Mutational analysis of the trans-activation-responsive region of the human immunodeficiency virus type I long terminal repeat.

0022-538X/88/030673-07$02.00/0

Copyright C)1988, AmericanSociety forMicrobiology

Mutational Analysis

of the

trans-Activation-Responsive Region

of

the

Human

Immunodeficiency Virus

Type

I

Long

Terminal

Repeat

JOACHIM HAUBERtAND BRYAN R. CULLENt*

Departmentof MolecularGenetics, RocheResearch Center,

Hoffmann-La

Roche Inc., Nutley, NewJersey 07110

Received30September 1987/Accepted4December1987

We usedsite-directed mutagenesis to delineate sequences within the humanimmunodeficiency virustypeI

(HIV-I) long terminal repeat (LTR) required for trans-activation by the viral tat gene product. We demonstratedthat sequences 3' to LTR position +44 are

dispensable

fortrans-activation but that almostallof

the mutations tested located between positions -17 and +44 greatly reduced trans-activation at both the

transcriptional and posttranscriptional levels. However, displacement of the HIV-1 LTR trans-activation-responsiveregion (TAR)3' by insertion ofupto 32 basepairsbetween the LTR TATA box and capsite had

little effect on trans-activation. An analysis of the DNase I hypersensitivity profile of the HIV-I LTR in

transfectedcultures suggested the presence of at least two DNase

I-hypersensitive

sites, includingone which

extends into the viralTARelement; however, neither of these sitesappeared to besignificantlyaffectedbytat

coexpression. These results allow moreprecise

delineation

of thesequences important for TAR function and

suggest that the TAR may be recognizedbyahost-specific DNA-binding protein rather than by the tat protein

directly.

The pathogenic human retrovirus human immunodefi-ciency virus type I (HIV-I) encodes not only the three structural genes (gag, pol, and env) common to all known replication-competent retroviruses but also at least four other, apparently nonstructural, gene products (4). Two of these, termedartltrs andtat,havebeenshownto actintrans to increasethe level of synthesis ofHIV-I-specific proteins (1, 5, 6, 8,16, 22,25, 30, 35, 39).Inthecaseoftat,evidence hasbeen presented forabimodalmechanism of action which involves both anincrease in the steady-state level of HIV-I-specificmRNAandanincrease inthe translational utiliza-tion of that mRNA (5, 39).

More

recently, it has been demonstrated that tat increases HIV-I mRNA levels by enhancingthe rateoftranscription rather thanby stabilizing HIV-I-specific mRNAs (12).

This

result is consistent with the predominantly nuclear subcellular localization of tat protein(12). Themeansbywhich thesecond, posttranscrip-tional action of tat on HIV-I gene expression is effected remains unknown. However, evidence has been presented suggesting that HIV-I mRNAs are poorly

utilized

by the cellular translational

machinery

in theabsence oftat

coex-pression (5, 8).

Inthis study, weattempted to address the mechanismof action of tat via mutational analysis of the HIV-I long terminalrepeat (LTR) sequence originally identified as the core trans-activation-responsive (TAR) region (31). This

sequence, whichextendsfrom -17to +80within theHIV-I

LTR, exhibits a number of striking inverted and direct

repeats (Fig. 1), and it has indeed been suggested that the

LTR sequence from + 1 to +59 can form a stable mRNA

stem-loop

structure important for the posttranscriptional

component ofHIV-Itrans-activation (22, 24).Alternatively,

directrepeats arefrequentlyobserved within transcriptional regulatory sequences such as enhancers or promoters (19),

*Correspondingauthor.

tPresent address: Howard Hughes Medical Institute,

Depart-ment of Medicine, Duke University Medical Center, Box 3025, Durham, NC 27710.

and therefore might also be involved in the transcriptional

action of tat. By using constructions in which the HIV-I

LTR was fused to areporter gene, the human interleukin-2

(IL-2) gene (5), we measured the effects of differenttargeted

LTR mutations on both the basal and tat trans-activated

levels of IL-2 protein production in transfected cells. For

severalmutations, we also quantitated the steady-state level

of the reporter gene mRNA and deduced the relative

trans-lational efficiencies of the mRNAs encoded by different

mutants. These results allow moreprecisedetermination of

the location of TAR and more accurate assessment of the roles of particular repeat sequences. Finally, we

demon-strated the existence of at least two sites of DNase I

hypersensitivity within the HIV-I LTR. The location of

these sites, including one site which extends into the TAR

region, was, however, not markedly affected by

coexpres-sion of the tatgene product.

