The human immunodeficiency virus type 1 long terminal repeat specifies two different transcription complexes, only one of which is regulated by Tat.

CopyrightX 1993,American SocietyforMicrobiology

The Human

Immunodeficiency

Virus

Type

1

Long

Terminal

Repeat

Specifies Two Different

Transcription Complexes,

Only

One of Which Is

Regulated by Tat

XIAOBIN LU,TERRY M.WELSH,ANDB. MATIJAPETERLIN*

HowardHughes Medical InstituteandDepartments ofMedicine andofMicrobiologyandImmunology,

University of

California,

San

Francisco,

San

Francisco,

Califomia

94143-0724

Received 18 September1992/Accepted 15 December 1992

The human immunodeficiency virus type 1 long terminal repeat sets up two different transcription complexes, which have been called processive and nonprocessive complexes. By mutating and substituting cis-actingsequences,wemappedelements of the humanimmunodeficiencyviruslongterminalrepeatthatare

responsible forcreatingeachtranscription complex.Whereasprocessivecomplexesareefficientlyassembledby upstreampromoter elements in the absence of theTATAbox, nonprocessive complexesabsolutely requirethe TATAbox.Moreover,the TATA box alonecansetupthesenonprocessivecomplexes, andnonprocessivebut notprocessivecomplexesaretransactivatedbyTat.Finally,astrongDNA-bindingsitebetweentheTATAbox andtrans-activation-responsive regioninterferes with either theassemblyormovement of thesenonprocessive complexes and diminishes the effects of Tat.Thus, Tataffectsa criticalstepinthe formation of elongation-competenttranscription complexes.

Human immunodeficiency virus type 1 (HIV-1) is the etiologic agent of AIDS. Besides structural proteins and

enzymes, its genome codes for six other proteins, two of which, Tat and Rev, are potenttrans activators that affect quantitativeandqualitative expression,respectively,ofviral proteins (8, 9, 30, 35).Botharealsorequiredforhighlevels ofproductiveviralreplicationand forcellularcytopathology (10, 13).Tat interacts withanRNAstem-loopfoundatthe5' end of all viral transcripts,which is called the trans-activa-tion-responsive region (TAR),togreatlyincreaselevels ofall viraltranscripts andproteins (7, 17, 34, 39).

By interacting with transcribed RNA, Tat could affect ratesofinitiation and/orelongationoftranscription, mRNA stability, and/or translation. In primate cells that support viralreplication, Tat primarilyincreases rates of transcrip-tion from the HIV-1longterminal repeat (LTR) (27, 32). In nuclearrun-on assays,effectsonboth initiation and

elonga-tion oftranscriptionwereobserved(12,18, 21). However,in

in vitro transcription systems, the effects of Tat on the elongation of RNA polymerase II predominate (20, 24). In

one such study, Tat-modified transcription complexes stalled artificially atposition +13 in TAR andacted

syner-gisticallywith transcription factor IIS (TFIIS) butnotwith TFIIF, which implies that TatactsanalogouslytoTFIIF late in theassembly ofelongation-competent transcription

com-plexes (20).

After the earliest nuclear run-on assays and elegant in vitro transcription studies, itwassuggested thattwo differ-ent types oftranscription complexes are assembled on the

HIV-1 LTR (18, 24, 25). One complex is processive and results infull-length transcripts thatarepolyadenylated (18).

The other complex is nonprocessive and results in

prema-turely terminated transcriptswhichconsist of the TAR RNA stem-loop (18). Whereas limited mutagenesis assays

sug-gested that cis-acting sequences that set up processive

complexesdonotinvolvethe TATA box (4),sequences3'to

*Correspondingauthor.

the TATA box andincluding TARweresufficienttogenerate short, nonpolyadenylated transcripts. Infact, the 85 nucle-otidesfrom positions -5 to +80werecalled the inducerof shorttranscripts (33). However,acorrelation betweenthese shorttranscriptsandnonprocessivecomplexesthataretrans activatedby Tat hasnotbeenmade.

Tocharacterize thesetwotranscription complexes in cells rather than in in vitro transcription systems and to define

sequencesin the HIV-1 LTR thatsetupeachtranscription complex,weperformedextensive mutageneses of theHIV-1 LTR.WhereasNF-KBandSP-1sequencesdirected

proces-sivetranscription in the absence ofaTATAbox,

nonproc-essivecomplexes required a functional TATA box. LBP-1 didnotcontribute to trans activation inthis invivoassay.

However, the TATA box was sufficient in the absence of

other transcription factors for setting up low levels of

nonprocessivecomplexes.Furthermore, only nonprocessive transcripts were trans activated by Tat. Finally, strong DNA-binding sites placed 3' to the TATA box interfered withtheassembly and/ormovementof thesenonprocessive complexes. Thus, sequences 3' tothe TATA boxmustbe free ofstrongDNA-binding proteins.

MATERLALSANDMETHODS

Cell culture. HeLa and COS cells were maintained in

Dulbecco's modified Eagle's medium supplemented with 10%fetalcalfserum, 100 U ofpenicillin, 100 ,ug of

strepto-mycin, and292 ,gofL-glutamineperml at370C.

