The human immunodeficiency virus type 1-specific protein vpu is required for efficient virus maturation and release.

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0022-538X/90/020621-09$02.00/0

The Human

Immunodeficiency Virus

Type

1-Specific

Protein

vpu

Is

Required for Efficient Virus Maturation and Release

THOMAS KLIMKAIT,l* KLAUS STREBEL,1 M. DAVIDHOGGAN,1MALCOLM A. MARTIN,' AND JAN M. ORENSTEIN2

Laboratory of Molecular Microbiology, NationalInstitute ofAllergy and Infectious Diseases, Bethesda, Maryland

20892,1

and

Department of

Pathology,

George

Washington University

Medical

School,

Washington,

D.C.200362

Received25July 1989/Accepted2October1989

A deletion mutation affectingvpu wasintroduced intoaninfectious molecular clone ofhuman

immunode-ficiency virustype1, and theresultantphenotypewasexamined afterinfection of humanT lymphocytes. The

absence ofvpuresulted inanaccumulation of cell-associated viral proteins and impairedthe release ofprogeny

virions. Both electron microscopic and biochemical analysesindicated thata large proportion of themutant particleswasattachedtothesurface of infectedcells.Significant variation in the size and shape of theseprogeny

virions was observed. In addition, intracytoplasmic particles, some of which formed aberrant budding structures, were visualized inT cellsinfected with thevpumutant. Indirectimmunofluorescenceanalysesof culturesinoculated with wild-typevirus withuseofa vpu-specificantiserum demonstrated thatvpuismainly

localizedtoaperinuclearregion in the cytoplasm of virus-producing cells.

Human immunodeficiency virus type 1 (HIV-1) contains numerous openreadingframes (ORFs), many of whichare

found in other lentiviruses. The vpu ORF, however, is

unique to HIV-1, not being present in the genomes of the

closely related primatelentiviruses HIV-2 and simian immu-nodeficiency virus (SIV) (10, 12). The vpugeneproductisa

small,81-amino-acid protein that is expressed in vivo (3, 16, 23). Because of its structural similarity to the membrane protein M2 encoded by influenza virus and the previously reported 4-to10-foldreduction inprogenyvirion production

associated withtheinterruptionof the HIV-1 vpuORF (23), wesuggested thatvpumight playarolein virion maturation,

efficient particle release from HIV-1-infected T lympho-cytes, orboth.

In this report, we have extended our analysis of vpu

functionduring productive virus infection of human T cells. We show that vpu is expressed only in the cytoplasm of

infected cells. In the absence ofvpu, an accumulation of cell-associated virion proteins associated with increased cytopathicitywasobserved. The results of cell fractionation studies and electron microscopic analyses indicate that the

vpumutantphenotype in infected T cells is characterizedby

adefectinvirion release.

MATERIALS AND METHODS

Cells and viruses. The human T-lymphocytic cell line A3.01(4)waspropagated in RPMI 1640 supplemented with 10% fetal bovine serum (FBS). Human peripheral blood leukocytes collected from healthy donors werepurified by centrifugation inlymphocyte separation medium (Organon Teknika, Durham, N.C.) and stimulated with 0.25 ,ug of phytohemagglutinin per ml in RPMI 1640 containing 10% FBS 3 days before infection. After HIV infection, the medium was supplemented with 10% interleukin-2. The

tissue culture mediumwaschangedeverythirddayorwhen the cell concentration exceeded 2 x 106/ml. HeLa cells (ATCCCCL2)orHeLa CD4 cells(a gift fromR.Axel)were

propagated in Dulbecco modified Eagle medium (DMEM) containing 10% FBS. Filtered supernatants, harvested

ap-*Correspondingauthor.

proximately 48 hposttransfection,wereusedasvirus stocks

for wild-type or mutant virus. Confluent cell monolayers

wereroutinely split 1 to5.

Constructionof the vpumutant. Adeletionmutant of the infectious molecular HIV-1 clone pNL4-3 (1) was

con-structedby site-specific oligonucleotide-directed mutagene-sis of the2.7-kilobase EcoRI-BamHI fragment cloned into M13. A deletion of 48nucleotidesimmediately following the initiator methioninecodon of the vpuORF plusa

7-nucleo-tideinsertionresulting inaminus-1 frameshift andpremature termination after 14 missense codons wasintroduced (Fig.

