Proc. Nati. Acad. Sci. USAVol. 86, pp. 821-824, February 1989Biochemistry

Bioassay for trans-activation using purified humanimmunodeficiency virus tat-encoded protein:Trans-activation requires mRNA synthesis

(gene expression/transcription)

REINER GENTZ*, CHEIN-HWA CHEN, AND CRAIG A. ROSENDepartment of Molecular Oncology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110

Communicated by Herbert Weissbach, October 28, 1988

ABSTRACT Expression of the human immunodeficiencyvirus tat-encoded protein (Tat) is required for virus replication.A genetic approach was used to facilitate the purification ofbiologically active Tat. A recombinant Tat protein containinga stretch of six histidine residues and a protease cleavage sitewas engineered and purified to >95% homogeneity in a singlestep by immobilized metal-ion chromatography with a specialaffinity resin that has selectivity for proteins with neighboringhistidine residues. A modified scrape loading method forintroduction of protein into cell monolayers was used todemonstrate that the purified Tat retained biological activity.Tat function was completely blocked in the presence of tran-scription inhibitors, which demonstrates the requirement ofongoing mRNA synthesis for trans-activation. These studiesindicate that the mechanism of trans-activation is unlikely toinvolve a direct action of Tat on mRNA stability, transport, ortranslation and provides the basis for a rapid assay that can beused to identify inhibitors of trans-activation. The methodsdescribed herein should be useful for the functional analysis ofother proteins that do not confer activity through a receptor-mediated pathway.

Human immunodeficiency virus (HIV) gene expression istightly controlled by numerous cellular (1, 2) and viral (3-6)trans-acting regulatory factors. The HIV genome aloneencodes at least three trans-acting regulatory proteins notpresent in other animal retroviruses (7-11). It is well estab-lished that expression of two of these proteins, referred to asTat (7) and Rev [regulator of virion expression, previouslyreferred to as art or trs (11)], is required for virus replication(12-15). The tat gene encodes a nucleolar protein (16, 17) thatis a positive regulator of virion gene expression. The cis-acting control elements responsive to Tat, referred to as TAR(18, 19), are present between nucleotides -17 to +44 (18, 19)in the long terminal repeat (LTR) and are thus present in the5' untranslated leader region of all viral messages. Mecha-nisms operating at both the transcriptional (18, 20-23) andposttranscriptional (20, 24, 25) level have been proposed toexplain tat function.The Tat protein contains at least three functional domains

(17): (i) a negatively charged region at the amino terminus, (ii)a cysteine-rich region, and (iii) a highly basic region. Studiesof tat mutants indicate that the amino-terminal residues andcysteines 22, 25, 27, and 37 are required for Tat function (17).The cysteine-rich region has been proposed to form a metalbinding "finger" common to several transcriptional regula-tory proteins. Frankel et al. have proposed an alternativestructure whereby the cysteines are involved in the formationof a metal-linked dimer (26). The presence of both the highly

charged region and the cysteine-rich cluster has hamperedprotein purification efforts.

In the present study we describe a simple and efficientmethod for purification of biologically active Tat protein.Using a modified scrape loading approach for introduction ofTat protein into cell monolayers, we demonstrate that trans-activation of HIV gene expression requires ongoing mRNAsynthesis.

MATERIALS AND METHODSPlasmid Construction. To construct the H6 tat gene, a

synthetic oligonucleotide encoding the amino acids picturedin Fig. 1 was synthesized with 5' Sal I and 3' Xho IIoverhangs. The oligonucleotide was ligated to an Xho II-HindIII fragment ofHIV (strain HXB2) that encodes the firstexon of the tat gene. The ligated material was cleaved withHindIII and Sal I and then cloned into the Xho I-HindIII sitepresent in the pDS Escherichia coli expression vector (27-29). For expression in mammalian cells, the same fragmentwas cloned into the Sal 1-HindIll sites immediately followingthe HIV LTR in the pHIV expression vector (Fig. 1).

Purification of H6 Tat Protein. The pDS expression plas-mids were used for expression of H6 tat in E. coli. In thisvector system, expression is driven from a bacteriophage T5promoter under control of a lac operator (27-29). PlasmidpDS/Histat was transformed into E. coli strain DZ291, whichcontains a plasmid encoding the lac repressor protein.Expression of Tat was induced in midlogarithmic cultures ofE. coli by addition of 1 mM isopropyl /-D-thiogalactoside.After a 4-hr incubation, cells were pelleted and lysed in 6 Mguanidine hydrochloride (pH 8.0). Lysis was continued inguanidine for at least 1 hr, then cellular debris was removedby centrifugation, and the cleared material was applied to themetal chelate affinity resin (30). Proteins were eluted with aguanidine pH step gradient, followed by analysis withSDS/PAGE.