Plasmid constructions. pHIVSCAT, pHIVSCATSV,pSV TAT, pSVTATZX, pHIVSYOCAT, and pHIVSTCCAT havebeendescribedpreviously (1, 18, 38). pHIVTCATand pHIVTCATSVwere derivedfrompHIVSCAT and pHIVS

CATSV, respectively, by deleting a fragment between the

EcoRV andXbaI restriction endonuclease sites which

con-tainsNF-KB andSP-1 sequences. Thesetwoplasmids

con-tain only the TATAbox, LBP-1, and TAR sequences. To construct pHIVSSVCAT, the following oligodeoxynucle-otides,which containthe72-bprepeatfrom the simian virus

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HIV-1 LTR-SPECIFIED TRANSCRIPTION COMPLEXES 1753 TABLE 1. Synthetic oligonucleotides

Oligonucleotide Sequencea

H1HTH2H3 5'-CATGATGCTGC ATATAA GCAGCTGCTTTTTGCATG-3'

D1DTD2D3 5'-CTAGAAGACTC TATTA CACCCCACATTCTGCATG-3'

H1DTH2H3 5'-CTAGATGCTGC TATTA GCAGCTGCTTTTTGCATG-3'

D1HTD2D3 5'-CTAGAAGACTC ATATAA CACCCCACATTCTGCATG-3'

A1ATA2A3 5'-CTAGAAGGGGGG CTATAA AAGGGGTGGGGGCGCATG-3'

H1ATH2H3 5'-CTAGATGCTGC CTATAA GCAGCTGCTTTTTGCATG-3'

A1HTA2A3 5'-CTAGAAGGGGGG ATATAA AAGGGGTGGGGGCGCATG-3'

A1ATH2H3 5'-CTAGAAGGGGGG CTATAA GCAGCTGCTTTTTGCATG-3'

H1ATA2A3 5'-CTAGATGCTGC CTATAA AAGGGGTGGGGGCGCATG-3'

H1HTH2A3 5'-CTAGATGCTGC ATATAA GCAGCTGCTTTTTGCATG-3'

HlHtH2H3 5'-CTAGATGCTGC TTATAT GCAGCTGCTTTTTGCATG-3'

H1HAH2H3 5'-CTAGATGCTGC AAAAAA GCAGCTGCTTTTTGCATG-3'

aSequences are from 5' flanking, TATA, and 3' flanking regions of the HIV-1 LTR, DRA, and AdML promoters.

40 (SV40) transcriptional enhancer, were annealed and

li-gated(45): AvaI

5'-CCGAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCA-3' 3'-CCCACACCTTTCAGGGGTCCGAGGGGTCGTCCGTCTTC-S'*

*5'-GAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACC-3'

3'-ATACGTCOGTACGTAGAGTTAATCAGTCGTTGGTCCA-5'*

*5'-AGGTGTGAAAGTCCCCAGGCTCCCAGCAG(GC(AGA-3'

3'-CACCTTTCAGGGGTCCGAGGGGTCGTCCGTCTTCAT-'5*

*5'-AGTATG}CAAAGCATG}CATCTCAATTAGTCAGCAACCAC-3'

3'-ACGTTTCOGTACGTAGAGTTAATCAGTCGTTGGTGGGCC-5' SmaI The ligated fragment was then inserted into pHIVSCAT between the AvaI and SmaIrestriction endonuclease sites. All replacements of the HIV-1 TATA box and flanking sequenceswere done byreplacing the region between the XbaI(at position -40)andSphI(atposition -10) restriction

endonuclease siteswith thesyntheticoligodeoxynucleotides

listed in Table 1.

Transient transfections and CAT assays. COS and HeLa cells whichweregrownin 150-mm and 100-mmpetridishes,

respectively, were transfected with 15 or 5 ,ug of plasmid DNA,respectively, by theDEAE-dextran transfection

pro-tocol (1, 18, 38). Transfected cells were harvested, and

chloramphenicol acetyltransferase (CAT) enzymatic assays were performed as described previously (1, 18, 38). Each

experimentwasrepeatedatleast fourtimesinduplicatewith similarresults.

RNaseprotection assays. From 5 to10 ,ug ofcytoplasmic

RNAwasassayed exactlyasdescribedbySelbyetal. (38).

The probes used in theseexperiments have been described

previously (38) and are depicted in Fig. 2A. For RNase

protection assays with

pHIVTCATSV

and pHIVDRCAT SV, gels were

exposed

to an imaging screen and

analyzed

with a PhosphorImager (Molecular Dynamics, Mountain

View, Calif.).

EMSAs. For

electrophoresis mobility

shiftassays

(EMSAs),

nuclear extracts from HeLa cells and purified recombinant

SP-1protein (akindgiftfrom Dr.

Tjian)

wereincubated with

a-32P-labeled

double-stranded

oligonucleotides

from the HIV-1 LTR and adenovirusmajorlate

(AdML)

promoter, 2

jig

of poly(dI-dC) per ml, and excess (100 ng) unlabeled

competitor oligonucleotides, as indicated in the

legend

to Fig.3. Theresulting

DNA-protein complexes

wereresolved on

nondenaturing

4%

polyacrylamide gels

and

exposed

to X-ray film. These EMSA conditionswere described

previ-ously(41).

RESULTS

Contributions

of transcriptional enhancer and promoter elementstobasalandtrans-activated levels of expression from theHIV-1 LTR. Toexamine the contributions ofcis-acting

sequences in the HIV-1 LTR to the formation of different

transcription complexes, a synthetic HIV-1 LTR was cre-atedbyplacinguniquerestriction sitesbetween theNF-KB, SP-1, TATA, and LBP-1 sequences (Fig. 1). This cassette-forming approach facilitated the mutagenesis ofeach func-tional unitand the replacement of DNA-binding sites from HIV-1 withthose from other promoters (Fig. 1). For analy-ses ofexpression, this syntheticHIV-1 LTR was linked to the CAT reporter gene(pHIVSCAT). Both CATenzymatic

assays and selected RNase protection assays were

per-formed on HeLa cells and Jurkat cells transiently cotrans-fected with these synthetic HIV-1 LTR target and Tat

effector plasmids. Two different effectors were used, and

they directed the synthesis of functional and nonfunctional

Tatproteins (pSVTAT andpSVTATZ, respectively). Since thedataobtained with Jurkat and HeLa cellswereidentical,

only the results with HeLa cellsarepresented.