MATERIALS AND METHODS

Construction of molecular clones. All molecular clones

usedin this work werederived from the vectorpXF3/ori(5),

which contains the simian virus 40 origin of replication

insertedinto the pBR322derivative pXF3. The

cytomegalo-virusimmediate-earlypromoter-based IL-2expression

vec-tor pBC12/CMV/IL-2 and the tat expression vector pBC12/CMV/t2have been previously described, as has the

internal control plasmid pBC12AI (5). All HIV-I LTR

mu-tants were derived from pBC12/HIV/IL-2, which contains

the human IL-2 gene undercontrol of a 728-base-pair (bp)

HIV-I DNA fragment (5). This HIV-I sequence is derived

from the replication-competent HXB-3 strain ofHIV-I (27)

and includes theentire LTR U3 region, as well as 84 bp of

the LTR R region (Fig. 1). pBC12/HIV-Xho was derived

frompBC12/HIV-IL-2byoligonucleotide-directed

mutagen-esis by using the procedure described by Morinaga et al.

(21). The oligonucleotide used, an 18-mer, introduced a contiguous 2-bp mutation at positions +10 and +11 (TG-*GA) and created a unique XhoI site (Fig. 1). pBC12/HIV/IL-2 and pBC12/HIV-Xho were then used to

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A

-40 C T CACAT C C TCCAT A T A A6CACC TGCTTTTTC C C T C TAC

TATA Pvu11 U3-J

+I 666TCTCTCTB6TTAGACCAGATCTGAGCCTGBGAUCTCT .40

AF agi11 Sac

Xho

+41 CT 66 C TA ACT A 6 6 AA C C CAC TCCT TA A6C C T CAATAA AGC0 4

31B

+81 CT TC9ta a9t

HindIII

B

Ic

+1 IC%I%

/-cap-G G U U C U C UC CU UACC CA6ACA6C C

. . I I I |II

CCCAABBBAUCAAUCBBUCUCUCB C

+59 4

FIG. 1. Sequence of the HIV-I LTR TAR region included in pBC12/HIV/IL-2. (A)Apartial sequenceof the LTRof the HXB3

strain of HIV-Iused in this report is shown(capital letters). This sequenceisidenticaltothe HXB3sequencepreviously published by

Ratneretal. (28),except atposition +52, wherewe observedaG

rather thananA. AG has been observed in this positionin some

other HIV-I isolates (32, 38). Also shown are the locations of

relevant restriction sites, including the XhoI site introduced by oligonucleotide-directed mutagenesis at position +9. The TATA

box and the 3' border of thecoreTARregion mappedinthis work

(3'B)areindicated. The location ofanimperfectdirectrepeatwhich isdiscussed in thetextis also shown(<-*). (B)Apossible secondary

structureinvolvingthe first59ntofthe HIV-I mRNAis shown. This

structurehasanestimated freeenergyof -37 kcal/mol(22, 24)and hasbeen shown to occurin a synthetic SP6 transcript byinvitro enzymatic analysis (22). The residual stem present in the

pD+45/+77mutant, which is deletedto thelocation indicated by 3'B, has a free energy of -17 kcal/mol as determined by the

procedureof Tinoco et al.(36).

generate series of internalHIV-I LTR deletion(pD)mutants

by cleavageatvarious restrictionenzymesites,followedby

bluntendingwith Klenow DNApolymeraseI andreligation.

Mutants generated by this procedure include pD-18/+7

(PvuII to XhoI), pD-18/+20 (PvuII to BglII), pD+9/+20

(XhoItoBglII),pD+9/+38(XhoItoSacl), pD+9/+77(XhoI

to HindIII), pD+25/+38 (BglII to

Sacl),

and pD+35/+38

(Sacl

sitedeletion). Ineachcase,the numbersin theplasmid

designationdescribe theinclusiveextentof the deletion(Fig.

1).Asecondsetof HIV-I LTRdeletionmutantswasderived

by processive BAL 31 digestion ofpBC12/HIV/IL-2 after

cleavagewithHindlll.After BAL31digestion (International

Biotechnologies, Inc.;slowform),the DNAwasbluntended

with Klenow DNA polymerase I and ligated to a Hindlll

linker(5'-GAAGCTTC-3').TheDNAwasthencleaved with HindIIIand with MstII, which cuts at a unique site in the HIV-I DNA at position -526. The resultant fragments of <608bpwereisolated fromapreparative2%agarosegeland recloned into the larger parental pBC12/HIV/IL-2 vector HindIII-MstII fragment. The resultant clones were

charac-terized

as to deletion size and tested for trans-activation

phenotype. Onlythetwo smallest deletions withanegative

phenotype (pD+42/+77andpD+44/+77)and the twolargest

deletions with a positive phenotype (pD+45/+77 and

pD+46/+77) are described. The final four LTR mutants,

termed

pl

mutants, were derivedby insertion of nucleotide

sequences at differentlocations within the LTR. p14/+7(a

4-nucleotide [nt] insertion at position +7) was derived by

filling in the XhoI site in pBC12/HIV-Xho. Similarly,

p14/+20 was derived by filling in the BglII site in

pBC12/HIV/LTR. pI8/-18 and p132/-18 were derived from

pBC12/HIV/IL-2 viaatwo-step process.Initially, the unique

HIV-I LTR HindlIl site was filled in to generate p14/+78.

