Genetic evidence that the Tat proteins of human immunodeficiency virus types 1 and 2 can multimerize in the eukaryotic cell nucleus.

(1)

Genetic Evidence that the

Tat

Proteins of

Human

Immunodeficiency Virus Types

1 and

2 Can

Multimerize

in

the

Eukaryotic Cell

Nucleus

HAL P. BOGERD,1 ROBERTA. FRIDELL,1WADES. BLAIR,2ANDBRYAN R.

CULLEN'

2.3* Howard HughesMedical

Institute'

and SectionofGenetics2 and DepartmentofMicrobiology,

Duke UniversityMedical Center, Durham, North Carolina 27710

Received28 January1993/Accepted29April 1993

Theformation of dimers or higher-order multimers is critical to thebiological activity of many eukaryotic regulatory proteins. However,biochemical

analyses

of the multimerizationcapacityof the Tat trans activator ofhumanimmunodeficiencyvirus types 1 (HIV-1) and 2 (HIV-2) have yielded contradictory results. We used the two-hybrid genetic assay for protein-protein interactions in the eukaryoteSaccharomyces cerevisiae (S. Fields and O.-K. Song, Nature

[London]

340:245-246, 1989) to examine the multimerization of Tatinvivo. BothHIV-1 and HIV-2Tat are shown to formspecific homo- but notheteromultimers in the yeast cell nucleus. Mutational

analysis

indicatesacriticalrole for the essentialcoremotif of Tat inmediatingthis interaction but demonstrates thatefficient Tat multimerization doesnotrequireanintactcysteine motif. These data raise the

possibilitythat themultimerization of Tat may be important for Tat function in higher eukaryotes.

Replication of human immunodeficiency virus type 1

(HIV-1) requires the functional expression of the viral nu-clearregulatory proteinsTatand Rev(reviewedinreference

9).The Tatprotein is a potent trans activator of transcription directed by the HIV-1 long terminal repeat promoter, and Rev is a posttranscriptional regulator of viral

structural-protein expression. While the mechanismsof action of Tat and Revarethereforedistinct,bothnevertheless relyonthe direct interaction of these regulatory proteins with viral RNA target sequences termed, respectively, thetrans

acti-vation response (TAR) element and the Rev response ele-ment (RRE).

The critical importance of Tat in the replication cycle of thispathogenic human retrovirus suggeststhat inhibitorsof Tat functionmightwell have asignificant beneficial impact

on HIV-1-induced disease. If Tat function requires the

specific dimerization or multimerization of Tat, this may representanattractivepotentialtargetforchemotherapeutic intervention. However, the biochemical evidence for Tat multimerization has remained contradictory. In particular,

while Frankel et al. (11) have proposed that Tat can form metal-linked dimers in aprocess mediatedby the essential

cysteine-rich domain of Tat, Rice and Chan (23) have presented data indicating thatthe Tat protein is found as a monomerin extracts ofexpressing cells.

Recently, Fields and Song (10) described anovelgenetic system in theyeastSaccharomyces cerevisiaethatpermits

the direct demonstration of specific protein-protein inter-actions. The assay uses the yeast strain GGY1::171 (gal4 gal80his3leu2), which contains achromosomally inserted GALl-lacZ fusion gene (13). Intothis strain are introduced two selectable plasmids that express two distinct hybrid

proteins. The first of these consists of the GAL4

DNA-bindingdomainfused to protein X, while the second consists ofatranscription activation domain attached to protein Y. If

proteins X and Y can interact, then the DNA-bound GAL4-Xhybridwillrecruittheactivation domain-Y hybrid

*Correspondingauthor.

protein to the GAL upstream activation sequence

(GALUAS),

resultinginactivation of 3-galactosidase (1-Gal)

expression.ThetestproteinsX and Ycanbe either different oridentical,andthis assaycantherefore be usedto demon-stratetheformation ofspecifichetero-orhomomultimers in the nucleus of yeast cells inculture (6, 7, 10,18).

