-transfected cells wasreleased into the medium

com-paredwiththe transfection withthewild-type provirus, the possibilitythatthismutationcausedgpl20todissociate from gp4lwasexamined. Such aputative association defect (15,

17) would be expected to reduce the amount of

virion-associated gpl20. To examine this possibility, HeLa cells were transfectedwith thepNL4-3, Q-267, Q-267/I-308, and I-308 proviral plasmidDNAsandthen labeled for 24 hwith [35S]methionine aspreviouslydescribed (31). Cell-free viri-onsgenerated duringthisperiodwerepelleted by

ultracen-trifugation (31), resuspended in complete RPMI 1640

[image:2.612.51.292.79.158.2]

me-dium, and then assayed for reverse transcriptase activity

TABLE 1. Virusdesignationsandrelativeinfectivity

Virus Infectivitya Reference(s)

pNL4-3 (wildtype) +++ 1, 33, 34

Q-267 33,34

Q-267/I-308 ++ 33

1-308 +++ 33

a ++, a 2-day delay in infection kinetics compared with thepNL4-3and I-308viruses.

(34).

Comparableamountsofprogeny virions(as determined by reverse transcriptase activity) were subjected to deter-gent lysis, and the labeled viral proteinswere

immunopre-cipitated with HIV-1 antibodies present in a mixture of AIDS patient sera. The HIV-1proteins wereseparated by reducing SDS-polyacrylamide gel electrophoresis (PAGE) (10% poly-acrylamide gel) and visualized by fluorography. This analy-sis (Fig. 3) demonstrated that the Q-267 mutation markedly reduced the amount of particle-associated gpl20 compared with that found in wild-type pNL4-3 virions. The

I-308

change, in the context of the

Q-267/I-308

virions, effecteda

substantial increase in the amount of particle-associated gp120 compared with that present in the Q-267 virions, suggesting that the restored infectivity associated with the Q-267/I-308 revertant (Table 1) is due to this increase in virion

gpl20.

Itis also

interesting

to note that

virtually

no

gp4l was detected in the Q-267 virions

(Fig.

3). While this could be due to the reduced processing of the Q-267gp160 (Fig. 2), it might also reflect a requirement for gp41 to associate with gp120 intracellularly in order to facilitate efficient

incorporation

ofgp4l into particles.

We

previously reported

that the

Q-267

mutation alone did not affect CD4 binding, as judged from the use of soluble

gp120

in the

binding

assay

(34). However,

it has recently been shown that the

binding

affinity

of virion-associated

gp120

for CD4 is less thanthat offree

gp120

(22).

To examine

CD4

binding

to virus-associated

gp120,

HeLa cells were

transfected

with the

pNL4-3,

Q-267/I-308,

or I-308

plasmid

DNA, and

radioactively

labeled

virions

were

pelleted

from the medium after

steady-state

labeling of the cells with

[35S]methionine

asdescribed earlier. The

particle-associated

gp120 of the Q-267mutantcouldnotbe

analyzed

because of the

extremely

low levels of

virion

gpl20

(Fig. 3)

caused

by

themutation. Each of the virion

pellets

was

resuspended

in

complete

RPMI 1640

medium,

and

100-,ul

aliquots

were

transferred into 1.5-ml screw-cap

Eppendorf

tubes

(Sarstedt).

The

CD4-immunoglobulin

G

(IgG)

form of CD4

(4, 5) (generous

gift

of

Genentech, Inc.,

South

San

Fran-cisco,

Calif.)

wasaddedtothree tubesat

final concentrations

of

500, 50, and 5 nM, while

a

fourth sample

served as an untreated control. All tubes wereincubated on ice for 1 h, and thevirionswere

pelleted

at

14,000

rpmina

refrigerated

(4°C) Eppendorf microcentrifuge (Brinkman)

for 1 h

(32);

supernatants were

collected

and transferred to new tubes.