Thisclone, which hasanNheI siteatposition +82 in place

of the HindlIl site, exhibits the same trans-activation

re-sponse as the wild-type clone pBC12/HIV/IL-2 (data not

shown). Subsequently,one orfour of the 8-nt HindlIl linkers

wereintroduced into the unique LTR PvuII site atposition

-18 toyield, respectively,pI8/-18 and pI32/-18. All of the

mutants weresequenced by the dideoxynucleotide sequenc-ing procedure (33) after being subcloned into the vector M13mp9 (20).

Cell culture and DNA transfection. COS cells were

main-tainedaspreviously described (5) and were transfected with

2.5

Rg

ofplasmidDNA per 100-mm(diameter) culture with

DEAE-dextran and chloroquine (5). Cell culture medium

and transfected-cell RNA were harvested at 72 h after

transfection. Secreted IL-2 levels were measuredby using

theIL-2-dependent murineT-celllineCTLL(5, 29).

RNAanalysis. TotalRNA waspreparedfrom transfected

cultures(5), and RNAsamples,asindicated in the text,were

then usedtoquantitatethe levelofRNA expressionby using

S1nucleaseprotection analysis (5).The S1probesused have

been previously described (5). The 750-nt 3'-end-labeled probe used (see Fig. 2) rescues a 175-nt fragment derived

fromthe RNAencoded by the IL-2expression vectors, as

wellas a156-ntfragment derived fromthe RNAencoded by

the internal referencevectorpBC12AI. Wehave previously

shown that the level ofRNAencodedbyRoussarcomavirus LTR-based pBC12AI is unaffected by tatcoexpression (5).

Theprobes used (see Fig. 3)wereuniquely5'end labeled eitheratposition +82 atthe wild-type HIV-I LTRHindlIl site or at +84attheintroducedNheI site ofpI32/-18. The probesextendtothe HIV-I LTRU3region Aval site (-159 inpBC12/HIV/IL-2). Transcripts initiating withinthe HIV-I LTRrescue aprobe fragmentwhoselengthisdeterminedby thedistance of the transcript initiation sitefromthe site of the label(5).The RNAderived from HIV-I-infectedH9cells (26; see Fig. 3)was agift ofM.Dukovich andW. Greene.

DNaseIhypersensitivity analysis. Nuclei from transfected

cultureswerepreparedat72h

posttransfection

before diges-tion with various amounts of DNase I (Worthington Diag-nostics; DPFF) (9, 10).TreatmentofthenuclearDNAwith therestrictionenzymeBamHI, agarosegel electrophoresis, Southern

blotting,

and filter

hybridization

with the

nick-translatedprobe werecarriedout asdescribedby Frittonet

al. (9, 10). A 261-bp XbaI-to-BamHI human IL-2 gene fragment derived from

pBC12/HIV/IL2

wasusedfor prepa-ration ofthe probeusedforhybridization.

RESULTS

Effect ofTARmutationson reporter geneprotein synthesis.

Theexpression vector

pBC12/HIV/IL-2

containsareporter

gene,the human IL-2 gene, under controlofa728-bpHIV-I

DNAfragmentwhich includes the entire456-bpHIV-I LTR U3

region,

aswellas84bpof the LTR R

region

(5;

Fig.

1).

Thecore TAR sequence has previously been shown to be

contained within an HIV-I LTR sequence

extending

from approximately -17 to +80(31; where the site of

transcrip-tion initiatranscrip-tion is +1). Ourinitialapproachtothe determina-tion ofthefunctional

significance

of different

regions

ofTAR wasprocessive5'deletion fromtheHIV-I LTRHindlllsite

at +80, with BAL 31 exonuclease, to determine the siteof

the 3' border of the TAR sequence. It has previouslybeen

shown that deletionofsequences 3' to

position

+37 results

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in loss of tat responsiveness by the HIV-I LTR (5, 39).

Numerousdeletionmutants werecharacterized, and thetwo

smallest deletions that lacked a significant tat response

(pD+42/+77 andpD+44/+77) and the twolargest deletions

which demonstrated tat responsiveness (pD+45/+77 and

pD+46/+77) were sequenced and characterized (Table 1). The results demonstrated that all four BAL 31 deletion

mutants exhibited approximately the same level of IL-2

protein expression in the absence oftat. However, pD+

42/+77andpD+44/+77 yieldedonly a small,abouttwofold trans-activation of IL-2 expression in the presence oftat.

The twomutantspD+45/+77 andpD+46/+77, which differ

by only 1 or 2 nt, respectively, from the negative deletion

mutant pD+44/+77, in contrast, yielded very significant

(-24-fold) trans-activationin response to tat.This isslightly lower than that observed for the wild-type clone pBC12/HIV/IL-2,thussuggestingsomerole forsequences 3' to position +44. Nevertheless, these results allowed place-ment of the 3' border of the core TAR element at nt +44 withinthe HIV-I LTR Rregion(Fig. 1).

