Contribution of NF-kappa B and Sp1 binding motifs to the replicative capacity of human immunodeficiency virus type 1: distinct patterns of viral growth are determined by T-cell types.

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Contribution of

NF-KB and

Spl

Binding

Motifs to the

Replicative

Capacity of

Human

Immunodeficiency Virus

Type 1: Distinct

Patterns

of

Viral Growth Are Determined by T-Cell Types

ELIZABETH K. ROSS,1 ALICIA J. BUCKLER-WHITE,2 ARNOLD B. RABSON,1t GEORGE ENGLUND, AND MALCOLM A. MARTIN1*

Laboratory of Molecular Microbiology, National Institute of Allergy andInfectious Diseases, Bethesda, Maryland

20892,1

andDivision ofMolecular Virology andImmunology, Georgetown University Schoolof Medicine,

Rockville, Maryland208542 Received 13 February1991/Accepted9May 1991

Startingwith areplication-incompetentmolecular clone ofhumanimmunodeficiency virustype1,lackingall the NF-KB and Spl binding sites present in the native long terminal repeat (LTR), proviruses containing reconstructed LTRs with individual or combinations of NF-KB and Spl elementsweregeneratedand evaluated fortheir capacity to produce virus progeny following transfection-cocultivation. Virusstocks obtained from theseexperiments exhibitedacontinuum ofreplicativecapacitiesindifferenthuman T-celltypesdependingon whichelement(s) was present in the LTR. For example, inexperiments involving proviral clones with LTRs containing one ortwoNF-KBelements (and no Spl bindingsites), ahierarchyofcellularpermissivitytovirus replication (peripheral blood lymphocytes = MT4 > H9 > CEM > Jurkat) was observed. Of note was the associated emergence of second-site LTR revertants which involved an alteration of the TATA box. These results suggest that the human immunodeficiency virus type 1 LTR possesses functional redundancy which ensures virus replication in different T-cell types and is capable of changing depending on the particular combination oftranscriptional factorspresent.

The retroviral long terminal repeat (LTR), generated dur-ing the early phase of virus infection by reverse transcrip-tion, contains several cis-acting elements that influence viral geneexpression. These include the transcriptional promoter and start site, the polyadenylation signal and addition site, and binding motifs for transcriptional activators. In some retroviral subfamilies, the LTR also contains targets for virus-encoded transactivator proteins.

During the past few years, the structure and function of the human immunodeficiency virus (HIV) LTR has been intensively investigated. Regulatory motifs (e.g., Spl, TATA)have been identified which bind cellular proteins that are constitutively expressed in most cells (7, 11, 20); these sequences appear to be critical for basal-level HIV gene

expression.OtherLTRelements (e.g., NF-KB and NFAT-1) may interact with factors synthesized only in activated T

lymphocytes (6, 21, 28, 37), thereby limiting vigorous virus

replication to a small subset of circulating peripheral blood

lymphocytes(PBLs) invivo. Still other regions (e.g., TAR) are unique to HIV and are required for high levels of LTR-directed gene activity (4, 8, 13, 18, 36, 41). Thus, the

interaction of virus-encoded (3, 40) and cellular regulatory proteins with one another and with the HIV LTR in both resting and activated human lymphocytes has the potential toeffect acontinuum of replicative capacities.

Todate, most of our knowledge of HIV LTR structure and function comes from studies of small DNA constructs con-taining the LTR linked to reporter genes and tested in HeLa cellsorin humanT-cell lines (2, 5, 15, 28, 32, 38). Although this approach has helped to elucidate the mechanism by which the HIV LTR is activated, the interaction of

virus-*Correspondingauthor.

tPresent address:Center for Advanced Biotechnology and Med-icine, Piscataway, NJ 08854.

encoded and cellular proteins with cis-acting regulatory

elements duringa productive infectioncannotbe evaluated in such systems. We (23) and others (14, 24) have electedto evaluateLTRfunction in the context of HIVreplicationand have previously reported that some of the cis-acting ele-ments may be dispensable, depending on the cell type infected. Forexample, the deletion of both NF-KB elements only modestly affects HIV replication in PBLs or in CEM andMT4cells (23). In contrast, mutation ofSpl motifscan drasticallyreducereplicativecapacityinsomehuman T-cell cultures. Inparticular,thedeletion of all threeSplelements either eliminated orgreatly reduced the capacity of HIV to infect CEM cells (two successes in fiveindependent exper-iments); replication of this mutant in PBLs and MT4 cells was only slightly reduced (30). Finally, the simultaneous elimination of the two NF-KB and all three Spl elements resulted in a provirus exhibiting no detectable biological activity (23); in this report, we refer to this irreversibly replication-incompetent proviral DNA aspNL-ZERO.

To understand more fully the contribution of individual LTR components to the replicative process and to the

complexinteractions of cellular and viralproteinswith their LTR targets, we analyzed the biological activity of pNL-ZEROinto which different combinations of NF-KB andSpl cis-acting elementswere added. Our results suggest a hier-archy of cellularpermissivity to virus infectionwhich very likely reflects the relative abundance oftranscriptional reg-ulatory factors and the presence of specificHIV LTRmotifs.