The specific HIV-1 Tatexpression plasmids used in this

experimentareshown inFig. 1. ThepGAL4-TATplasmidis predicted to express a fusion protein consisting of the N-terminal GAL4 DNA-binding domain (16) (amino acids

[aa]1to117)fusedtoaa2to86of Tat(20).ThepVP16-TAT plasmid expresses a fusion protein consisting ofa protein nuclear localization signal derived from the simian virus 40

largeTantigen(15)fusedtothe acidic activation domain(aa

412to 490)of the VP16 transcription factor (28), fused, in turn, tothefull-length86-aa Tatprotein. Plasmids

express-ingtheGAL4DNA-bindingdomain(pGAL4) and the VP16 activation domain (pVP16) in a nonfusion form served as

negative controls, while fusions of these domains to the 130-aa Tatproteinof HIV-2(14) (pGAL4-TAT2and pVP16-TAT2)or tothe 116-aa Revproteinof HIV-1 (20)

(pGAL4-RevandpVP16-Rev) providedcontrols forspecificity.

Yeastcellsweretransformed

(1,

25)withanAlkali-Cation Yeast Transformation Kit (Bio 101, Inc., La Jolla, Calif.),

with 1 ,g of each of the pGALA and pVP16 expression plasmids. Double transformants were selected on

supple-mented synthetic dextrose plates lacking histidine and leucine. After 3days,coloniesweretransferredto a

supple-mented nonrepressing synthetic sucrose medium lacking

histidine and leucine and incubatedovernight at30°C with

shaking.Equivalent optical density(OD)units(-2 ml)of the double-transformant cultureswerecentrifuged, andthe cell

pellet was resuspended in 500 p,lof complete Zbuffer, as describedpreviously (1). Cellswerethenpermeabilized by

the addition of 25p,lof chloroform andvortexing.The1-Gal substrate chlorophenol

red-13-D-galactopyranoside

was added to afinal concentration of 4 mM, and sampleswere incubated at roomtemperature for-30 min. After

centrifu-gationto removecellulardebris,the

A595

of thesampleswas determined.

5030

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A

ADH Terminator

EcoRI

2p

PGKPromoter SV40NLS

ion

-PGKTerminator

ORI

FIG. 1. Plasmids encoding Tat fusion proteins. (A) The yeast

expression plasmid pGAL4-TATencodesafusionprotein consisting

of the DNA-binding domain of GAILA fused to the HIV-1 Tat

protein.Expressionis directedbytheyeastalcoholdehydrogenase (ADH) promoterand terminator(10, 30). (B)Theexpression

plas-midpVP16-TATencodesatripartitefusionprotein consistingofa

synthetic 8-aa nuclearlocalization signalderived from the simian

virus 40largeTantigen(SV40NLS) (15),the acidicactivationmotif

of VP16 (28), and, at the C terminus, the full-length HIV-1 Tat

protein. Expression is directed by the yeast phosphoglycerate

kinase(PGK) promoterand terminator(21). 2>L, yeast plasmid2p.m

origin of replication; HIS3 and LEU2, yeast selectable markers; ORI, prokaryotic origin of replication; Amp, bacterial ampicillin

resistance marker.

An analysisof thevarioushybrid proteinsfortheirability

to form homo- or heteromultimers ispresented inTable 1. BothTat and Tat2werefound tomultimerizeefficiently, as

determinedby the high level of 1-Gal activityobserved in yeast cells expressing GAL4-Tat plus VP16-Tat or

GAL4-Tat2 plus VP16-Tat2, respectively. This interaction was

highly specificinthatnoothercombination of thesevarious GAIA and VP16hybrid proteinswas able to give rise to a

significantlevel of 1-Galactivity.Thesenegativeresults also demonstrate that neither the HIV-1 nor the HIV-2 Tat protein contains a transcriptional activation domain that is

able tofunctionwhen bound to the GALuASinyeastcells. Althoughboth HIV-1and HIV-2Tatwerefoundto form homomultimersintheyeastcellnucleus,the datapresented

TABLE 1. 3-Galactivity inS. cerevisiae strainsexpressing various combinations ofhybridproteins

1-Galactivity (mU/mlofextract)'

Protein

GAL4 GAL4-Tat GAL4-Tat2 GAL4-Rev

VP16 <1 <1 <1 <1

VP16-Tat <1 1,160 13 <1

VP16-Tat2 5 10 275 <1

VP16-Rev 22 <1 <1 2

aDeterminedasdescribed inthe textafter transformationintoyeaststrain GGY1:171.U, OD units.

in Table 1 indicate that these two related proteins are unable to form a mixed multimer. This result, although perhaps surprising given the significant level of aminoacid sequence homology between HIV-1 and HIV-2 Tat (14), strongly attests to the specificity of these interactions. It is also of interest that the HIV-1 Rev protein did not give rise to a detectable level of homomultimer formation in this assay. This does not reflect any instability of the hybrid GAL4-Rev andVP16-Revproteins in yeast cells, as these were readily detectable byimmunoprecipitation or Western immunoblot analysis (2). Although all negative results should be inter-preted cautiously, these data are nevertheless consistent with theproposal that Revmultimerizationin vivo is medi-ated by binding to the viral Rev response element RNA targetsite (19) and may also be facilitated by a mammalian cellcofactor that could belacking in yeast cells (3).