Detergent

lysates

of the viral pellets and corresponding supernatant samples were prepared by adding 1 ml of lysis buffer

(50

mM

Tris-hydrochloride [pH

7.4], 300 mM NaCl,

0.1%

Triton

X-100)

to each sample and then subjecting the

samples

to vortex

mixing.

Protein A-agarose beads

(Be-thesda Research

Laboratories)

were then added to the

lysates

ofthe viral

pellets

toprecipitate the

gpl20

which was associatedwith

CD4-IgG,

since the IgGportion of CD4-IgG allows it to bind

protein

A

(5).

After 1 h of

mixing

at 4°C, beads

containing CD4-gpl2O complexes

were

pelleted,

and J. VIROL.

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[image:2.612.306.549.90.148.2]

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NOTES 3641

co x cc

0 0 0

N N N N N NK

C D v C-DC

-j N - N N N N

a C) - C- a a 0 c

cells 090

.0

0

00

*so

*

a_s -gpl2O

gp120 0

p66- _ _ _ _

Ip55-

IV

_ Mr

gp4 j ;

p39-B

200' C

co

150-100

co 6-=~ 50

-S s

p24

0 2 4 6

time(hrs)

a

0.-0

I..

200-

150-100

-50

-pNL4-3

:_-a-267/~

O2

-0-267/i-308

0 2 4 6

time(hrs)

FIG. 2. Intracellularprocessingandrelease of thewild-typeand

mutant envelope glycoproteins. (A) HeLa cells were transfected

with the pNL4-3, Q-267, or Q-267/I-308 proviral plasmid DNA, pulse-labeled for 30minwith [35S]methionine,and chasedforupto

6 h in the absence of radioactive label. Polyclonal anti-gp120 antibodieswereusedtoimmunoprecipitatelabeledgp160andgp120 envelope glycoproteins present in the cells and released into the

mediumatthe indicated times. Theprecipitated glycoproteinswere

resolvedbySDS-PAGE and visualizedbyfluorography. The faint band around 120 kDaatthe 0 timepoint representsanendoplasmic

reticulum-derived envelope protein not producedfromproteolytic processingofgp160(31). (B) Quantitationof thegp120bands shown inpanelAasdescribed in thetext.

the remaining supernatants were transferred to protein A

beadspretreatedwith the AIDS patient serumforasecond round ofprecipitationtomeasureunbound

gpl20.

TheAIDS patientserumwasalso used toimmunoprecipitateany

gpl20

which was present in the supernatant samples following

virus pelleting in the microcentrifuge to detect

gpl20

re-leased from thevirions as aresult of CD4binding (14, 21, 23).Allprecipitateswerewashed twice with thelysis buffer, resuspendedinsample buffer, and resolvedbySDS-PAGE. Labeled gpl20 was visualized by fluorography, and the

amountofgp120in the variousfractions(boundtoCD4,not

[image:3.612.70.306.77.463.2]

bound,andreleased)wasquantitated by usingtheFujixBas

FIG. 3. Incorporation of envelope glycoproteins into virions. HIV-1 virions present in supernatants from metabolically labeled HeLa cells transfected with the wild-type pNL4-3 or the Q-267,

Q-267/I-308,or1-308mutantproviral DNAwerepelleted by ultra-centrifugation. The virus pellets were resuspended in RPMI 1640 medium, and lysates from comparable amounts of virions were

immunoprecipitated with protein A-agarose beads pretreated witha

mixture of AIDS patient sera. The precipitates were resolved by

SDS-PAGE and visualizedby fluorography. The virion-associated gp120 and gp4lenvelope glycoproteinsaswellasthe virus-encoded

p66 reversetranscriptase and p55, p39, and p24 Gag proteinsare

indicated. Theorigin of the protein bandsimmediately above p24 is unknown.