In these cotransfections, wild-type target and effector

plasmids yielded 600-fold-higher CAT activities than were observed with thewild-type targetplasmidalone, i.e., 600-foldtransactivation,whichwassetas100%transactivation (Fig. 1B).WhenNF-KB sitesweremutatedbytwomutations of three nucleotides, an almost identical fold increase in transactivationwasobserved(data not

presented)

(28). On the otherhand,whenthese sequenceswerereplacedby the

72-bprepeatwhich is thetranscriptionalenhancer ofSV40, six-fold-higher basal levels of expression and three-fold-lower levels of trans activation were observed (Fig.

1B).

Although basal levelswere

increased,

this strong

transcrip-tional enhancer, which is active in HeLa

cells,

could not abrogate the effects of Tat. Infact,thehighestabsolute CAT

activities were observed with this

target-effector

plasmid

combination.Thus,astrong

transcriptional

enhancercannot set up a transcription complex on the HIV-1 LTR that is

independent of Tat. This observation extends those for

heterologous promoters, with whichbasal levels of expres-sion andfoldtransactivationwereincreased and

decreased,

respectively, and suggests that

transcriptional

enhancers

only

partially

substitute for Tat

(32, 33,

36).

To determine the effects of SP-1 and LBP-1 on trans activation by Tat,we

exchanged

SP-1 sites with those for NF-Y and theoctamerfromthe DRApromoter

(Fig.

1A).

Y and 0 elements represent essential

cis-acting

sequences for VOL. 67,1993

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Aval -150 ScaI TaqI StyI -100 SmaI -50

IIII

Im

I

,I

CTGATGCATCC

TAAAGAdTG GAcAA

CATTCtA

GGArCTTTC

TTCGTGGGGACT

cGsGCC

T

CGGTGGGCCGGGCGACTGGGGGA

XbaI PvuII SphI BglII SacI +50

FCTAGX

I

I

C

I

GTGGCGTCIC TGCTATAT TTTTCACTGACTGGGTCTCTCTGGTTAGAC GAGCC TCTGGCTAACTAGGGAACCC

IXx,73>s>> sZ

-150 -100 -50 -1 +1 150

I I I I

I I I I

'I

i

TARA

9~~~

pHIVSCAT

NF-KB SP-1 TATA LBP-1

pHIVSSVCAT

pHIVSYOCAT

pHIVSTCCAT

I

TAR

72bp REPEAT

I

TAR

*-I3

*-3Amh

1 2 3 200 400 600 200 400 600 50% 100%

FIG. 1. Mutations and substitutionsofsequencesinthe HIV-1 LTR.(A) Sequenceof thesyntheticHIV-1 LTRandreplacements andmutations ofcis-actingelements inthis HIV-1 LTR. At the top of thefigureis thesequenceof the HIV-1 LTR from HIV-1SF2.ChangesintheprimaryDNA

sequencethatwererequiredtointroduceconvenientrestrictionendonuclease sitesareshown in boxesabove thewild-typesequence.Onlyseven

nucleotideswerechanged, mostly with conservative substitutions. Bars underneath thesequencedenote well-characterized DNA-binding sites in

theHIV-1LTR.Solid bars, cross-hatchedbars, theopenbar, and the hatched barrepresentNF-KB,SP-1, TBP,andLBP-1DNA-bindingsites,

respectively. Below thesequenceof theHIV-1 LTRaredrawn four plasmid constructions. pHIVSCAT is the parental constructionand contains

synthetic restriction endonuclease sites. pHIVSSVCAT contains the 72-bprepeatfrom theSV40 transcriptional enhancer;pHIVSYOCATcontains

Y and 0 boxes from the human MHC class II DRApromoter; and pHIVSTRCAT contains mutations ofLBP-1 DNA-binding sites and

compensatorymutationsthat maintainbase-pairing in thestemof TAR.(B) CATenzymaticassaysfromtransient transfections of these plasmid

constructions in thepresenceoffunctionalandnonfunctional Tateffectors in HeLa cells.Target and effectorplasmidsareshowntothe right and

below the panels, respectively, and levels of expression (fold trans activation) are presented above the panels. Cotransfections with the

nonfunctional Tat(pSVTATZX)areshownfirst,followedby those with thefunctional Tat (pSVTAT). Thenextpanelrepresentsacalculation of

the increasein the level ofexpression in the presenceof the functional Tat (pSVTAT) over that in thepresence of thenonfunctional Tat (pSVTATZX) (foldtransactivation[FOLD-TA]). Finally,foldtransactivationisexpressedas apercentageof thelevel obtained with pHIVSCAT (100%). Theseexperimentswereperformedthree timesinduplicate. Standarderrorsof themean aredenotedbyerrorbars.