Introduction of Purified H6 Tat Protein into Cell Monolay-ers. The purified His/Tat protein and the mutant proteincontaining the Cys-25 mutation were introduced into cellmonolayers by using a modified scrape loading procedure(31, 32). Briefly, 5 x 105 cells (35-mm dish) were transfectedwith plasmid pU3R-III (7) and incubated for 40 hr. Afterincubation, medium was removed, and 1 ml of phosphate-buffered saline (PBS) containing the indicated amount ofpurified Tat (see Figs. 3 and 4) was added to the dish. Cellswere immediately scraped from the surface with a rubberpoliceman and then resuspended in fresh media and centri-fuged. The pelleted material was taken up in 2 ml of fresh

Abbreviations: HIV, human immunodeficiency virus; LTR, longterminal repeat; CAT, chloramphenicol acetyl transferase; DRB,5, 6-dichloro-l-/8-D-riboforanosyl benzimidazole.*Permanent address: Central Research Unit, F. Hoffmann-La Roche& Co., Basel, Switzerland.

821

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Proc. NatL. Acad. Sci. USA 86 (1989)

media containing 10%o fetal calf serum, and incubation wascontinued an additional 4 hr. Identical results were obtainedif the centrifugation step was omitted and cells were justdiluted in fresh media following scraping.

RESULTSPurification of Tat Protein. To further our understanding of

trans-activation, a genetic approach was used to facilitatepurification of Tat protein. In the past, a variety of geneticapproaches have been used for protein purification. Exam-ples include fusion of the coding sequence to Staphylococcusprotein A (33), ,B-galactosidase (34), or polyarginine (35). Thehybrid proteins are then purified on their respective substrateaffinity columns. Often, the fusion proteins are large and thuslose biological activity. For purification ofTat, a recombinantprotein with the structure pictured in Fig. 1 was engineered.An oligonucleotide encoding six histidine residues followedby a glutamic acid residue was ligated to the 5' end of the tatgene and cloned into a pDS E. coli expression vector (27-29).Inclusion of the glutamic acid residue following the histidinemoiety and preceding the Tat initiation codon creates aunique Staphylococcus V8 protease cleavage site that can beused for removal of the added histidines. Expression of Tatwas induced by addition of 1 mM isopropyl ,8-D-thiogalacto-side. Proteins were extracted with 6 M guanidine hydrochlo-ride (pH 8.0), and Tat was purified by a single columnchromatography step using a quadridentate metal chelateadsorbent affinity resin consisting of a nitrilotriacetic acidderivative that has selectivity for proteins containing neigh-boring histidine residues (30). Proteins were eluted using apH step gradient (Fig. 2A), and fractions 6-11, whichcontained the Tat protein, were combined and dialyzedagainst PBS and analyzed by SDS/PAGE (Fig. 2A). Aminoacid analysis of the pooled fraction demonstrated >95%homogeneity (results not shown). The higher molecularweight band shown in Fig. 2A was found to represent dimerformation. Removal of the six-histidine moiety was accom-plished by dialyzing the protein against 1% formic acid,followed by treatment with Staphylococcus V8 protease in 50mM ammonium acetate (pH 4.0), and then a final dialysisagainst PBS. Treatment with protease resulted in the appear-ance of a faster migrating form of the protein, which sug-gested removal of the six-histidine moiety (Fig. 2B). Amino-terminal sequencing of this protein confirmed accurate totalcleavage at the glutamic acid residue (data not shown). On thebasis of our results, we estimate that at least 20-50 mg of Tatcan be purified from 1 liter of E. coli by using a singlechromatography step with the affinity matrix described.

Activity of the H6 Tat Protein Expressed in Vivo. Thebiological activity of the His/Tat protein was assessed firstby cloning of the H6/Tat gene into the eukaryotic expression

V8 Protease Cleavage

Met Arg Gly Ser(His)6Gly Ser Val Asp Glu tat (First Exon)1 66

AMP ori- ---1I CT L

pDS-His/tat ID,

AMP or

pHIV-His/tat (Eukaryotic)

FIG. 1. Engineering and purification of the His/Tat protein. Asynthetic oligonucleotide encoding the amino acids shown wassynthesized with a Sal I and Xho II overhang, ligated to the Xho II-

HindIII portion of the tat gene (encoding the first exon), and clonedinto the Sal I-HindIII site of the pDS expression vector. Foreukaryotic expression, the same fragment was ligated into the pHIVexpression vector (17).