2000Bio-ImageAnalyzer. The relative binding of the differ-entgpl20swasdeterminedbycalculating thepercentgpl20

which had bound to CD4-IgG as follows: [(bound +

re-leased)/(bound

+ notbound + released)] x 100.

AtthehighestconcentrationofCD4-IgG (500 nM), 12% of

the

Q-267/I-308

gpl20 was bound to CD4-IgG, compared

with39 and42% for the pNL4-3 and I-308gpl20s,

respec-tively (Fig. 4 and Table 2). These values include the small amountof the virion-associated gp120 (6to7% of the total) whichwasreleased from the pNL4-3 and 1-308 virionsas a

result of the CD4-IgG binding. Only 2%of the

Q-267/1-308

gpl20bound toCD4 when the CD4-IgGconcentrationwas

reduced 10-foldto50 nM, while 12% of the pNL4-3 and9%

of the I-308 gpl20s were associated with CD4-IgG. The reducedCD4bindingof theQ-267/I-308gpl20aswellasthe

previously demonstrated reduced level of virion-associated

gpl20 (Fig. 3)mayboth contribute tothe delayedinfection kineticsdisplayedbythis virus(Table 1). AlthoughtheI-308 change alone did not affect gp120 binding to CD4, this substitutionmightstillplayarole in thecontextof the

Q-267

mutation,inview ofarecentreportindicatingthatchanges

inV3mayinfluence the interaction ofgp120with CD4(35).

Given the results of the current study, our previous conclusion that the

Q-267

glycosylation site mutation im-pairs envelopefunctionatapost-CD4bindingstep(34)must

be amended since the N-to-Q substitution at position 267 affects multiple aspects of HIV-1 envelope structure and function. The reducedassociation of

gp120

with virionsas a

consequence of the

Q-267

mutation would appear to be

primarily responsiblefor theinabilityoftheQ-267virionsto

establishaproductive infection, inview of thehigherlevels

of gpl20 present on the infectious wild-type,

I-308,

and

Q-2671/-308

virusparticles. It should be noted thatanamino

A

~00

-gpl60

-gp 120

0

4 N rN

l-

6D

z C

IC o a

medium

time(hrs) 0

0

2

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3642 NOTES

viriongpl20 viriongp120 gpl20released boundtoCD4 notboundtoCD4 fromvirions

pNL4-3

Q-267/1.308

1-308

4W0

....

b,AN

5e

7t

nMCD4-IgG ° ¢ ° o ga 0

0 o 0

FIG. 4. Binding of CD4tovirion-associatedgpl20. HeLa cells transfected with thewild-type pNL4-3orthemutantQ-267/I-308or

I-308 proviral plasmid DNA were metabolically labeled with [35S]methionine for 24 h. Labeled virions werepelleted from the medium by ultracentrifugation and resuspended in RPMI 1640 medium, and CD4-IgGwasaddedto100-,ul aliquots ofeach virus suspensionattheindicated concentrations. Afterincubationat4°C for 1 h, virions were pelleted in an Eppendorf microcentrifuge, supernatantswereremoved, anddetergentlysates ofthepellet and supernatant sampleswere prepared. CD4-gpl20 complexes in the pellet samples (virion gpl20 bound to CD4) were recovered by adding proteinA-agarose beadswhich bind tothe IgGportion of CD4-IgG. Viriongp120notboundtoCD4wasimmunoprecipitated from thepelletlysatesbyconductingasecond round ofprecipitation with proteinA-agarose beads pretreated with a mixture of AIDS patientsera.gp120released from virionsas aresult ofCD4binding

was immunoprecipitated from the supernatant samples with the AIDSpatientserum.Allproteinswereresolvedby SDS-PAGE and visualized by fluorography. Quantitation of the data is shown in Table2.

acid

substitution

in Cl

(N-128), capable

of

restoring

infec-tivity

tothe

Q-267

envelope

mutantof HIV-1

(34),

increases the level of virion-associated

gp120

(unpublished data).