1754

B

pHIVSCAT

pHIVSSVCAT

pHIVSYOCAT

pHIVSTCCAT

I

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HIV-1 LTR-SPECIFIED TRANSCRIPTION COMPLEXES 1755

the transcription of this human major histocompatibility (MHC) class II gene and have been studiedextensively (15,

42). As reported previously, mutations or deletions of all

SP-1 sites profoundly reduced basal levels oftranscription from the HIV-1 LTR, and little or no trans activation was

observed (data not presented) (16, 17). However, target

plasmids with Y and 0 elements instead ofSP-1 sites were

efficiently trans activated by Tat (Fig.1B). In fact, the fold

transactivation was 90% of that observed with thewild-type targetplasmid. Similar results wereobtained with mutations

of the three LBP-1 sites (Fig. 1B). Since two of these sites

are located in TAR, compensatory mutations had to be

introduced in the distal TAR to maintain base-pairingin the stem. Again, Tat functioned efficiently on this mutated HIV-1 LTR, and only twofold-lower levels of trans

activa-tion than with the wild-type targetplasmid were observed.

Thus, SP-1 sites are not absolutely requiredfor the effects of

Tat, and other transcriptional activators with disparate DNA-bindingoractivation motifs can functionallysubstitute for SP-1. Moreover, although mutations of LBP-1 sites had

deleterious effects in an in vitro transcription system (16,

17), theywerecompletely dispensable fortransactivation in

vivo. This LBP-1 phenotype has also been described

previ-ously (23). We conclude that none of the well-described and well-studied cis-acting motifs in the HIV-1 LTR, i.e., its

transcriptional enhancer (two tandemly repeated NF-KB

sites) or upstream or downstream promoter elements (SP-1 andLBP-1), areessential for trans activation byTat.

Functional TATA box is essential for trans activation by Tat. Since none of the upstream or downstream promoter elements were absolutely required for trans activation by Tat, we also replaced the HIV-1 TATA box region with

those fromthe DRA (42) and AdML (11, 43)promoters (Fig. 2A). The first TATA sequence was chosen because it does notbindTATA-binding protein (TBP) (data not presented) and because it is dispensable for DRA promoter function (2, 42a). On the other hand, the AdML promoter TATA box is

probablythebest-studied TATA box in biology and assem-bles RNA polymerase II and associated factors by first

bindingTBP(5, 26, 44). Although initially the entire cassette frompositions-40 to + 1 in the HIV-1 LTR wasreplaced by sequences from these other two promoters, other plasmid constructions with

only

parts of these other TATA box regions were also made (Fig. 2A).

To our surprise, the TATA box region from neither the DRA norAdML promoter was trans activated by Tat (Fig.

2B, D1DTD2D3 andA1ATA2A3). In the case of the DRA promoter, this defect mapped to the TATA box itself. This

informationwasobtained from two additional plasmid

con-structions. First, when sequences flanking the DRA pro-moterTATAbox were placed around the HIV-1 LTR TATA

box, trans activation levels only twofold lower than wild-typelevelswereobserved(Fig. 2B,D1HTD2D3). However, when the DRA promoter TATA box replaced the HIV-1 LTR TATA box, little to no trans activation by Tat was

observed (Fig. 2B,H1DTH2H3).The trans activation levels observed with the DRA promoter TATA box weresimilar to

those observed with plasmid constructions in which the TATA sequence was replaced by the same number of A

residues(Fig.2B,H1HAH2H30)orthe TATA sequence was

inverted (Fig. 2B, HlHtH2H3). We conclude that a func-tional TATAbox is absolutelyessential for trans activation by Tat.

A more complex picture emerged with the TATA box

region from the AdML promoter. When the entire HIV-1 TATAbox cassette was replaced with that from the AdML

promoter, the trans activation effects of Tat were 10- to 20-fold lower than with the wild-type target (Fig. 2B, A1ATA2A3). However, when we placed just the TATA sequence from the AdML promoter into the HIV-1 LTR, almost wild-type levels of trans activation were observed (Fig. 2B, H1ATH2H3). Thus, the AdML promoter TATA box is functional in the HIV-1 LTR and supports the effects of Tat. However, sequences in its flanking region abrogated the effects of Tat.

To find these deleterious sequences, four additional plas-mid constructions were made (Fig. 2A, A1HA2A3, A1ATH2H3, H1ATA2A3, andH1HTH2A3). The first con-struction (A1HA2A3) tested the AdML promoter 5'- and 3'-flanking sequences in the context of the functional HIV-1 TATA box. The second construction

(A1ATH2H3)

tested the contribution of AdML promoter 5'-flanking sequences. The third construction

(H1ATA2A3)

tested the contribution of AdML promoter 3'-flanking sequences. The final con-struction(H1HTH2A3) tested the contribution of the AdML promoter initiator sequence. As shown in Fig. 2B, since A1ATH2H3 but not H1ATA2A3 approached wild-type

lev-els of trans activation, AdML 3'-flanking sequences were responsible for the decreased levels of trans activation by Tat. Moreover, the AdML initiator sequence functioned better than the HIV-1 initiator sequence, which mapped the

defect tosequences between the AdML TATA box and the initiator. We conclude that whereas sequences upstream and downstream from the HIV-1 LTR can be mutated or re-placed, the TATA box is absolutely essential for trans activation byTat. Furthermore, mutations in its3'-flanking sequences can be deleterious to the function of Tat (see below).

SP-1 binds to 3'-flanking sequences of the AdML TATA

box. To identify the nature of the 3'-flanking defect of the

AdML promoter, we performed EMSAs with nuclear

ex-tracts from HeLa cells with oligonucleotides that

corre-sponded to the HIV-1 and AdML TATA box regions. Althoughfive Tresidues are found at this site, we reasoned

that they were insufficient to cause appreciable bending of

the DNA. Rather, we expected that an essential protein

which is absent inAdML binds to these sequences from the

HIV-1 LTR and thatthis protein helps to set up a

transcrip-tion complex that is responsive to Tat. To our surprise, just

the opposite was observed. Whereas no protein bound to

this site with oligonucleotides from the HIV-1 LTR, very

strongbinding wasobserved with oligonucleotides from the

AdML promoter (Fig. 3A, lane 2). Additionally, excess unlabeledoligonucleotides from the AdML promoter but not

from HIV-1 competed for this binding (Fig. 3A, lanes 3 and

4). Furthermore, oligonucleotides which contained se-quences that supported trans activation by Tat did not compete for thisbinding (Fig. 3A, lanes 5 and 6).