A number of HIV-1-encoded proteins are predicted to form homomultimers, including the Gag protein, which

multimerizes to form the virion capsid, and Pol, which is known to beactive as a dimer (29). Luban et al. (18) have previously demonstrated that thespecificmultimerization of HIV-1Gagcanbereadilydetectedby the two-hybrid system in yeastcells. As one approach to determining whether the

observedmultimerization of Tat in yeast cells islikelytobe physiologically relevant, we therefore constructedaseries of

additionalplasmids that woulddirectthesynthesis of hybrid

proteinsconsisting of the GAL4DNA-bindingdomain or the VP16activation domainfusedtothe HIV-1proteinGag(aa

2to500),Pol(theC-terminal 848 aa encoded bythepolopen

reading frame, i.e., lacking protease but including all of reversetranscriptase andintegrase),Vpr(aa2 to96), or Vif

(aa2 to 191) (9). Ineach case, the relevant segment of the HIV-1 genome was excised by polymerase chain reaction

(PCR) (22) and substituted in place of the tat gene in the yeastexpressionplasmidsshown inFig.1.The relative level of1-Gal activityobserved in yeastcellsexpressingeach of these HIV-1 proteins as both GALA DNA-binding domain and VP16 activation domainfusionproteinswasthen deter-mined (Table2). Inaddition,we alsoincluded in this assay

expression plasmids that encode the hybrid proteins GAIA(1-147)-SNF1 and SNF4-GAL4(768-881) that were

originally shown by Fields and Song (10) to activate the

13-Galgeneinthe yeast strain GGY1::171throughformation ofaheteromultimer onthe GAL4 DNA-binding site.

Inspection of Table 2 shows that both HIV-1 Gag and HIV-1 Polcanindeed form thepredictedhomomultimers in yeastcells. In addition, the HIV-1Vprproteingave riseto strong 1-Gal activity, suggesting that Vpr likely has a

tendencytoform homomultimers in the HIV-1-infected cell

and/or in HIV-1 virions (8). In contrast, the HIV-1 Vif protein failedtoinduce detectable1-Galactivitywhentested in this yeast two-hybrid system. That the observed 1-Gal

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TABLE 2. Homomultimer formation by selected HIV-1 proteinsa

DNA-binding domain Activationdomain 13-Galactivity" (mU/

hybrid hybrid ml ofextract)

GAIA VP16 3

GAL4-Tat VP16-Tat 2,598

GALA-Gag VP16-Gag 128

GALA-Pol VP16-Pol 809

GAL4-Vpr VP16-Vpr 1,796

GALA-Vif VP16-Vif <1

GAL4(1-147)-SNF1 SNF4-GAL4(768-881) 1,736

a All the indicated fusion proteins were expressed from derivatives of

pGAL4andpVP16,asdescribedin thetext,exceptforGA14(1-147)-SNF1

andSNF4-GAL4(768-881),whichwereexpressed asdescribed previously

(10).

IActivitiesweredeterminedasdescribedin thetextaftertransformation

into yeaststrainGGY1::171. U,OD units.

activitywasindeed due tohomomultimer formationon the

GALUAS

was demonstrated by the

finding

that all of the

indicated GAL4DNA-bindingdomain fusionproteinsfailed toinduce 3-Gal activitywhen expressedinthe presence of

the pVP16 control plasmid. Similarly, all of the indicated

VP16 activation domain fusion

proteins

were also inactive whenexpressed togetherwith thepGAL4vector.