Previous reports

have also shown that mutations

in

Cl

and C2

disrupt

the association of

gp120

with

gp41

(15, 17).

Our studies suggest that the

interaction

of the

Cl, C2,

and V3

regions

of

gp120

stabilizes the

noncovalently linked gp120

and

gp41

(17). However, it

mustbe remembered that subse-quent to the

binding

of

virions

to the CD4 receptor,

gpl20

undergoes

conformational

changes (26)

that may be

required

for its

dissociation

from

gp4l, thereby allowing

the latterto

mediate

the fusion of viral and cellular membranes. Thus, the interaction of

Cl, C2,

andV3 may be both

complex

and

delicately balanced. Following cleavage

from gp160, the

noncovalent association

linking gp120 and

gp41 must

be

sufficiently stable to maintain structural integrity of this

TABLE 2. Amountof thewild-typeand mutantgpl20sbound to CD4asdetermined fromquantitation ofthegp120bandsshown

inFig. 4

%Bound' atCD4-IgGconcn(nM) of: Virus

500 50 5

pNL4-3 39 12 3

Q-267/I-308 12 2 0

I-308 42 9 1

aExpressed as

gpl20

bound +

gpl20

released/total

gpl20

(bound + not

bound+released).

complexyetcommensurately unstabletopermit its dissoci-ation following the binding of virion-associated

gp120

to

CD4.

Wethank DimiterDimitrov forproviding the conditionsusedfor pelleting HIV-1 particles in the Eppendorf microcentrifuge and KlausStrebel for criticalreview of themanuscript.

REFERENCES

1. Adachi, A., H.E.Gendelman, S. Koenig, T.Folks, R.Willey,A. Rabson, and M. A. Martin.1986.Production ofacquired immu-nodeficiency syndrome-associatedretrovirus in human and non-human cellstransfected with aninfectious molecular clone.J. Virol. 59:284-291.

2. Allan, J. S., J. E. Colligan, F. Barin, M. F. McLane, J. G. Sodroski, C. A.Rosen, W. A. Haseltine, T. H.Lee,and M.Essex. 1985. Major glycoprotein antigens that induce antibodies in AIDS patients are encoded by HTLV-III. Science 228:1091-1094.

3. Bergeron, L., N. Sullivan, andJ. Sodroski. 1992. Target cell-specific determinants of membrane fusion within the human immunodeficiency virus type gpl20 third variableregion and gp4l amino terminus. J.Virol. 66:2389-2397.

4. Byrn, R.A., J.Mordenti, C. Lucas,D. Smith, S.A.Marsters, J. S. Johnson, P. Cossum, S. M. Chamow, F. M. Wurm, T. Gregory,J. E. Groopman, and D. J. Capon. 1990. Biological properties ofa CD4 immunoadhesion. Nature (London) 344: 667-670.

5. Capon,D.J., S. M.Chamow, J. Mordenti, S.A. Marsters,T. Gregory,H.Mitsuya, R. A. Byrn,C.Lucas, F. M.Wurm,J.E. Groopman, S.Broder, and D. H.Smith. 1989. DesigningCD4 immunoadhesions forAIDStherapy.Nature(London) 337:525-531.

6. Cordonnier, A.,Y.Riviere, L.Montagnier, and M. Emerman. 1989. Effects of mutations in hyperconserved regions of the extracellular glycoprotein of human immunodeficiency virus type1 on receptorbinding. J. Virol. 63:4464-4468.

7. Dewar, R. L., M. B.Vasudevachari,V. Natarajan,and N. P. Salzman.1989.Biosynthesis and processing of human immuno-deficiency virustype 1envelope glycoproteins: effects of

mon-ensinonglycosylation andtransport.J. Virol.63:2452-2456. 8. Dubay, J. W., S. J.Roberts, B. Brody, and E. Hunter. 1992.