Several other BAL 31 deletion mutants were also exam-ined and yielded the expected trans-activation phenotype; i.e., all of the deletions tested extending 5' to +45 were inactive, whereas all of the deletions terminating3' to +44

were active (data not shown). One extensive deletion,

pD+9/+77, whichremovesessentiallythe entire HIV-I LTR Rregion,including thesequencesproposed toformastable mRNA stem-loop, was examined in detail (Table 1). As expected, this deletion was not responsive to tat. Interest-ingly, this mutation still demonstrated thesamebasallevelof IL-2 expression seen with the wild-type construction pBC12/HIV/IL-2.

TheHIV-ILTRcontains anumberof direct and inverted

repeats. An almost perfect 14-bp direct repeat occurs

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be-tweenpositions-2and+12andagainbetween +30 and +44

TABLE 1. Levelsofexpression fromtheHIV-ILTR mutants in thepresence orabsence oftata

IL-2production

Clonetransfected (U/ml) Avg

trans-activation'

-tat +tat

BC12/HIV/IL-2 96 6,148 64

pD+46/+77 192 6,148 24

pD+45/+77 128 3,072 23

pD+44/+77 128 192 2.1

pD+42/+77 9 192 1.8

pD+9/+77 128 192 1.4

pBC12/HIV-Xho 96 6,148 64

pD-18/+7 128 192 2.0

pD-18/+20 96 192 1.5

pD+9/+20 192 768 4

pD+9/+38 192 192 1.0

pD+25/+38 96 96 1.0

pD+35/+38 128 192 1.4

pI4/+7 192 6,148 32

p14/+20 192 1,536 8

p18/-18 128 4,096 28

p132/-18 256 3,072 14

a Culturesweretransfected with equimolaramountsofeachHIV-I con-struction,together with eitherthetatexpressionvectorpBC12/CMV/t2orthe

negativecontrol vectorpXF3/ori. SecretedIL-2levelsweredeterminedat72 hposttransfection, and results obtained inarepresentative experiment are

shown. Mock-transfected cultures yielded no detectable IL-2activity (<2

U/mI).

bThe averagelevel of trans-activation ofHIV-I LTR-drivenIL-2

expres-sion due to tatcoexpression is shown(averageoftwo tosix independent transfections).

(Fig. 1). This particularrepeat is of interest because ofthe above observation that the 3' border of the second repeat element precisely coincides with the 3' border of the TAR

core sequence. To address the significance of these and

other reportedrepeatelements, we constructed a number of additional mutants between HIV-I LTR positions -17 and

+38 within pBC12/HIV/IL-2. Initially, weused

oligonucle-otide-directed mutagenesis(21)togenerate a2-bp changeat positions+10 and +11within repeatelement 1(Fig. 1).This

LTR mutant, termed pBC12/HIV-Xho, demonstrated a

trans-activation phenotype indistinguishable from that of

the parental clone (Table 1). Subsequently, we used

pBC12/HIV-Xho and pBC12/HIV/IL-2 to generate a series

ofdeletion mutations between the different restriction

en-zyme sites present within the TAR region (Fig. 1). All of

these deletion mutants yielded anessentially negative

trans-activation phenotype (i.e., c twofold), except for pD+9/

+20, which demonstrated a low butsignificant

trans-activa-tion of about fourfold (Table 1). Of particular interest is the

small-deletion mutant pD+35/+38, which lost 4 bp from

within the 3' repeatelement and which was trans-activation

negative. Allof thedeletion mutants retained essentially the

same basal level of expression as the parental clone

pBC12/HIV/IL-2.

A final set ofmutants was constructed which contained

smallinsertions within the TAR sequence. Insertion of 4 bp

atposition +7 (pI4/+7) had little effect on the

trans-activa-tion phenotype; however, insertion of4bp atposition +20

(pI4/+20) resulted in a significantly reduced level of

trans-activation (about eightfold). Two mutants, pI8/-18 and

p132/-18, contained different size insertions 5' to the cap

siteatposition-18adjacentto the TATAbox (Fig. 1).Both

of these mutants demonstrated levels of trans-activation

slightly but significantly lower than that of the wild-type

construction. In the case of pI32/-18, in particular, this

reductionappeared to result in part from amodestly(two- to

threefold) higher level of basal IL-2 expression.