1). The mutated EcoRI-BamHI fragmentwasreinserted into

the infectious pNL4-3 proviral clone; the mutation was

confirmedby sequencing, using the dideoxy-chain termina-tion method (19).

Transfection of HeLa cells. HeLa cells were grown to

about 70%confluency ina25-cm2 flask. Beforetransfection, the medium was replaced with 5 ml ofDMEM containing

10% FBS. Calciumphosphate-precipitated plasmidDNA(20

,ug)

was addedto the cellsfor 4

h,

after which the medium

was removed and the cells were subjected to a glycerol

shock for 2min(9). The cultureswerethen washed twicein DMEM and maintained in 5 ml ofDMEM supplemented with 10% FBS.

RTassay. Reversetranscriptase (RT) assays oncell-free mediumsamples (8)wereperformedaspreviouslydescribed

(24). Samplesfor intracellular RTactivitywerepreparedas follows. Infected-cell suspensions (1 ml) were pelleted and suspendedin 1 mlofphosphate-bufferedsaline (PBS). Cells were disrupted by three cycles of freezing on dry ice and thawing ina37°Cwaterbath. Aftervortexingand sedimen-tation of cell debris, 10-,ul samples of supernatant were subjected toRT assay.

Western immunoblot analysis. Cells were pelleted and

lysed in CHAPSbuffer(50mM Trishydrochloride [pH 8],5 mMEDTA, 100 mMNaCl,0.5% CHAPS [Behring Diagnos-tics, LaJolla, Calif.]) containing0.5% sodiumdeoxycholate. Insoluble material was removed by centrifugation, the

ly-sates were boiled for 5 min in anequal volume ofsample buffer (14), and the proteins were separated on 10 to 20% sodiumdodecyl sulfate-polyacrylamide gradient gels. After 621

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622 KLIMKAIT ET AL.

A

1 2 3 4 5 6 7 8 9 kb

.~~~~~~~~~~~~~~~~~~~~

vpu

| _ rev -_

_~~~~~~~~~~~~~~~4

_gagInet

Y

%,pr

B

SA

5961 +

...CATCTCCTATGGCAGGAAGAAGCGG sis

6-revlit

AGACAGCGACGAAGAGCTCATCAGAACAGTCAGACTCATCAAGCTTCTCTATCAAAGCAGTAAGTAGTACATGTA 66so

rev 'Pt tat

Pt-,.--VpU

wts M 9 P r Z v a Z v a L V v a Z Z Z a Z v v gr s Z v

ATGCAACCTATAATAGTAGCAATAGTAGCATTAGTAGTAGCAATAATAATAGCAATAGTTGTGTGGTCCATAGTA 13s

AULATGCTTTCTTT- CAATAGTTGTGTGGTCCATAGTA

wtFiz z Y Rx x L R Q R X Z D R L Z D A ZZ H X A X ATCATAGAATATAGGAAAATATTAAGACAAAGAAAAATAGACAGGTTAATTGATAGACTAATAGAAAGAGCAGAA 62t

AUF N H R I *

Wt > D S G N H S H GC V S A L V N M G V H M G H1 H A P

GACAGTGGCAATGAGAGTGAAGGAGAAGTATCAGCACTTGTGGAGATGGGGGTGGAAATGGGGCACCATGCTCCT6285

L-.,env

wt VN D Z D D L

TGGGATATTGATGATCTGTAGTGCT...

6305

FIG. 1. (A)Genetic map ofHIV-1 indicatingtheposition ofthevpugene in the viral genome.(B)Nucleotidesequenceof the vpu gene present inthe parentalwild-type (wt)plasmid pNL4-3 and the deletionmutantvpuDEL-1(AU).Thebeginningof the firstcodingexonofrev

and thesplicedonor site(SD)atthe end of the firstexonsofrevandtatareindicated. SA marks theputativespliceacceptor for vpu. The deduced amino acid sequences forwild-typeanddeleted vpuarealso shown. Thebeginningof theenvcodingregionis marked. Numbers indicate the nucleotide distance from the beginning of the5' long terminal repeat.

transferto

0.22-gim-pore-size

nitrocellulosemembranes, the

filterswereincubated with specific antiseraand

251I-labeled

proteinAandthen subjected toautoradiography (21).