Interest-ingly,unlabeledoligonucleotides which contained three SP-1 sitesbut notthree LBP-1 sites also competed for this binding

(Fig. 3A, lanes 7 and 8).

Toprove that thisprotein was indeed SP-1, two additional experimentswereperformed.First,excess unlabeled AdML oligonucleotide competed specifically for the binding of

HeLa nuclear extracts to SP-1 sequences from the HIV-1

LTR(Fig. 3B, lanes 4 and5). Second, purified SP-1 madein HeLacells with thevaccinia virus expression systembound

specifically

tothis site in theAdML promoter (Fig. 3C,lanes

2 through 7). We conclude that SP-1 binds to sequences between the TATA box and the initiator in the AdML

promoter and interferes with either the assembly or move-ment of transcription complexes along the DNA template. VOL.67, 1993

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A

TCAGATGCTGC ATATAA GCAGCTGCTTTTTGCCTGTACT CIAGATGCTGC ATATAAGCAGCTGCTTTTICTGAGTACT

CATGATGCTGC ATATAAGCAGCTGCTTTTTGCATGCACT

f.TLAMGACTC TATTACACCCCACATTCTGCATGACT

CTAGATGCTGC TATTAGCAGCTGCTTTTTGCATGCACT

C.TAGAAGACTC ATATAACACCCCACATTCTGCATGCACT

,CAAGAAGGGGGGCTATAAAAGGGGTGGGGGCGCATGCGTC

C2TGATGCTGC CTATAAAAAGCTGCTTTTTGCATGCACT

IA1AGGGGGGATATAAAGGGGTGGGGGCGCATGCGTC IAG^AGGGGGGCTATAAAAAGCTGCTTTTTGCATGCACT

C.TGATGCTGC CTATAAAAGGGGTGGGGGCCATGCGCT

CTAGATGCTGCATATAAAAAGCTGCTTTTTGCATGCGCT

100 200 300

TCAGATGCTGC TTATATGCAGCTGCTTTTTGCATGCACT

TCAGATGCTGCAAAAAAGCAGCTGCTTTTTGCATGCACT

200 400 600 800 50 100%

+ pSVTATZX + pSVTAT I FOLD-TA % FOLD-TA

FIG. 2. Substitutions of the TATA boxregionin the HIV-1 LTR. (A) Diagrammatic representation ofsubstitutions and sequencesof substituted TATA boxregions.At the topof thisfigureisdepictedthesyntheticHIV-1 LTRwithwild-typesequencesflankedbyconvenient restriction endonuclease sites(see Fig. 1A).TheTATA boxregion has been divided into four convenientsections, labeled 1, T, 2, and 3.

Whereas Tdenotesthe TATA boxitself, regions 1, 2,and 3contain 5'- and3-flankingsequencesandthe initiatorsequence, respectively.

Thesemotifs in theHIV-1 LTRareshownassolid bars;those from the DRA and AdMLpromotersareshownascross-hatched and hatched

bars, respectively. Capitallettersdenotetheorigin of thesequence(H, HIV; A, AdML; D, DRA)andarefollowed by its location; thus, Hi

represents5'-flankingsequencefrom theHIV-1 LTR.H1HtH2H3 contains the inverted TATA box from the HIV-1 LTR, andH1HAH2H3

containsareplacementof six A residues for the TATAsequence.To theright of the bar diagramsaretheprecisesequencesof theseTATA

boxregions; restriction endonuclease sites areunderlined. Three different sequences aregiven for thewild-type plasmid construction;

however, theyallgavethesame levels ofexpression (datanotpresented). (B) CAT enzymaticassaysfromtransienttransfections ofthese plasmidconstructionsin thepresenceoffunctional andnonfunctional Tat effectorsin HeLacells.Panelsare asdescribed in the legendtoFig. 1B.Different barshadingsreflect the donorof theTATAboxregion, and onlythewild-type HIV-1LTR isdepictedas asolid bar. These

experimentswereperformeduptofive times and intriplicate. Standarderrorsof themean aredenotedbyerrorbars. 1756

pHIVSCAT

H1HTH2H3

D1DTD2D3

H1DTH2H3

D1HTD2D3

T 2 3

A1ATA2A3

H1ATH2H3

A1HTA2A3

A1ATH2H3

H1ATA2A3

H1HTH2A3

HlHtH2H3

H1HAH2H3

B

H1HTH2H3 D1DTD2D3 H1DTH2H3 D1HTD2D3 A1ATA2A3 H1ATH2H3

A1HTA2A3

A1ATH2H3 H1ATA2A3 H1HTH2A3 HlHtH2H3 H1HAH2H3

M-MENEM

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HIV-1 LTR-SPECIFIED TRANSCRIPTION COMPLEXES 1757 B

a".1i.a

<-.s -

r6-) ,- -Z:6-N

:4 i 6 X

6-\>

-_....~

FIG. 3. EMSAs with nuclearextractsfromHeLa cells, purified SP-1, and oligonucleotides fromthe HIV-1 LTR and AdMLpromoter.(A)