Effect of TAR mutations on reporter gene mRNA levels. In

the previous section, weexaminedthe levelofreporter gene

expression at the protein levelfor anumber ofHIV-I LTR

mutants in the presence or absence of tat. It has been

suggested that tat-mediated enhancement ofHIV-I-specific

geneexpression is due to both anincreasein HIV-I mRNA

levels and an increase in theutilization ofthat mRNAbythe

cellular translationalmachinery (5, 39). To examinealsothe

phenotype of the HIV-I LTR mutants at these levels, we

performed an internally controlled S1 nuclease protection

assay (5) to quantitate the level of HIV-I-specific IL-2

mRNA in cultures transfected with several of the HIV-I

mutantsdescribed above(Fig. 2). Inaddition, we examined

the parental vector pBC12/HIV/IL-2 and a control,

tat-nonresponsive construction (pBC12/CMV/IL-2) which

con-tains the IL-2 gene driven by the active human cytomegalo-virus immediate-early promoter (5). The results (Fig. 2;

Table 2) demonstrated that pBC12/HIV/IL-2, pD+46/+77,

andpD+45/+77all respondedto tatwith a markedincrease

in IL-2 mRNA steady-state levels. In contrast, pD+42/+77

yielded only a slight increase in IL-2 mRNA, whereas pD+9/+77 was almost nonresponsive to tat. The

cytomeg-alovirusimmediate-earlypromoter was, as expected,highly

transcriptionally active in both the presence and absence of tat.

The S1 protection analysis presented in Fig. 2 used a 3'-end-labeled probe specific for the 3' noncoding region of

the IL-2 reporter gene mRNA. Thisprobe has theadvantage

that it also allows determination ofthe level ofan internal

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FIG. 2. Analysis of the effect of TAR mutations on the

trans-activation of HIV-I mRNA expression.COS cellsweretransfected

with the differentvectors indicatedin the absence (-)or presence

(+) of the tat expression vectorpBC12/CMV/t2. Total RNA was

harvested 72 h aftertransfection, and 4-pug sampleswereused in the

quantitative Si nuclease protectionassayvisualized here (except for

lanes 14and 15;seebelow). Levels of IL-2 mRNA (175-ntprotected

probe fragment) and reference mRNA(Ref; 156-nt protected probe fragment) in the culture are proportional to the level of rescued probe (5; Table 2). All of the cultures except the negative control (lane 1) werealso transfected with equalamountsof the reference plasmid pBC12AI. Lanes: 1, cellstransfected with pBC12/CMV/t2 alone(negative control); 14, 2 ,ug ofpositive control IL-2mRNA; 15, 20 jig of positive control IL-2 mRNA.

control insulin mRNA encoded by the tat-nonresponsive Rous sarcomavirusLTR-based reference plasmidpBC12AI

(5, 12). We also examined (Fig. 3)the RNA levelsencoded

by PBC12/HIV/IL-2 and p132/-18 in the presence or

ab-senceoftatby using Si probes5' endlabeledatthelocation of the HIV-I LTRHindlllsite(Fig. 1).Theseprobesareable

tomaptranscription initiation (cap) sites used by the HIV-I

transcripts (5). These data confirmed the large increase in

HIV-I-specific mRNA induced by tat cotransfection in

pBC12/HIV/IL-2-transfected cells (Fig. 2) and also

demon-strated that the cap site usedin the pBC12/HIV/IL-2-trans-fectedcellswasthesame asthat in HIV-I-infected H9 cells.

In contrast, the site of transcription initiation used in

pl32/-18-transfected cells, which contain a 32-bpinsertion

mutation between theHIV-I LTRTATA boxand thenormal

cap site, was displaced 5' by -32 bp into the inserted

sequence. In addition, pl32/-18-specific mRNA levels

ap-peared somewhatlower than thoseencodedbypBC12/HIV/

IL-2 in both the presence and absence of tat (Table 2).

Nevertheless, these results clearly demonstrated that

shift-ing the site of transcription initiation within the HIV-I LTR

does not necessarily significantly affect trans-activation by

the viral tatgeneproduct (Table 1; Fig. 3).

Effect oftatonreportergenemRNAtranslational efficiency.

Ithas beenproposed that the reported enhancement in the

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translational efficiency of HIV-I-specific mRNAs in the presence of tat is due to relief of a specific translational

TABLE 2. Effect ofHIV-I LTR mutations on indicator gene mRNAandprotein levels

IL-2

IL-2 mRNA IL-2protein protein!

Clonetransfecteda U(%)b

U/mIL

(%) mRNA

ratio' -tat

pBC12/HIV/IL-2 203 (5.4) 48 (0.4) 0.072

pD+46/+77 360(9.6) 64 (0.6) 0.054

pD+45/+77 250(6.7) 48 (0.4) 0.059

pD+42/+77 232(6.2) 96(0.8) 0.126

pD+9/+77 237(6.3) 48 (0.4) 0.062

p132/-18 127 (3.4) 128 (1.0) 0.31

pBC12/CMV/IL-2 3,749 (100) 12,286 (100) 1.00 +tat

pBC12/HIV/IL-2 2,581 (56) 3,072 (25) 0.45 pD+46/+77 2,491 (54) 1,536 (13) 0.24 pD+45/+77 1,849(40) 768 (6.3) 0.16

pD+42/+77 794(17) 192 (1.6) 0.09

pD+9/+77 512(11) 48 (0.4) 0.04

p132/-18 521(11) 1,536 (13) 1.11

pBC12/CMV/IL-2 4,629 (100) 12,286 (100) 1.00

aEquimolar levels ofthe referenceplasmid pBC12AIwere also

cotrans-fectedineachculture.Inaddition, the negative control plasmidpXF3/oriwas

cotransfected inthe -tatcultures,andpBC12/CMV/t2wascotransfected in

the +tat cultures. A culturetransfectedwithpBC12/CMV/t2 alone yieldedno

detectableIL-2 mRNA(Fig. 2)orprotein (<2U/mI).