Immunohistochemistry. In a modification ofa previously describedmethod(18), cells(3 x 105)were washed in PBS andcentrifugedontoglass slides(5 min at 250 x g),usinga

Cytospin centrifuge (Shandon Inc., Pittsburgh, Pa.). The slides were air dried, and the cells were fixed either in

ice-coldacetone for 5 min or in

3.7%

paraformaldehyde for 10 min. The dried samples were blocked for 1 h in PBS

containing

5% bovine serumalbumin. The seraused forthe

primary antibody reaction were preadsorbed with

acetone-fixed uninfected A3.01

cells

and cleared by centrifugation (9,000 x g for1 min). The anti-vpu serum raised in rabbits

againstavpufusion protein synthesizedinEscherichia coli (F.Maldarelli, manuscriptinpreparation)and serum from an HIV-1seropositive individualwere each diluted 1:50. After

reaction with the primary antibodies, thecells were

exten-sivelywashed for 1 h with PBS containing 1% bovine serum

albumin. Fluorescein isothiocyanate-conjugated goat

anti-rabbitimmunoglobulin G(IgG) (Organon Teknika,

Malvern,

Pa.) for the anti-vpureaction ortetramethylrhodamine

isothio-cyanate-coupled anti-human IgG antibodies, developed in goat

(Qrganon

Teknika), were used in concentrations rec-ommended by the manufacturer as secondary antibodies. Aftera40-min incubation, the slides were washed twice in

PBS, rinsed in distilled water, and analyzed by using a fluorescence-activatedlightmicroscope (Zeiss, Oberkochen,

FederalRepublic ofGermany).

Electron microscopy. At different times after infection,

samples of the infected lymphocyte cultures containing

ctlution B

A

101

103

10 3x10 6x10 104

4 3x10

FIG. 2. Endpoint dilution titration of wild-type (A) and vpuDEL-1(B) virus. Filteredculture mediumfromwild-type-and vpuDEL-1-infected lymphocyte cultures was adjusted to equivalent RT activity(approximately6x 106cpm/ml). The indicated dilutions of supernatantwereusedtoinfect A3.01 cellsinsixfoldreplicas.An autoradiogram of a 32P-RTassay performed on cell-free superna-tants16days postinfectionis shown.

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A.

E - 15

0

v-.0 CD

iF.1

A

?*

RT

C.)

0

S

_ _

...

_ :1.

EuI

B

0

to

I

1

a: c.

10

S

10

AU

(x10Qcpm)

15

10

5.

5 7 9

-m

n

m

wt

F

1-1111 0

1-

_ I I I I I I time 11 14 17 5 7 9 11 14 17 (daysp1.)

..

49

-gP160

0 -gp120

__v . -p55

-t -p41

t(daysp.i.)

B. wt 'U

;

v . ...J;X

4W~ ~ ~ ~ \.

FIG. 3. Cytopathicity of wild-type (wt) and vpuDEL-1

(A&U)

virus. (A) Cell viability of A3.01 cells after infection with wild-type pNL4-3 or vpuDEL-1 was monitored at the indicated times by trypan blue exclusion. RT activity in corresponding supernatant sampleswasalsodetermined. (B) Cell fusionwasassayed in HeLa CD4 cellstransiently transfected withequivalentamounts of wild-type or vpuDEL-1 plasmid DNA as described in Materials and Methods. The formation of syncytia was scored under a phase-contrast light microscope 20 h after transfection (magnification, x62). Syncytia are marked witharrowheads. Supernatant RT activ-ity for vpuDEL-1 was only 1/10 the amount produced in the transfection with wild-type virus.

approximately 106cells werepelleted by low-speed centrif-ugation (320 x g for 10min) and fixed for 15 min at room temperature in5mlofa2.5%glutaraldehyde solutionin 100 mM cacodylate buffer as previously described (17, 20). Samples were stored at4°C until they were processed for

ultrastructural studies.

RESULTS

Molecularcharacteristicsof the vpu deletion mutant. A vpu deletion mutation(vpuDEL-1),identical to thatpresentin a naturally occurring HIV-1isolate designatedNY-5 (2),was introducedinto theinfectious molecularclonepNL4-3 (1)as described in Materials and Methods. Unlikeour

previously

describedlinker insertion mutant, which had the

potential

to encode the amino-terminal 32 residues of vpu (23), the deletionpresent in vpuDEL-1 allowed

only

the

synthesis

of a15-residue missense

peptide.

The deduced missense amino acids ofthe mutated vpu lack anyhomologywith the authentic

protein

(Fig.