With theexceptionof the combination of GAL4-Tat and VP16-Vpr, which gave alow but detectable level of 1-Gal

activity, none of the indicated GAL4hybrid proteinswere abletoactivate 1-Galexpression, i.e., toform heteromulti-mers,whencoexpressedwith any of the other VP16hybrids

(2). These findings further confirm the specificity of the

observedhomomultimer formation. However, it should be noted thatwehave notexaminedtheexpression,stability, or

subcellular location of the

majority

of the hybrid proteins

indicated in Table2, sothatnegativedata could be mislead-ing. Thiscaveat also appliesto differences in the observed

level of 1-Gal inducedby each homomultimer pair. How-ever,with thisqualification inmind, itappearsclear that the abilityofboth Tat and Vpr toform homomultimers in the yeastcell nucleus and henceactivate 1-Galexpression isat least comparable to that exhibited by the two HIV-1 pro-teins, Gag and Pol, that are known to multimerize in vivo and is also similar tothe activity displayed by the

GAL4-SNF1/SNF4-GALA hybrid

pair described by Fields and Song(10).

Mutational analysis of

the

86-aa Tat protein (12, 17, 24, 27), incombinationwithanalysisof sequencehomologies in Tatproteinsofbothprimateandnonprimatelentivirusorigin

(5), has permitted the definition of at least three distinct essential domains in Tat. In the domain map proposed by Carrolletal. (5),theseare acysteine-richsequence extend-ingfromaa22to31,a coremotifextending fromaa32to47, andabasic motifextendingfromaa48to 57. Thecysteine motif is conserved in allprimate immunodeficiencyvirus Tat

proteins,whilethecoremotif is conserved in Tat proteins of both primate and ungulate origin (5). Genetic evidence suggeststhatthese motifsarecritical for mediating essential protein-protein interactions in vivo and, in particular, may be important for the functional interaction of Tat with its hypothetical cellular cofactor (12, 17, 24, 26, 27). The basic domainis,in contrast,believed to be important only for the direct interaction of Tat with the viral TAR element (26). While sequences both C- and, particularly, N-terminal to these essential motifs are important for full Tat activity in vivo (27), they display little evolutionary conservation and donot appeartorepresent discrete functional domains.

CYS-RICH

N-TERM CORE BASIC C-TERM

_t//X/4s21 1

21

r

31

47

57

72

12

22

32 48

22 57

%I-Gal

Activity

J 100

86

<0.1

36

108

104

=1 106

J 39

J 4

<0.1

[image:3.612.329.536.69.238.2]

34

FIG. 2. Multimerization capacity of Tat deletion mutants. A seriesof nestedN-and C-terminaldeletion mutants of the 86-aa Tat protein were generated in the context of thepGAL4-TAT expres-sionplasmid byPCR. The ability of the indicated deletion mutants of Tat to multimerize with the full-length Tat fusion protein ex-pressedby thepVP16-TATplasmid was determined in yeast cells by measurementofp-Galactivity,as described in the text, and is given as apercentageof theactivity obtained with the full-length pGAL4-TATplasmid. A proposed domain organization of the HIV-1 Tat protein(5)is indicated at the top of the figure and is explained in the text.

Inorder to define the sequences important for the multi-merization of Tat in yeastcells, we introduced a set of nested

deletion mutations that were designed to sequentially re-moveTatfunctional domains starting from the protein N or C terminusintothepGAL4-TATplasmid(Fig. 2). Deletion of Tat sequencesC-terminal to aa 57 had no detectable effect on the efficiency of multimerization. Further deletion of aa 48to57,i.e.,the basicdomain, produced only a modest, ca. threefolddrop in activity. However, the additional deletion of the core motif resulted in a level of 1-Gal activity

indistinguishablefrombackground(Fig. 2). While deletion of

the N-terminal21 aaof Tat produced only a -2-fold drop in

13-Gal

activity, the additional removal of the cysteine motif

(aa22 to31)led to a level of

1-Gal

activity that, while clearly

above

background,

was nevertheless -25-fold lower than that seen with the full-length GAL4-Tat protein. Further deletion of the core motif again resulted in the loss of all detectable activity. Afinal GAL4-Tat deletion mutant that

retained only Tat aa 22 to 57, i.e., the three essential Tat motifs, gave a level of

13-Gal

activity that was only ca. threefold lower than that seen with the full-length Tat fusion

protein(Fig. 2).Analysis of the level of expression of these

various GAL4-Tat fusion proteinsby Western blot analysis demonstrated that all deletion mutants were expressed at, or slightly above, the level of the full-length GAL4-Tat protein

(2). From thesedata, it therefore appeared that Tat multi-merization was mediated by the core motif and, to a perhaps

slightlylesser extent, thecysteine motif. All other parts of Tat, including the basic domain, were dispensable for

mul-timerizationin the yeast cell nucleus.