bIL-2 mRNAlevelsweredetermined by scanning of the autoradiographs

shown in Fig. 2 and 3 with an LKB soft-laser scanner. The values are

corrected forslightdifferences observed intheintensity ofthereference RNA signal. Activity is giveninarbitrary units.

' SecretedIL-2protein levelsweredetermined by bioassay (29).

dTheratio ofIL-2 proteintoIL-2 mRNAdetermined in each culture is expressed relativetopBC12/CMV/IL-2, which is arbitrarilyset at1.00.

1 2 3 4 5 6

147- 123-

110-

90-76 -

67-FIG. 3. Analysis of the HIV-I mRNA transcription startsitesin

transfectedcells.RNA from transfectedCOScellswasharvestedat

72 hposttransfection, and 10-p.g samples wereused in the

quanti-tativeS1 nuclease protectionassay visualizedhere. The size of the

rescued probe fragment wasdetermined with reference to

MspI-cleavedpBR322DNA size markers andmeasuresthe distance of the

transcriptionstartsite from the site oflabelingatposition+82(see

Materials and Methods fordetails). Lanes: 1, 0.5 jigofpoly(A)+

mRNA from HIV-I-infected H9 cells(a giftof M. Dukovich and W.

Greene); 2,pBC12/CMV/IL-2-transfectedCOS cells(negative

con-trol); 3,pBC12/HIV/IL-2+pXF3/ori; 4, pBC12/HIV/IL-2+pBC12/ CMV/t2; 5, p132/-18 + pXF3/ori; 6, p132/-18 + pBC12/CMV/t2. Thenumbersonthe leftindicatemolecular size in nucleotides.

1 2 3 4 5 6 7 8 9

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inhibition (5, 8).Toexaminethisquestion,wecomparedthe ratiosofIL-2

protein

to IL-2 mRNA levels produced after transfection of cultures with several of the HIV-I LTR constructs(Table2). This ratio isameasureof theefficiency of translational utilization of each mRNA molecule by the

cell (5).

Aspreviously described (5),theparental pBC12/HIV/IL-2

constructionyieldedanIL-2 mRNAwhich,inthe absence of

tat, wastranslated much lessefficiently (-10-to25-fold)by

the cell thanwas the IL-2 mRNAencodedby the

cytomeg-alovirus immediate-early vector. All of the deletion (pD)

mutantsyielded similar low translational efficiencies, includ-ing the extensively deletedmutant pD+9/+77. In the

pres-enceoftat, the threetat-responsive clones

(pBC12/HIV/IL-2, pD+46/+77, and pD+45/+77) all showed significant increases in mRNAtranslationalefficiency. In contrast, the translational efficiencies ofthe RNAs encoded by the two essentially nonresponsive clones pD+42/+77 and pD+9/ +77wereunaffectedbytat.This suggeststhat the transcrip-tionalandposttranscriptionaleffectsoftat aretightlylinked. The mutant

p132/-18

producedaninteresting phenotype.In the absence of tat, the mRNA encoded by pI32/-18 was

present at a slightly lower steady-state level (Fig. 3) yet

encoded a higher level ofIL-2 protein than did the other

LTR mutants tested (Table 2). In the presence of tat, the

level of this mRNA increased only modestly, to a level

essentially

identical to that ofthe tat-nonresponsive clone

pD+9/+77.

The level ofIL-2 proteinencoded by p132/-18

mRNA was,however, -32-foldhigherthan that encodedby

pD+9/+77

mRNA, thus

suggesting

a large difference

be-tween the translational efficiencies of these two similar mRNAmolecules.

Does tat affect the location of DNase I-hypersensitive sites

within the HIV-I LTR? The trans-acting gene product tat

enhancesHIV-I-specificgeneexpressionin partby

increas-ing the steady-state level ofHIV-I-specific mRNAs (5, 22, 25, 39).Thisincrease, which is dueto anincrease in therate

of HIV-I mRNAtranscription (12), mustbemediatedbythe

sequences located within the LTR TAR sequence. A

prop-erty which can be associated with shifts in the level of

transcription ofa gene is a shift in the pattern of DNase I hypersensitivity (7, 13).This isbelievedtobe duetochanges

in the componentsofthechromatin which enclosethe gene.

We therefore examined the effect oftat onthe

hypersensi-tivity profile

ofthe HIV-I LTRwithintransfected cells

(Fig.

4).