1). The

FIG. 4. Accumulation ofHIVproteins inlymphocytes infected with the vpu mutant. At the indicated times, cells and culture mediumwereseparated bycentrifugation (9,000xgfor 3 min). The supernatantfractionwasassayed forRTactivity, andthelysedcell pellets were electrophoretically separated and analyzed by immu-noblotting, using human anti-HIV-1 serum. vpu is notidentified in the blotsince thepredominantp17gagprotein comigrates with vpu and obscures it(23). wt,Wild type;

AU,

vpuDEL-1mutant.

splice donor sites for the first coding exons oftat and rev

immediately5'ofthe vpu gene areintact, and the envgene

located 3' from the vpu mutation is preserved (Fig. 1).

Western

analysis of transfection lysatesor radioimmunopre-cipitated lysatesofvpuDEL-1-infectedTcellsindicatedthat noneoftheotherHIVORFs is affectedby thevpudeletion.

The banding

patterns

and relative band intensities of

wild-type and mutant

proteins

were indistinguishable (data not

shown).

Theinfectivity of mutant virionsisunaffectedby the dele-tion. The infectivity of vpuDEL-1 virus

preparations

was

compared

with that ofthe

isogenic

wild-type virus in

end-point

titration

experiments.

Wild-type

and

vpuDEL-1

virus stocks were

prepared

by

filtering

supernatants from HeLa

cells 48 h after transfection. After normalization for RT

levels, A3.01 cellsin 96-well microdilution plates

(103

cells

per well) were infected with serial dilutions of each virus stock; each dilutionwasplatedinsixreplicas.Theinfectious

titerof each stockwasdetermined

by

RTassayoftheculture

supernatant.

Wild-type

and vpuDEL-1 HIV-1 had similar infectivities, although vpuDEL-1 consistently

generated

lower virus

yields

at each dilution

(Fig. 2).

It should be

emphasized

that in this

infectivity

assay,

comparable

amounts(RT activity) ofcell-freefiltered virus were exam-ined;aggregatedorcell-associatedformsof

vpuDEL-1 (see

below) were

probably

eliminated from the test inoculum.

Furthermore, immunoblot

analyses

of

wild-type

or

vpuDEL-1particles, obtained

by

pelleting

viruspresent ineachfiltrate

by

ultracentrifugation,

were

indistinguishable,

indicating

similarprotein

compositions

(data

not

shown).

In contrast, infection ofA3.01 cells with

vpuDEL-1

re-sulted in amore

rapid

onsetof

cytopathicity

in

comparison

with wild-typevirus. Greater cell

killing

was evident in the

r%

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624 KLIMKAIT ET AL.

As _ofratio_RT

-

0-, 1.6

ID 1.2

0

°.S0.8

= 0.4

0

.B

Bs

:0

*5

50F

A

0- AU

--*wt

5 0 5

5 10 15 t

(daysp.i.)

wt

AU

[/,1

2L 0

1c

FIG. 5. Cell-associated RTactivity. Samples of A3.01 cell cul-tures infected with equivalent amounts (3 x 106 cpm of

32P-RT

activity per ml) ofwild-type (wt) or vpuDEL-1 (AU) virus were

sedimented at the indicated times after infection. (A) Ratio of cell-associated RT activity, liberated byfreezing and thawingthe cellpelletororiginally present in themedium,plottedasafunction of timepostinfection.Thecell-associated viruswasfurther fraction-ated (B) byseparating virions loosely attached tothe cell surface from intracellular virusbyvortexingandcentrifugationtogenerate thesurface fraction. Thepelletedcellswerethenresuspendedin the original volume and disrupted by freezing and thawing (cytosol fraction).

vpuDEL-1-infected cultures after day10 eventhough

signif-icantly less progeny virus (as measured by RT

assay)

was produced (Fig. 3A). Similarly, vpuDEL-1

proviral

DNA induced larger and more numerous

syncytia

in transient transfections of HeLa CD4+ cells than did the

wild-type

provirus(Fig. 3B)yet

generated

only

10%

of theRT

activity

observed in the lattertransfection.

Lack of vpu leads to an accumulation of viralproteinsand cell-associated HIV particles.The effect ofthe vpu mutation

onthe

synthesis

ofHIV

proteins

and the releaseofprogeny

virions into the

supernatant

medium is depicted in Fig. 4.