Mutations in the leucinezipper of the humanimmunodeficiency virus type 1 transmembrane glycoprotein affect fusion and infectivity.J. Virol. 66:4748-4756.

9. Dubay, J. W., S. J. Roberts, B. H. Hahn, and E. Hunter. 1992. Truncation ofthehumanimmunodeficiencyvirus type 1 trans-membraneglycoprotein cytoplasmic domain blocks infectivity. J. Virol. 66:6616-6625.

10. Earl, P. L., R. W. Doms, and B. Moss. 1990. Oligomeric structureof the humanimmunodeficiency virustype1envelope glycoprotein. Proc.NatI.Acad.Sci. USA 87:648-652. 11. Freed, E. O., D. J. Myers, and R. Risser. 1989. Mutational

analysis of the cleavage sequence of the human immunodefi-ciency virustype 1envelope glycoprotein precursorgpl60. J. Virol. 63:4670-4675.

12. Freed, E.O., D. J. Myers, and R. Risser. 1990.Characterization of the fusion domain of the humanimmunodeficiencyvirus type 1 envelope glycoprotein gp4l. Proc. Natl. Acad. Sci. USA 87:4650-4654.

13. Freed, E. O., D. J. Myers, and R Risser. 1991. Identification of the principal neutralizing determinant of human immunodefi-ciencyvirus type 1 as afusiondomain. J. Virol.65:190-194. 14. Hart, T. K., R. Kirsh, H.Ellens,R. W. Sweet,D. M. Lambert,

S. R. Pettaway, J. Leary, and P. Bugelski. 1991. CD4-HIV-1 interactions:binding of soluble CD4 (sT4) to HIV-1 and HIV-1 infectedcells inducessheddingof envelope gpl20.Proc. Natl. Acad. Sci. USA88:2189-2193.

15. Helseth, E., U. Olshevsky, C. Furman, and J. Sodroski. 1991. Human immunodeficiency virustype 1gpl20 envelope glyco-protein regions importantforassociation with the gp4l trans-membraneglycoprotein.J.Virol. 65:2119-2123.

16. Helseth, E., U.Olshevsky,D.Gabuzda, B. Ardman, W. Hasel-J.VIROL.

on November 9, 2019 by guest

http://jvi.asm.org/

(5)

NOTES 3643

tine, and J. Sodroski. 1990. Changes in the transmembrane regionofthe humanimmunodeficiencyvirus type 1gp4l enve-lope glycoprotein affect membrane fusion. J. Virol. 64:6314-6318.

17. Kowalski, M., J. Potz, L. Basiripour, T. Dorfman, W. C. Goh, E. Terwilliger, A. Dayton, C. Rosen, W. Haseltine, and J. Sodroski. 1987.Functional regions of the envelope glycoprotein of human immunodeficiencyvirus type 1.Science 237:1351-1355. 18. Lasky, L. A., G. M. Nakamura, D. H. Smith, C. Fennie, C.

Shimasaki,E. Patzer, P. Berman, T.Gregory, and D. J. Capon. 1987. Delineation ofaregion of the human immunodeficiency virustype1gpl20 glycoprotein critical for interactionwith the CD4 receptor. Cell 50:975-985.

19. Leonard, C., M. Spellman, L. Riddle, R. Harris, J. Thomas, and T.Gregory. 1990.Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 human immunodeficiencyvirusenvelope glycoprotein (gpl20) expressed in Chinese hamster ovary cells. J. Biol. Chem. 265:10373-10382.

20. McCune, J. M., L. B. Rabin, M. B. Fienberg, M. Lieberman, J. C.Kosek, G. R. Reyes, and I. L. Weissman. 1988. Endopro-teolytic cleavage of gpl60 is required for the activation of humanimmunodeficiencyvirus. Cell53:55-67.