COS cells were transfected with

pBC12/HIV/IL-2

in

the presence or absence of the tat

expression

vector

pBC12/CMV/t2,

and

permeabilized

nuclei were preparedat

72h aftertransfection (9, 10). The nuclei weretreatedwith

various levels of DNase I, and the nuclear DNA was then

isolated and cleaved with BamHI. This restriction enzyme

cutsthe4.7-kilobase

pBC12/HIV/IL-2

vector at auniquesite

located atposition+765 relativetotheHIV-I LTR cap site.

The isolated DNA was subjected to electrophoresis on a

1.1% agarose gel and transferred to a nitrocellulose filter.

The

hypersensitive

sites were visualized with a probe

pre-pared against

a

261-bp

XbaI-to-BamHI

fragment

derived fromthe IL-2 indicator gene coding region.

Two

hypersensitive

siteswereobserved within the HIV-I LTRby thisprocedure. The location and intensity of these

sites were not markedly affected by tat coexpression,

al-though the faster-migrating band did appear slightly more

diffuse in the presence of tat protein (Fig. 4, lane e). No

specific

bands were seen after probing of DNase I-treated

nuclei derived from mock-transfected COS cells or when

naked pBC12/HIV/IL-2 DNA was used (data not shown).

-tat

j

7M

MA4l4 .

+tat

k b

-4.4 -- 2.3

--2.0

--

1.35--0.S7- - - TATA TAR

FIG. 4. DNase I-hypersensitive chromatin sites in the HIV-I LTR. Theautoradiograph visualizes DNase I-hypersensitive sites located within the HIV-1 LTR in COS cells transfected with pBC12/HIV/IL-2 with (+tat) and without (-tat)the tatexpression

vector pBC12/CMV/t2. Tatcoexpression resulted in a64-fold in-crease in IL-2 expression. The slower-migrating band maps to

position -175 to -125 in the HIV-I LTR, the site of a possible negative regulatory element (NRE).Thefaster-migrating bandmaps toHIV-I LTRposition -45 to +25, the siteof theTATAboxand the TAR element(Fig. 1). Lanes:a, no DNase I; b,c,d,e,andf, 128, 256, 512, 1,024, and 2,048 U of DNase I, respectively. kb, Kilobases.

The location of the two DNase I-hypersensitive sites was

carefully mappedin relationto DNA size markers

(HindIII-cleaved phage A DNA and HaeIII-cleaved XX174 DNA)

which were visualized by reprobing of the blot with the appropriate nick-translated phage DNA probes. This mea-surement indicated that thelargerbandwas910 + 25 bp in size,whereasthe smaller bandwas775 ± 35bpin size. This

correlates with the HIV-I LTRlocations -175 to -125, the

possible site ofa negative regulatoryelement(11, 34a),and

to -45 to +25 bp, which coincides with the HIV-I LTR

TATAbox and part of the TARelement.

DISCUSSION

In this study, we used site-directed mutagenesis ofthe

HIV-I LTR, combined witha transient-expression assay in

the HIV-I-permissive (8, 18) monkey cell line COS, to

examine the roles of specific LTR sequences in

trans-activationby theviraltatgeneproduct. Because this

trans-activationevent occurs atleast in partatthetranscriptional

level (12) and repeat structures have been shown to be

important in the regulationofexpression from several tran-scriptioncontrolregions (19),wefocusedparticularlyonthe

functionalsignificanceof inverted and direct repeats present

within the core TAR sequence (Fig. 1). Initially, we used

processive deletion mutagenesis to define the 3' border of

the core TAR element at position +44 relative to the cap

site. These dataconfirmpreviousresultsshowingthat the 3'

borderofTARlaybetween +31 and +54(22).Subsequently,

we used deletion andinsertion mutagenesisto examine the

roles of different areas within the TAR sequence between

-18 and +44 (Table 1). These studies revealed that most

mutations of this region dramatically reduced the

trans-activation response. Two minor mutations at about +10,

pBC12/HIV-Xho andpI4/+7, which disrupt the first of the

twodirect repeat elements noted in Fig. 1, were, however,

found to have little or no effect. Also of interest is

pD+9/+20, which is the largest internal TAR deletion

mu-tation to permit some level of residual trans-activation.

None of the TAR mutations, including extensive deletions

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http://jvi.asm.org/

[image:5.612.318.558.70.196.2]

such aspD-18/+20 andpD+9/+77, were found to affect the

basal activity of theHIV-I LTR promoter significantly. This

observation argues against the hypothesis that the TAR element is a cis-acting negative regulator of HIV-I-specific

gene expression.

The results presented heredefineasmall, -60-bp element

which is critically important for tat-mediated trans-activa-tion of the HIV-I LTR and which retains a number of interestingstructural features. These include the two 14-bp imperfect direct repeats noted in Fig. 1, which are in turn

separated by a 10-bp palindrome (5'-TAGACCAGAT-3').