A1 2 3 4 5 6 7 8 9 10 11 12

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FIG. 2. Purification and modification of the H6 Tat protein. (A)tat expression was induced in mid-logarithmic-phase cultures of E.coli by addition of 1 mM isopropyl 3-D-thiogalactoside. H6 Tatprotein was purified from lysed E. coli by using a pH elution from themetal chelate affinity resin. Proteins were visualized by staining withCoomassie blue. Lanes: 1, column load; 2, flow-through; 3, pH 8.0wash; 4, pH 6.0; 5 and 6, pH 5.0 ascending peak; 7 and 8, pH 5.0 peak;9 and 10, pH 5.0 descending peak; 11 and 12, pH 2.0. (B) The purifiedTat was dialyzed against 1% formic acid, then cleaved with Staphy-lococcus V8 protease in 50 mM ammonium acetate (pH 4) anddialyzed against PBS. Proteins were analyzed by SDS/PAGE andvisualized by staining with Coomassie blue. Lanes: 1, uncleaved Tat;M, molecular weight markers; 2, Staphylococcus V8 protease-cleaved Tat.

vector pictured in Fig. 1. Cotransfection of plasmid pHIV-His/tat with plasmid pU3R-III (7), which contains the bac-terial chloramphenicol acetyltransferase (CAT) gene undercontrol of the HIV LTR, led to a marked increase in CATactivity, demonstrating that the His/Tat protein is functionalwhen expressed in vivo (Fig. 3A). Moreover, the similar levelof trans-activation observed with cotransfection of plasmidpHIV-tat, which expresses authentic Tat protein encoded bythe first exon, indicates that the histidine moiety has littleeffect on Tat function.

Biological Activity of Purified H6 Tat Protein. To examinewhether purified Tat retained biological activity, a recentlydescribed method, referred to as scrape loading (31, 32), wasmodified for introduction of Tat into cell monolayers. In thisprocedure, either a monolayer culture of HeLa cells trans-fected 40 hr prior with plasmid pU3R-III or a monolayerculture ofa HeLa cell line that contained integrated pU3R-IIIDNA was rubbed from the surface of the dish with a rubberpoliceman in the presence of purified Tat. It is thought thatprotein passes into the cell through holes created during themechanical process of scraping from the dish. Following thescraping procedure, cells were centrifuged, replated in fresh

822 Biochemistry: Gentz et al.

Proc. Natl. Acad. Sci. USA 86 (1989) 823

DNA, ug/ml

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I?

tat + +

His/tat HIS/tat-CyS25

0 30 20 10 5 2.5

Conceration, pg/ml

FIG. 3. Activity of the His/Tat protein. (A) Plasmid pHIV-His/tat (curve 2) and pHIV-tat (curve 1) (expresses authentic Tatprotein) were co-transfected (17) with plasmid pU3R-III (7) intoHeLa cells, and CAT assays (36) were done 40 hr posttransfection.The percent acetylation (15-min reaction) of [14C]chloramphenicolper microgram of transfected DNA is shown. (B and C) The activityof the purified His/Tat protein and the mutant protein containing theCys-25 mutation was assessed by introduction of the protein into cellmonolayers by using the modified scrape loading procedure. Fifteen-minute CAT assays are shown.

media, and CAT assays were performed 4 hr after addition ofprotein. Just scraping of the cells in the absence of proteinhad no effect on LTR-directed CAT gene expression (Figs.3B and 4A). However, CAT gene expression was markedlyelevated (12- to 35-fold) in cells that received Tat. Cells thatreceived purified His/Tat protein that contained a substitu-tion of serine for Cys-25, which has previously been shownto inactivate Tat function (17), demonstrated no increase inLTR-directed CAT activity (Fig. 3B). The trans-activationresponse was linear with respect to protein concentration and

A

0 30 20

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30 20 20

+Act D +DRB

(5ug/ml) (40ug/ml)

B

tat (30ug/ml)

+DRB(40ug/ml) -tat -DRB 0.5 1 2 6

0

Wash away DRB (Hours)

FIG. 4. Trans-activation requires ongoing RNA synthesis. HeLacells that constitutively express CAT from an HIV LTR werepretreated for 15 min with the indicated transcription inhibitors andthen scraped in the presence of Tat as described in the legend to Fig.3. (A) CAT assays were done 4 hr after the addition of protein. (B)The transcription inhibitors were washed away at the 4-hr time point,and CAT assays were performed at the time intervals shown. Act D,actinomycin D.

was easily detected with as little as 2.5-5 gg of Tat per ml(Fig. 3C).