EMSA with the oligonucleotide containing the TATA box region and nuclearextractsfromHeLacells. The nuclearextract, radiolabeled

oligonucleotide, andexcessunlabeledcompetitor oligonucleotides usedaregiven above the gel.Lane 1, noextract; lane 2,nocompetitor

oligonucleotide;lanes 3to9,100ngofcompetitoroligonucleotide containing only AdMLsequences(lane 3),HIV-1 5' and AdML TATA box

and3-flanking sequences(lane 4), AdML 5'-flanking and TATA box and HIV-1 3'-flankingsequences(lane5), HIV-1 5'- and 3'-flanking

sequences and AdML TATAbox (lane 6), three LBP-1 DNA-binding sites (lane 7), and three SP-1 DNA-binding sites (lane 7). The

oligonucleotideswereidenticaltothecassettesused in the construction of the synthetic HIV-1 LTR (Fig. 2A)andaredenoted by lowercase

letters. Arrows pointtoSP-1 andanonspecific proteincomplex (NS),sonamed because unlabeled specificand nonspecific oligonucleotides

didnotcompetefor itsbinding. Free probe referstoradiolabeled oligonucleotides thatwerenotcomplexed withprotein. (B) EMSA with the

oligonucleotide containing the three SP-1 sites and nuclear extracts from HeLa cells. Lanes are labeled as in panel A. Radiolabeled

oligonucleotide containingthe threeSP-1 sites from theHIV-1 LTRwasincubatedwith nuclearextractsfrom HeLa cells (lane 2)and 100ng

of thesameunlabeledoligonucleotide (lane 3)andthosecontaining the AdML TATA boxsequences(lane 4), HIV-15'-flankingand AdML

TATA boxand3'-flanking sequences(lane 5),AdML5'-flanking and TATA box and HIV-1 3'-flankingsequences(lane 6), HIV-1 5'- and

3-flankingsequencesand AdML TATA box(lane 7), andSP-1 sites from thetumornecrosis factor alphapromoter(lane 8).Noextractwas

added in lane 1.(C)EMSA with theoligonucleotide containing theAdML TATAboxregion and purified SP-1 from HeLa cells infected with

avaccinia virus which directs thesynthesisof SP-1. Lanesarelabeledasinpanel A. SP-1 withan excessof bovineserumalbumin (BSA)

wasincubatedwith radiolabeled oligonucleotide containingAdML TATAboxsequencesandnospecific oligonucleotide (lane 2)or100ng

of thesameunlabeledoligonucleotide (lane 4)andthosecontaining three SP-1 sites (lane 3) and HIV-15'-flanking and AdML TATA box and 3-flankingsequences(lane 5).The radiolabeledoligonucleotidewasalsoincubatedinthe absence ofSP-1 (lane 1), in the absence of BSA (lane 6),which stabilized the dilute SP-1proteininsolution, and in theabsenceofZn2+ (lane 7), which didnotdestabilize this binding.

The TATA boxregionof HIV-1 functionsefficientlybecause

nonuclearproteinbindsstronglytoits3'-flankingsequences

or toTAR DNA.

TATA box alone can set up nonprocessive transcription complexes that are trans activated by Tat. Having demon-strated that whereas upstream and downstream promoter elements increaseexpressionfrom the HIV-1LTR butdo not affect the response to Tat and that the TATA box and its immediate 3'-flanking sequences areabsolutelyessential for

transactivationby Tat,wewondered whether theTATAbox by itself could assemble nonprocessive transcription

com-plexes. Previously,weand others demonstrated thepresence

oftwodifferent transcription complexesthatwereidentified bytheirhybridization topromoter-proximaland-distal frag-ments in nuclear run-on assays (12, 18, 21). In studies of steady-state RNA, they were reflected in full-length poly-adenylated and shortnonpolyadenylated transcripts,

respec-tively (18). Since the half-lives of these RNAspecies were

similar(31),weexaminedtransientlytransfected cells for the

presenceof thesetwodifferenttranscripts.Sincehigherlevels of RNA could be achieved inCOS cellsby using replicating vectors, these studies were carried out with targets and effectors that containedtheSV40 originofreplication.

SinceLBP-1 didnotcontribute totransactivation in either

HeLa or COS cells, we removed only NF-KB and SP-1

sequences from the HIV-1 LTR and cotransfected these

plasmids with functional and nonfunctional Tat effector

plasmids. As depicted in Fig. 4A, wild-type targets and effectorsgavehigh levels of short transcripts in the absence

of Tat and correspondingly high levels of full-length

tran-scriptsin thepresenceof Tat(Fig. 4A, lanes 3, 4, 7, and 8). These RNA levels approached those observed in CAT

assays (Fig. 4A). Likewise, with an HIV-1 LTR which

containedonly the TATA boxbut lackedNF-KB and SP-1

sequences,low levels of these shorttranscripts,whichwere

efficientlytrans activated by Tat,were observed (Fig. 4A,

lanes 3 and 4). However, since levels of expression were

low, these gels had to be exposed to a phosphorimaging screen to demonstrate the presence ofspecific bands (Fig. 4A). Weconclude that the TATA box alone is sufficient for setting upnonprocessive complexes that aretransactivated byTat.Thus,the main role of upstream promotersequences

istoincrease theloadingof RNApolymeraseII,which results

in higher levels oftranscriptionandtrans activationbyTat. Perhaps most importantly, these results suggest that any

TATA box withanopen3'-flankingsequencecanassemblea

transcription complexthat istransactivatedbyTat. What abouttranscription complexesthatareformed in the absence of a functional TATA box? To detect these

com-plexes, similar RNase protection assays were performed

with RNA from COS cells cotransfected with plasmids in

which the HIV-1LTRwassubstituted with the DRATATA

box region and plasmids carrying functional and nonfunc-tional Tateffectors. With this substituted HIV-1 LTR, only