Only part ofthe previously noted (22, 24) imperfect 24-bp inverted repeat wasfound tobe important fortat-mediated trans-activation of HIV-I gene expression; however, this

could still form the basis fora 9-bp hairpin loop structure

(Fig. 1B). The limited mutational analysis described here

appears to focus attention on the second ofthe two direct

repeats (Fig. 1), since all deletions involving this element,

including pD+35/+38 and pD+44/+77, which affect this

element onlyslightly, resultin almost totalabrogationof the

trans-activationresponse. Itwill beofinterest to determine

whether syntheticcopies of this element canconfer a

trans-activation response on heterologous promoters when

pres-entin cis.

Ithaspreviously been shown that thefunction ofthe TAR

element isbothposition andorientation dependent (22, 25),

thus distinguishing TAR from inducible enhancer elements

which have been described for other systems (19). One possible function ofTAR, byanalogy toothertranscription

control regions(3, 15, 34),isas abindingsite foravirus-or

host-specifictranscription factor.OccupationofTARbythis

protein would, in turn, stabilize binding of transcription

factors to LTR U3 region sequences. In simian virus 40

late-gene trans-activation by simian virus 40 T antigen, stable binding of transcription factorsto Tantigen-binding

site II and the simian virus 4072-bp repeatelement occurs

only when the two binding sites are correctly and closely

spaced (3). Insertionofas little as 42 bp totally abrogated functional binding, and even a 4-bp insertion produced a significant effect(3). To testwhether the proximity ofTAR tothebodyoftheHIV-ILTRtranscriptioncontrol

region

in

the LTR U3 regionwascritical forfunction,wechangedthis

distance by insertion of8 or32 bp between the TATAbox

and the core TAR region (pl8/-18 and

p132/-18).

These

mutations resulted in only a modest decrease in

trans-activation and therefore suggest that the position ofTAR

within theHIV-I LTR is flexible to atleast somedegree.

Because trans-activation appears to involve

transcrip-tional activation of the HIV-I LTR (12), we examined the

DNase I hypersensitivity profile of the LTR and asked

whetherthis was affected by tat coexpression. Our results

(Fig. 4) revealed two areas of

hypersensitivity

within the

HIV-I LTRwhich mappedto -45to +25 bp (3'

site)

andto

-175 to -125bp (5' site) butwere notmarkedlyaffectedby tat coexpression. The location of the 3' site extends from

-15 bp 5'to theTATAbox into theTAR region sequences

and the HIV-I LTR R region. The 5' site does not accord

with any knownimportant structurewithin theHIV-I LTR;

however, deletion of this sequence has been associated with

elevated expression from the HIV-I LTR, and this may,

therefore, be a negative regulatory element (11, 34a). No signal was noted atthe site ofthe HIV-I LTR Spl

binding

sites (14) (about -50 to -80) or at the location of the

inducibleHIV-Ienhancer element(23)(about -80to-105).

However,becauseonlyasubfraction of the introducedDNA maybefully transcriptionally activein this

transient-expres-sion assay, it ispossiblethat this assaycannotdetectaweak

but

significant signal

at these sites.

Itis of interesttocomparethe in vivo DNase I

hypersen-sitivity analysis presented

here with a recent

study

on the locationof DNase

I-protected

sites

(footprints)

in theHIV-I

LTR after in vitro incubation with a HeLa cell nuclear

extract (11). This report identified three

protected sites,

of which the most 5',

extending

from -173 to

-159,

appears

coincident with the 5' site noted in

Fig.

4. A second

weakly

protected site,

locatedat -97 to

-78,

wasnot notedin our

data. Of

particular

interest isathird

protected

site,

extend-ing

from -42to +28 in theHIV-ILTR

coding

strand andas

faras +52 in the

noncoding

strand

(11).

This

protection

of

the TAR sequence

closely

mirrors both thelocation ofthe

DNase-hypersensitive

3' site noted in

Fig.

4

(-45

to

+25)

and the location of the sequences

required

for tat

trans-activationasdefined

by

the above mutational

analysis.

The

closecorrelation between these in vivo dataand the

previ-ously

published

in vitroDNase I

footprint analysis strongly

suggests the existence ofa cellular TAR

sequence-binding

protein

in cells that do not express the tat gene

product.

Recent reports indicate that a number ofviral and cellular

trans-acting transcriptional

activators, including

the

trans-activator (tatl or

p40x)

encoded

by

the human retrovirus

humanT-cell

lymphotropic

virus type

I,

mayactvia cellular

sequence-specific DNA-binding proteins (2, 13a, 17, 37).

The

evidence for a constitutive cellular TAR

sequence-binding

protein

may suggest a similar mechanism of actionfor the

HIV-I tatgene

product.

ACKNOWLEDGMENTS

WethankAnn Perkinsfor technical assistance and LisaNieves andSharon Goodwinforsecretarialsupport. Wealso thankKlaus JantzenandHans-PeterFritton forhelpfuldiscussions.

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