Cells andmediawere collected at the indicated timesafter

infectionwithwild-typeorvpuDEL-1 virus and analyzed by immunoblotting (Fig. 4B) or RT assay (Fig. 4A),

respec-tively. Duringtheearly phase (day 9) oftheinfectioncycle,

lysatesfrom

vpuDEL-1-infected

cells contained nearly five times more viral protein than comparable samples from a

wild-type virusinfection. Even atlatertimes,theamountof

on

B

~4. .

4~~~~~4

'w kj.' ,- :r

10~~~~

.. . ^ e j; or ., R~~~~~~~~r'v

.. ^< SX :E ;0S

*8'

iaS

t',01

^X^s~~~~~

FIG. 6. Low-magnification transmission electron micrographs otA3.01cellsintectedwith wild-type (A) or vpuDEL-1 (B) virus showing virions associated with the cell surface. Magnification, x9,000.

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HIV-1-SPECIFIC PROTEIN vpu 625

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cell-associated HIV proteins in cells infectedwith the vpu

mutantwasconsiderably higher. Acorrection for the lower

number of viable cells in the vpuDEL-1 infection at the indicated time points would result inan evengreater

accu-mulation of proteins. In contrast, and as reported earlier

(23), significantly more(fivefoldintheexperiment shown in Fig. 4) wild-type virus than vpuDEL-1wasreleased into the

medium.

N

FIG. 7. (A and B) vpuDEL-1 virions closely associated with the plasma membrane of virus-producing cells, particles with condensed nucleoids, while still attached. Magnification, x65,000. (C) Extra-cellular cluster ofvpuDEL-1 particles of various sizes and shapes, onecontainingatleastfive nucleoids. Magnification, x91,000.

Ina completely independent experiment, cellswere

sep-arated from the medium by low-speed centrifugation at

various times after infection and disrupted bythree cyclesof freezing and thawing, after which theRTactivitypresent in the medium orreleasedfrom pelleted cells wasmeasured.

Most of the RT activity generated by wild-type HIV was

released into the medium by day 11 (Fig. 5A). In contrast,

highlevels(>50%) of theRTactivitywerecell associated in

thevpuDEL-1 infection. Tofurtheranalyze the cell-associ-ated progeny virions in vpuDEL-1 infections, samples of culturesexposed towild-type virusorthevpumutant were

harvestedbefore peakRTproduction, and the resuspended cell pellet was subjected to vigorous vortexing for 10 s.

Virions releasedbythistreatmentwereseparatedfrom cells by centrifugation (surface fraction), and the remaining cell pelletwasresuspendedinmedium,frozen and thawedtwice, andcentrifugedafinal time(cytosol fraction). Nearly75% of

thecell-associated RT invpuDEL-1 infectionswasreleased by vortexing (Fig. SB). A simultaneous trypan blue exclu-sion assay indicated that most of the vortexed cells were viable (data notshown). These results suggest that a large

proportion of the cell-associated vpuDEL-1 particles may

have beenlooselyattached tothe cell surface.

Electron microscopic examination ofvpuDEL-l-infected T cells.Approximately 10i A3.01 cellswereinfected withequal

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626 KLIMKAIT ET AL.

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amounts (based on RT activity) of wild-type or vpuDEL-1 at amultiplicity similar to that used for the experiment shown

in Fig. 3; samples were collected on day 7, 9, or 10 and examined by transmission electron microscopy. Although the percentages of virus-producing cells were comparable (by transmission electron microscopy and indirect

immuno-fluorescence) in the two cultures, up to fivefold more parti-cles were associated with the surface of cells infected with vpuDEL-1 (Fig. 6). A characteristic electron microscopic

feature of the vpuDEL-1 infection was the presence of progeny particles, physically attached to the plasma

mem-brane of infected cells, that contained prematurely con-densed nucleoids (Fig. 7A). In some instances, chains of vpuDEL-1 virions were observed extending from the plasma membrane (Fig. 7B). In addition, a greater proportion of the vpuDEL-1 particles appeared aberrant compared with wild-type HIV,varying in size and shape, and in some instances appeared to harbor multiple nucleoids (Fig.7C).