The data presented in Fig. 2 are striking in that the Tat sequences important for multimerization map precisely to that part of the Tatprotein that is known to be essential for Tat function in vivo yet not required, at least directly, for

binding to the TAR element (4, 12, 17, 24, 26, 27, 31). Several single-amino-acid point mutations within the cys-teine and core motifs that inactivate Tat function in vivo

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>

w

wi a:

-j 0 0 0

1:.<<,

O co F_

< <_cs < < -J

CD

FIG. 3. Multimerization activity of Tat missense mutants. A

series ofmissense mutationswereintroducedinto the Tatgenein

the contextof the GAL4-Tat fusionprotein. Each Tat mutation is

described in the text. Analysis of the abilityofthese mutants to multimerize withawild-typeTatproteinfusedtothe VP16

activa-tion motifwasdetermined inyeastcellsasdescribedinthetextand

isgivenas apercentage of the activityobtainedwith theparental GAL4-Tatfusionprotein.

have beendefined. Theseincludemutation ofcysteine21 to

serine(C21S), cysteine37 to serine(C37S),andlysine41 to

alanine(K41A) (22, 25).We thereforeintroduced these three mutations, togetherwith two othersknown tohave littleor noeffectonTatactivity (glutamicacid 9 toglutamine [E9Q]

andcysteine31 to serine

[C31S]),

into theGAL4-Tatprotein

and assessed the effect of these point mutations on the

multimerization ofTat. Surprisingly, none of these

muta-tions had a greater than twofold effect on this process, as

measuredby productionof,B-Gal (Fig. 3).To further testthe importanceof the HIV-1Tatcysteinemotif for

multimeriza-tion in yeast cells,we used PCR to derive aTat mutant in

whichfive of thesevencysteineresidues(at positions 22, 25, 27, 30, and 31) were changed to serines. Remarkably, a

GALA-Tathybrid bearingthis5Cx5S mutationwasfoundto multimerizeaseffectivelyasthewild-typeGAL4-Tatprotein

(Fig. 3).

We have used a genetic approach in yeast cells, the two-hybrid system of Fields and Song (10), to assess the potential of the Tat proteins of HIV-1 and HIV-2 to form specific multimers in the yeast cell nucleus. The data pre-sented inthis article demonstratethat bothHIV-1 and HIV-2

Tat can indeed form such homomultimers and further

sug-gestthattheaffinityof thisinteraction is at leastcomparable,

in terms ofinducedp-Galactivity,to thepreviously reported Gag-GagandSNF1-SNF4interactions(10, 18) (Table 2). A setof Tat deletion mutantswereusedtomapthe HIV-1 Tat sequencesinvolved in multimerization tothecoreand,to a

lesserextent,thecysteine motif(Fig. 2). Surprisingly, how-ever, multimerization of Tat remained efficient even after

five of the sevencysteine residues of Tatweremutated to

serines (Fig. 3). These data appear to be inconsistent with

the hypothesis that Tat multimerization results from the

formation of metal-linked dimers, in which these cysteine

residues act asessential metalligands (11).

Although the observationsreported inthis articleclearly

demonstrate that HIV-1 Tat can form specific

homomulti-mers in the nucleus of the eukaryote S. cerevisiae, we caution that these data do not address whetherTat multim-erization is, in fact, important for Tat function inthenucleus of mammalian cells. We also note that nonfunctional Tat proteins that, from this analysis, should remain fully able to multimerize invivo, such as the K41A mutant of Tat (Fig.3), do not exert atrans-dominant negative phenotype (27).Such atrans-dominant phenotype would bepredicted if the K41A protein were able to form inactive mixed multimers with the wild-type Tat protein. Despite these caveats, these genetic

data do clearly demonstrate that sequences within Tat that areknown to be critical for biologicalactivity caneffectively

mediate ahighly specific protein-protein interaction in vivo. Inhibitors of this interaction in yeast cells might therefore also provecapable of effectively inhibiting the activity ofTat in infected human cells.

We thank S.Fields,B.Kohorn,M.Malim,and L. Davisforyeast strains and vectors. We also thank S. Goodwin for secretarial support.

This research was supported bythe Howard Hughes Medical Institute.

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