Trans-activation Requires Ongoing mRNA Synthesis. Themechanism oftrans-activation has proven to be complex withboth transcriptional and posttranscriptional mechanisms be-ing proposed (18-25). The availability of biologically activeprotein allowed us to examine whether the posttranscrip-tional component of trans-activation involves a direct orindirect mechanism. For these studies, HeLa cells thatconstitutively express CAT from the HIV LTR were pre-treated with the transcription inhibitors actinomycin D or

5,6-dichloro-1-p-D-riboforanosyl benzimidazole (DRB) (37)15 min prior to introduction of Tat protein. After addition ofprotein, cells were replated in the appropriate media, eithercontaining or lacking the inhibitor, and CAT assays wereperformed after a 4-hr incubation. As shown in Fig. 4A,LTR-directed CAT gene expression was markedly stimulatedin the presence of Tat alone, in accord with results obtainedby using the transfected cell lines (Fig. 3B). However, nostimulation ofCAT gene expression was observed with thosecells treated either with actinomycin D or DRB. Analysis ofCAT-specific mRNA at 0 and 4 hr revealed that significantlevels of mRNA were still present at 4 hr (data not shown),indicating that lack of trans-activation does not reflect a lossof CAT mRNA following addition of the transcription inhib-itor. In the case ofDRB treatment, Tat activation was evidentwithin 1 hr after removal of the inhibitor and increasedsteadily with time (Fig. 4B).

DISCUSSIONIn this report we describe the use of a genetic approach tofacilitate purification of biologically active HIV Tat protein.By using an inducible E. coli expression system, largeamounts of pure Tat were easily obtained with a singlecolumn chromatography step with the quadridentate metalchelate affinity matrix. In vivo expression of the modifiedHis/Tat protein in mammalian cells and scrape loading ofpurified protein into cell monolayers clearly demonstratesthat the histidine moiety and the additional amino acidspresent within the recombinant protein do not interfere withTat activity and that the purified protein retains biologicalactivity. Therefore, this material should be useful for thestructural analysis of Tat protein. The ability to remove thehistidine moiety following cleavage with Staphylococcus V8protease will facilitate those studies that require authenticprotein encoded by the first exon.To further our understanding of Tat function, the require-

ment of ongoing mRNA synthesis for trans-activation wasexamined. Using two transcription inhibitors, each having adifferent mechanism of action, we observed that trans-activation did not occur in the absence of mRNA synthesis.This finding may be interpreted in several ways. If trans-activation, or a component of the mechanism, operates at theposttranscriptional level, as suggested in numerous reports(13, 16, 19, 24, 25), it is likely to do so in an indirect manner.Our results clearly indicate that Tat does not act directly withthe mRNA to facilitate trans-activation through a transport,stability, or translational mechanism. With regard to thetranscriptional component of trans-activation, the resultscannot distinguish between a direct action of Tat or thepossibility that Tat induces expression or interacts with a

cellular protein that, in turn, activates viral gene expression.Since tat expression is essential for virus replication (13,

14), therapeutic and prophylactic strategies designed tointerfere with trans-activation are desirable. The systemdescribed herein, whereby trans-activation is easily mea-sured as early as 3 hr after addition of protein, provides thebasis for a rapid assay to identify inhibitors of Tat function.Potential inhibitors of trans-activation could simply be incu-

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Biochemistry: Gentz et al.

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Proc. Natl. Acad. Sci. USA 86 (1989)

bated with Tat protein added to cells, and the amount ofindicator gene activity (either CAT enzyme or a secretedindicator product) could be measured in the presence andabsence ofthe test compound. By using this approach, a largenumber of potential Tat inhibitors could be screened in arelatively short time period. The purified protein should alsofacilitate the study of structure-function relationships be-tween Tat and its cis-acting control elements as well aspossible interactions with host cell factors. We have used asimilar approach to purify large quantities of both the HIVRev protein and the HTLV-1 Tax protein (A. Cochrane, S.Ruben, C.-H.C., R. Kramer, C.A.R., unpublished observa-tions). In both instances biological activity was retainedwithout removal of the histidine moiety. Thus, the methodsdescribed herein for protein engineering, purification, andintroduction into cells should be applicable for the functionalanalysis of other proteins that do not confer activity througha receptor-mediated pathway.

We thank E. Hochuli for the nitrilotriacetic acid chelate chroma-tography resin, D. Stueber for supplying plasmid constructs, Y. C.Pan for protein sequencing, and Tom Curran and Alan Cochrane forhelpful discussion.

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