Another common feature of vpuDEL-1-infected A3.01 cells was the presence of intracytoplasmic vacuoles

contain-ing large numbers of virions in all stages of assembly,

including highly aberrant forms (Fig. 8). Atypical detached

single, double, and multimeric budding structures that

formed rosettelike structures were also found (Fig. 8C). Besides virions, the cytoplasmic vacuoles frequently

con-tained small vesicles similar to multivesicular bodies

typi-cally associated with Golgi elements (Fig. 8D).

vpuDEL-1-infected peripheral blood leukocytes exhibited a similar

phenotype (data not shown).

vpu is located in the cytoplasm of virus-producing cells. Acetone-fixed and permeabilized A3.01 cells, 9 days after

infection with wild-type HIV-1, were simultaneously

incu-bated with a monospecific vpu antiserum raised in rabbits and human serum with HIV-1 seroreactivity and then

exam-inedfor the presence of viral proteins by indirect

immuno-fluorescence as described in Materials and Methods. The human anti-HIV-1 serum,whichreactedwell with HIV gag and env but not vpu in immunoblots (data not shown), gave strongcytoplasmic fluorescence (Fig. 9A). The signal with theanti-vpuserum wasalso strong and could be localized to theperinuclear region ofthecytoplasm;no nuclear reactiv-ity wasapparent (Fig. 9B). In control infections with vpu-DEL-1, thereaction withthe serumfromtheHIV-1-infected individualwassimilarto thewild-typereaction (Fig. 9C),but no reactivity was observed with the anti-vpu serum (Fig.

9D). When cells fixed withparaformaldehyde to study

sur-face reactivity were examined with the same antibodies, intense staining was observed with the human anti-HIV-1

serum(Fig. 8E), presumablybecauseofthepresence of gag or env protein on the plasma membrane, but no reaction above background levelswasobservedwith the vpu antise-rum(Fig. 8F).

DISCUSSION

We have recently reported that the HIV-1 vpu gene product is anintegralmembrane

protein

that is

expressed

at

anti-HIV-1 anti-vpu

FIG. 9. Localization of vpu by indirect immunofluorescence microscopy. Wild-type (A, B, E, and F)- or vpuDEL-1 (C and D)-infected lymphocyteswerefixed withacetone (A toD)orin3.7% paraformaldehyde-PBS (E and F). Samples were simultaneously incubated with human anti-HIV-1 serum and an anti-vpu serum raised in rabbits. Tetramethylrhodamine isothiocyanate-conjugated goat anti-human IgG antibody and a fluorescein isothiocyanate-coupled mouse anti-rabbitIgG antibody were used as secondary antibodies, and fluorescence of the same fields was evaluated. Magnifications: AandB, x504;CtoF, x332.

highlevels invirus-producing Tlymphocytes but is largely, if not completely, absent from progeny virions (23; this report). Mutations affecting the synthesis of vpu lead to a marked reduction in virus particle production and an asso-ciated intracellular accumulation of HIV structural proteins

(23). Inthis study, vpu was found only in the cytoplasmof productively infected cells by indirect immunofluorescence andappearedtobeconcentrated ina subcellularorganelle,

most likelytheGolgicomplex.

Oneofthe moststriking findings of this work camefrom the electron microscopic analyses, which suggest that vpu

playsacriticalroleduringthe release of virusparticlesfrom infectedTcells.Inthe absenceofvpu, increased numbers of

virus aggregates and budding structures, including many mature particles characterized by condensed cores, were found associated with or evenattached totheplasma mem-brane ofvirus-producing cells. Concomitantly, budding of progeny vpu mutant particles from intracytoplasmic mem-branes into vacuoles was also seen, explaining in part the intracellularaccumulationof HIVproteinsobservedin met-abolic labeling (22) and cell fractionation (Fig. 5) experi-ments. Thus, in additiontoresultinginimpaired release of

FIG. 8. Intracytoplasmic vacuoles withincells infectedwithvpuDEL-1-containing particles invarious stages ofassembly. Shown are budding vacuolar particles (A to D) as well as highly aberrant structures containing multiple nucleoids of aberrant shape (A and C; arrowheads)orinapparentcores(A).Matureand aberrant virionscontainingeccentricdensematerialor anucleoid withahollowappearance (AandC)canbeseen. Incompletelyseparatedadjacentbuddingunits formastalklikestructure(B)ordetachedrosettelike structures(C; stars)consistingofsingle andmultiplebuds locatedwithinvacuoles ofamultinucleated giantcell. (D)Two vacuoles that contain mature virionsas well assmall smooth vesicles (arrows). Three typical budding structures are presenton the plasma membrane on the right. Magnifications: AandB, x110,000;C, x64,000;D, x80,000.