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FIG. 4. RNaseprotectionofRNA from COScellscotransfected withplasmids carryingtheTATA boxregionalone andplasmidsin

whichthe HIV-1 LTRwasreplaced bytheTATA box from the DRA promoter in the presence of plasmids carrying nonfunctional or

functional Tat effectors.(A) Phosphorimagesof RNaseprotectionof cotransfected COS cells. In both panels, thetargets are givenon

top, and thepresenceof nonfunctional (-)and functional(+) Tat effectors is depicted below the brackets. Foldtrans activation in CATenzymaticassaysisgivenbelow thephosphorimages.Lanes1, 2, 5,and 6 represent thewild-typesituation,inwhich theHIV-1 LTR targetswerecotransfected with both forms of Tat. In lanes 3 and 4are

similarexperimentswithplasmidsin whichsequences5'totheTATA box in the HIV-1 LTRwereremoved. In lanes 7and 8arepresented

cotransfections withplasmidsinwhich the HIV-1 LTRwasreplaced

bythe TATA box from the DRA promoter.Long(LT)and short(ST) transcripts correspond respectively to full-length polyadenylated transcripts and short nonpolyadenylatedtranscripts, which contain

onlyTARsequences and are55, 57, and 59nucleotides in length.

HIV-1 transcription initiates atallthree G residues 23to 26

nucle-otides 3'tothe TATAbox(18). (B)Diagrammatic representationof RNA probes. Below the picture of the HIV-1 LTR are given the

transcribed antisense RNAprobeand hybridizing speciesfrom the HIV-1 LTR. The probe is 220 nucleotides long and hybridizes to

full-length, polyadenylatedtranscriptsof 78to80bporthree short,

nonpolyadenylated transcripts of 55to59 bp. pA, poly(A) site.

full-length transcripts were observed, and these were not

affectedbyTat (Fig. 4A, lanes 7 and8). We conclude that whereas nonprocessive complexes require a functional

TATA box, processive complexes, which are formed by

upstream promoter sequences, do not require a functional

TATAbox and do notrespondto Tat.

SP-1placed betweenthe TATA box and TARinterfereswith assembly and/or migration of nonprocessive transcription

Probe - -- - 220bpprobe

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)tCLttl tranIscripts I )1|)

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FIG. 5. RNaseprotectionof the HIV-1 LTR substituted with the

TATA boxregionfrom the AdML promoter.(A) Autoradiogramsof

RNase protection of RNA from COS cells cotransfected with the

plasmidsin which HIV-1 LTRwasreplaced bytheAdMLTATA

box region and plasmids with nonfunctional or functional Tat effectors. Lanes are labeled as in Fig. 4. Lanes 1, 2, 5, and 6 represent the wild-type situation, in which the HIV-1 LTR was

cotransfected with both forms of Tat. In lanes 3, 4, 7, and 8 are

RNAs from similar cotransfections with plasmids in which the HIV-1 LTRwasreplaced bythe AdML TATA boxregion.Whereas RNAprobes in the left-hand panel were full-length, those in the

right-hand panelwere shorter by 20 nucleotides at their 3' ends because ofdigestion oftemplate DNAwithBglII. Note that two smaller bands in lanes 3 and 4arereducedtoasingleband in lanes 6and 7. Thus,the shorttranscriptsfrom theplasmidin which the HIV-1 LTRwasreplaced bythe AdML TATA boxregioninitiated

correctly and were shorter at their 3' ends. (B) Diagrammatic representation of RNA probes. Both full-length and short,

60-nucleotide-long probes are depicted, with the corresponding

ex-pected hybridizingbands.pA, poly(A)site.

complexes. Finally, we wanted to know how strong DNA-binding proteins placed between the TATA box and TAR affect nonprocessive complexes that aretrans activatedby Tat. To this end, we performed RNase protection

experi-ments with RNA from COS cells cotransfected with plas-midsin whichthe HIV-1 LTRwasreplaced with the AdML

TATA box region and plasmids carrying functional and nonfunctional Tat effectors.AsshowninFig. 5A, lanes 1, 2, 5, and6, thesamepatternsasinFig. 4A, lanes1, 2, 5, and

6, were observed with the wild-type target and effector plasmids. However, with the plasmid which contained the TATA boxregionfromthe AdMLpromoter, lower steady-state levels of short and full-length transcripts were ob-served. In addition, whereas the sizes of full-length tran-scriptswerethesame aswith thewild-type HIV-1 LTR, the

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HIV-1 LTR-SPECIFIED TRANSCRIPTION COMPLEXES 1759

short transcripts were smaller (Fig. 5A, lanes 3, 4, 7, and 8). Since the full-length transcripts were of comparable sizes, we reasoned that they initiated from the same site. By making the RNA probe shorter at the 5' end by 20

nucle-otides,we alsoobservedthat these short transcripts initiated

correctly (Fig. 5A, lanes 7 and 8). However, they were

shorter at their 3' ends. Moreover, this shorter probe re-duced two short transcripts obtained with the longer probe to one transcript (Fig. 5A, lanes 3, 4, 7, and 8). From

inspection of the stem-loop structure, which has a bulge at the bottom, we conclude that these differences between

wild-type and substituted targets can be explained if

tran-scription complexes do not migrate as far with constructs which contained the AdML TATA box region than with

thosecarrying thewild-type HIV-1 LTR. Thus, SP-1 inter-fereswith themigrationofnonprocessive transcription com-plexes; only a few reach the 3' border of TAR, and none goes beyond TAR.