21. McKeating,J. A., A. McKnight, and J. P. Moore. 1991. Differ-ential loss of envelope glycoprotein gpl20 from virions of humanimmunodeficiency virustype 1isolates:effecton infec-tivity and neutralization. J.Virol. 65:852-860.

22. Moore, J. P., J. A. McKeating, Y. Huang, A. Ashkenazi, and D. D. Ho. 1992. Virions of primary humanimmunodeficiency virus type 1isolatesresistanttosolubleCD4 (sCD4) neutraliza-tion differ in sCD4 binding and glycoprotein retention from sCD4-sensitiveisolates.J.Virol. 66:235-243.

23. Moore, J. P., J. A. McKeating, R. W. Weiss, and Q. J.Sattentau. 1990. Dissociation of gpl20 from HIV-1 virions induced by soluble CD4. Science 250:1139-1142.

24. Oldshevsky,U., E. Helseth,C.Furman, J. Li, W. Haseltine, and J.Sodroski. 1990. Identification of individual human immuno-deficiencyvirus type 1 gpl20 amino acids important for CD4 receptorbinding. J.Virol. 64:5701-5707.

25. Pinter,A., W. J. Honnen, S. A.Tilley,C.Bona,H.Zaghouani,

M. K.Gomy,andS. Zolla-Pazner. 1989.Oligomericstructure of gp4l,the transmembrane protein of humanimmunodeficiency virustype1. J.Virol. 63:2674-2679.

26. Sattentau,Q. J., and J. P. Moore. 1991. Conformationalchanges in the human immunodeficiencyvirus envelope glycoproteins bysoluble CD4binding.J. Exp.Med. 174:407-415.

27. Schwaller, M.,G.E. Smith, J. J.Skehel,and D. C.Wiley. 1989. Studieswithcrosslinkingreagents on theoligomericstructure of the envglycoproteinofHIV. Virology 172:367-369.

28. Stein,B.S.,andE. G. Engleman. 1990.Intracellular processing of the gpl60 HIV-1 envelope precursor. J. Biol. Chem. 265: 2640-2649.

29. Veronese, F. D., A. L. DeVico,T. D. Copeland, S.Oroszlan, R. C.Gallo,and M. G.Sarngadharan.1985.Characterizationof gp4l as the transmembrane protein coded by the HTLV-III/ LAVenvelopegene. Science 229:1402-1405.

30. Willey,R.L., J.S.Bonifacino, B.J. Potts,M.A. Martin,and R. D.KIausner. 1988.Biosynthesis, cleavage, and degradation of the human immunodeficiency virus type 1envelope glyco-proteingpl60.Proc. Natl. Acad. Sci. USA 85:9580-9584. 31. Willey, R.L.,T.Klimkait,D.M.Frucht,J. S.Bonifacino, and

M. A.Martin. 1991. Mutations within the human immunodefi-ciencyvirustype1gp160 envelope glycoproteinalter its intra-cellular transportandprocessing. Virology184:319-329. 32. Willey,R.L., M. A. Martin, and K. Peden.Unpublisheddata. 33. Willey, R.L.,E. K.Ross,A.J.Buckler-White,T.S.Theodore, and M.A.Martin.1989. Functional interaction ofconstantand variable domains of human immunodeficiency virus type 1 gpl20. J. Virol. 63:3595-3600.

34. Willey, R. L., D. H.Smith,L.A.Lasky, T. S.Theodore,P. L. Earl, B. Moss,D.J. Capon,andM. A. Martin. 1988. In vitro mutagenesisidentifiesaregion withintheenvelopegeneofthe humanimmunodeficiency virus that is critical for infectivity.J. Virol.62:139-147.

35. Wyatt, R., M.Thali, S.Tilley,A.Pinter,M.Posner,D.Ho, J. Robinson, and J. Sodroski. 1992. Relationship of the human immunodeficiency virus type 1 gpl20 third variableloop to a component of the CD4 binding site in the fourth conserved region.J.Virol. 66:6997-7004.

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