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628 KLIMKAIT ET AL.

hdex

4- vpu

A

2

I

,

p3A

FIG. 10. Hydropathicity profiles of the deduced amino acid

sequences for HIV-1 vpu, foot-and-mouth disease virus p3A, and

influe'nza virus M2 proteins. The relative hydropathicity index of each residue is shown as afu'nction of amino acid position in the

proteinandwascalcu'latedbythemethod ofKyteandDoolittle(13).

The border between hydrophobicdomain andhydrophilicC

termi-nusof eachprotein isindicatedbythe verticalbroken line.

virions from thecell surface oflymphocytes, theabsenceof

vpu results inthe budding and maturation of virus particles

at membrane sites ordinarily restricted from such activity.

Perhaps the high concentrations ofvpu associated with the

Golgicomplexincells infected withwild-typeviruspreclude

particleproduction atthese intracytoplasmic membranes.

The electron microscopic studies also demonstrated that

vpuDEL-1 infections ofT lymphocytes were characterized

bytheproductionofaberrant virusparticles, including some

with multiple nucleoids and others that were several times

larger than wild-type virions and contained uncondensed

nucleoids. Thiswasaprominentfeatureofintracytoplasmic

vpuDEL-1 virus production, in which multimeric budding

structures werefound. One explanation linking all of these

phenomenais that vpumediatesacritical and terminal step

of thebuddingprocess; in its absence, individual immature

progenyparticlesfailtoseparatefrom oneanotherandfrom

the plasma membrane. This perturbation of virus release

could lead to aretardation ofHIV maturation and

second-arilyresult inthe accumulation ofhighintracellular

concen-trations of viral

piroteins

as well as the diversion of virus

production to intracellular sites. A paradox of vpuDEL-1 infectionswasthe earlierappearance ofvirus-induced

cyto-pathicity thatwas accompanied by significantredu'ctions in

progenyvirionproduction. The intracellular accumulation of

potentiallytoxic viralproteins invpuDEL-1-infectedT

lym-phocytesmight explain the observed reduced cellviability.

Similarly,

high

concentrations ofvirionstructuresassociated with essential cellular membranes could alter their

compo-sition and impair function by altering permeability and

transport properties. In addition, the potentially increased concentrations ofgp120 and

gp41

on the surface of vpuDEL-1-infected T lymphocytes could contribute to the morerapid killing of other

CD4+

cells in the culture by cell fusion.

The evolution of small viral proteins like vpu may not be unique to HIV-1, since a number of other RNA viruses encode proteins that appear to functionduring final steps of virion production and show similarity to vpu. For example,

the size, hydropathicity, and domain structure of the mem-brane-associated influenza virus M2protein (15, 26) andthe

foot-and-mouth disease virus protein p3A (5) are strikingly similar to those of vpu (Fig. 10). Ahydrophobic N-terminal

anchor is followed by a very hydrophobic C terminus, with

at least 18 charged residues in each case. Other possible

homologs of vpu include the small NB protein of influenza B virus (25) and the SH protein of simian virus 5 (11). These proteins have been suggested to beinvolvedin the processof virus assembly (11, 25, 26). In contrast, neither HIV-2 nor SIV encodes a structural homolog of vpu (10, 12). It remains to be shown whether these two primate lentiviruses utilize another viral protein to functionally replace vpu.

Alterna-tively, a putative host cell

protein

may serve as a virus release factor in HIV-2 or SIV infections and obviate the requirement for a virus-encoded vpu-like protein.

The intracytoplasmic production of virus particles charac-teristic of a wild type HIV-1 infection of monocytes-macro-phages (6, 7, 17) is reminiscent

of

the vpuDEL-1 infection of T cells described above. What is puzzling, however, is that HIV-1 andHIV-2 both produce intracytoplasmic virus par-ticles in infected monocytes even though only HIV-1 has the capacity to encode vpu. Possible explanations include (i) the presence of an inhibitor of vpu in monocytes-macrophages or (ii) the fact that vpu function in human T cells requires the synthesis of or the interaction with a cellular cofactor that is not expressed in monocytes. Experiments are in progress to more fullydissectthe role of vpu in both cell systems and to develop antagonists that specifically abrogate vpu function during the acute infection of T lymphocytes.

ACKNOWLEDGMENTS

We thank Charles Buckler for computer analyses and Ronald Willey for valuable discussions.

T.K. was supported by afellowship from the Deutsche Forschungs-gemeinschaft, Bonn, Federal Republic of Germany.

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