These data stronglysuggestthat nonprocessive complexes

assembled at theTATA box are sluggish and susceptible to

prematureterminationand that Tat completes the process of making these complexes processive at TAR. Furthermore, they also argue against the notion that Tat reaches back to the TATA box toincrease initiationof HIV-1 transcription,

sincelevels oftrans-activated full-lengthtranscripts with the

AdMLTATA boxregion should have more closely resem-bledthoseobservedwith the wild-type target if that were so.

DISCUSSION

Thedatafrom these studies confirm the existence of two different transcription complexes which are set up on the HIV-1 LTR. Whereas processive complexes are formed

independentlyof theTATAsequence and do not respond to Tat, nonprocessive complexes require a functional TATA sequence and relatively free 3'-flanking sequences and are transactivatedbyTat. It is veryattractivetospeculate that

the relativeamounts ofthesecomplexes determinethe level

oftransactivationby Tatand thattranscriptional enhancers

and other acidicactivators such as ElA affecttheseratios (22, 32, 33, 36).

CAT enzymatic studies were confirmed by analyses of

transcripts from the HIV-1 LTR, and short but not long

transcripts correlated with trans activation by Tat. More

importantly, when no short transcriptswere observed, Tat

hadnoeffect. Onthe otherhand,even with a minimal TATA sequence, short transcripts predominated and were trans activated by Tat. Thus, Tat affects a generic RNA poly-merase II complex that is able to move, albeit sluggishly, alongthe DNAtemplate. Thebrittlenessof thistranscription complex was confirmed by studies with the AdML pro-moter,whoseTATA sequence functionedin thecontext of the HIV-1 LTR but whose 3'-flanking sequences inhibited

trans activationby Tat. This was due to the binding of SP-1 between the TATA box and TAR. The fewer and shorter

transcripts

observed with thisstrongDNA-binding sitemost likely reflect a barrier to movement of RNApolymerase II along thetemplate. However,its effects on the assemblyof

transcription complexes cannot be ruled out. It should be noted that Tat functioned less well on targets which con-tained SP-1binding sites 3' tothe TATA box on

nonrepli-catingvectors in HeLa cells than onreplicating vectors in

COS cells (compare Fig. 2A and 5), which could be due either to amounts of SP-1 insufficient to saturate all cognate

bindingsites or toits removal from DNAduringreplication.

Furthermore, since Tat did not increase overall levels of

expressionbut only effected a qualitative change from short to long transcripts in this hybrid promoter, it is unlikely that Tatcan reach back and increase the loading of RNA

poly-merase II. Finally, since DRA promoter 3'-flanking

se-quences could functionally replace those from HIV-1, the only requirement for these sequences is their inability to bind proteins tightly.

Ourdata extend and support those of previous studies on transcriptional regulation of HIV-1 gene expression (3, 18, 20, 24, 25, 29, 33). For example, initial studies with trans-fected or intrans-fected cells and in vitro transcription systems

revealedastrongpolarityof HIV-1 transcription, which was

modifiedby Tat (12, 18, 19, 21, 24). Whether called

antiter-mination, increased elongation, attenuation, or processivity, all refer to the simple notion that although many transcrip-tioncomplexesfrom HIV-1 LTR cannot copy the entire viral

genome, this situation is reversed by the addition of Tat. What this study adds is the precise mapping of sequences in the HIV-1 LTR that specify different transcription com-plexes and the notion that any TATA sequence that can bind

TBPcan set up transcription complexes that are intermedi-atebetweenabortive initiation and competent elongation.

Recent studies on cis-acting elements in the HIV-1 LTR and the role of the TATA box in trans activation by Tat

support these conclusions (3, 29). Furthermore, our study clearlysuggests that the same transcription complexes that are sensitive to 5,6-dichloro-1-3-D-ribofuranosylbenzimida-zole(DRB)and that are trans activated by Tat in vitroresult

in theformationoftheseshort transcripts in vivo (25). Also, the inducer of short transcripts consists primarily of the TATA box and its3'-flankingsequencesthat are free or only

looselybound by DNA-binding proteins (33). We conclude

that short transcripts that are generated by the inducer of

short transcripts are also the substrate for Tat.

The situationwith the HIV-1 LTR is similar to that found in theheat shockpromoterofDrosophila melanogaster (37) and several mammalian proto-oncogenes, such as c-myc,

c-myb, and c-fos (40).In thefirstexample, RNApolymerase

II isarrested just 3' of the site of initiation of transcription and is released following heat shock. In the second case,

variousactivationandproliferation signals alter the polarity oftranscription so that sequences distal to these genes are

copied. From these and other examples, it appears that Tat

mimics a cellular protein(s) that, by associating with the

transcription complex, increasesitsabilityto elongate. Cer-tainly Tat can function after several phosphodiester bonds have beensynthesizedandaftersignificanttranscription has

occurred. Whether these effects of Tat are assimple asthe

phosphorylationofC-terminal domainsofRNApolymerase

II,which is required for thetransitionof RNA polymerase II frominitiation to elongation (6), or more complex, such as

interactionswithbasal transcription factors such as TFIIE,

TFIIF, TFIIH, and TFIIJ (14, 44), or both remains to be

established.

ACKNOWLEDGMENTS

We aregrateful to MichaelArmaninifor expertsecretarial assis-tance; Shaw-Yi Kao, Mark Selby, and Sandra Tong for helpful suggestions and criticisms; and other members of the Peterlin laboratoryforcarefulcommentsonthemanuscript.

Thiswork wassupportedbythe HowardHughes Medical Insti-tute.

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