MATERIALS ANDMETHODS

Construction ofproviral DNAs containing LTR mutations and generation of virus stocks. pNL-ZERO is a replication-incompetentderivative of the infectiouscircularlypermuted proviralclonepILIC(23); pNL-ZERO lacks the two NF-KB 4350

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and three Spl sites and was previously designated pTA. pNL-ZERO DNA was digested with BamHI and SphI to generate a 2.1-kb fragment containing the LTR. This

frag-ment was subcloned into M13 and subsequently used to produce the reconstructed LTRs, shown in Fig. 1, by oligonucleotide-directed mutagenesis (47) as modified by

Kunkel(22). Full-lengthcircularlypermutedproviralDNAs were reconstituted by ligating the LTR-containing 1.7-kb

XhoI-SphI fragment from M13 recombinants to a similarly

digested7.4-kb cleavage productofpNL-ZERO. Following the verification of the mutations prepared in vitro by

nucle-otidesequenceanalysis(34), thereplicative capacities ofthe

different LTR mutants were evaluated by transfecting a derivative of A3.01 (9)CEMcells, designated 12D7 (gener-ously providedbyGuido Poli, Laboratory of Immunoregu-lation, National Institute of Allergy and Infectious Diseases) with 3 ,ug of DNA (per 6 x 106 cells) in the presence of DEAE-dextran as previously described (23). Forty-eight hours later, thetransfected cells werecocultivated with2 x 106 MT4(25)cells and monitored for virus production by a

32P-reversetranscriptase (RT) assay (46). Virus stocks were

prepared from the supernatants of transfection-cocultures

when RT levels exceeded 3,500

32p cpm/,lp.

Infections. PBLs, the A3.01 (9)clonal derivative ofCEM

cells, MT4, H9 (31), and JE6.1 (45), a clonal lineof Jurkat cells (2 x 106 cells of each type), were infected with

equivalentamounts of virus (300,000 cpm of32P-RT activi-ty). Priortoinfection, PBLs werestimulated with phytohe-magglutinin (0.25 ,ug/ml) for 3 days; upon infection, the

PBLs were maintained in medium containing 10%

interleu-kin 2. Infected cells were maintained in T25 Teflon flasks (Costar)in RPMI 1640medium(10 ml)containing 10%fetal calf serum, and progenyvirion production was detected by

RT activity.

PCR analysis of genomic RNA.Aliquots(100 ,ul)ofculture supernatants from selected infections were digested with

RNase-free DNase (0.05 U; PromegaBiotec) for 15 min at

37°C and then incubated for10min at68°Ctoinactivatethe DNase. First-strand cDNA synthesis from viral RNA was

performed as described by Morgan et al. (26). The cDNAs werethen amplified in aPerkin-Elmer/Cetus DNAThermal

Cycler with thefollowing primers: (i)a plus-sense oligonu-cleotide spanning positions 9073 to 9092

(HIVBRU

coordi-nates[27])with 9nucleotidesatits5' endbearinganEcoRI site (5'-ATCGAATTCTGGAAGGGCTAATTCACTCC-3'; gift from Jonathan Silver), and (ii)aminus-sense oligonucle-otide spanning positions 9504to9521 with12nucleotidesat

its 5' end bearing botha PstI and an RsaIsite (5'-AACTG

CAGGTACCAGGCAAAAAGCAGCTGC-3').

The

amplifi-cation protocol consisted of denaturation at 94°C for 30 s,

annealing at

60°C

for 30 s, andextension at72°C for2 min.

Digestion of the polymerase chain reaction (PCR) product with

EcoRI

and PstI, ligation into

pIBI30

(International Biotechnologies, Inc.), transformation of Escherichia coli

DH5-aMCR (Bethesda Research Laboratories), and selec-tion of recombinants by colony hybridization by using a

5'-end-labeled

oligonucleotide probe spanningpositions 143 to 182 of the

HIVNL4-3

(1, 27) LTR were all done as

previously described (33). Hybridization-positive

recombi-nants were sequenced by the method ofSangeretal. (34).

RESULTS

We previously described the construction ofa circularly permuted infectious molecular clone of HIV containing a

single LTR which was used to investigate the role(s) of

NF-KBand

Spl

elements

during

virus

replication

in human T cells (23). Viruses in which the two NF-KB binding sites were deleted

replicated efficiently

in

phytohemagglutinin-stimulatedhumanPBLs, aswellas in MT4 and CEMcells. HIV

preparations containing

one or twomutated

Spl

bind-ing

sites were also

replication

competent but exhibited variablegrowth kineticsdependingonthe cell typeinfected. Deletion of all three

Spl

sites resulted in virus which

replicated

efficiently

in MT4 cellsand in PBLs but

inconsis-tently

andwith

delayed

kinetics inCEMTcells unless

they

were exposed to tumor necrosis factor

alpha (30).

In con-trast, the deletion of68

bp

ofU3 sequences

encompassing

the two NF-KB and the three

Spl

sites

generated

an HIV

provirus

which failed to

replicate following

transfection-cocultivation. This

replication-defective

HIV

provirus,

des-ignated pNL-ZERO,

wasused ina series of

experiments

as a basal-level

proviral

DNA target to which NF-KB or

Spl

siteswere added either

individually

orincombination.

Generation ofmutant LTR HIV-1 stocks by transfection-cocultivation.

Circularly permuted

HIV

proviruses

contain-ing

a

single wild-type

orreconstructed LTR werereleased from

plasmid

vectors

following digestion

with BamHI and

ligated

to regenerate "conventional"

full-length

proviral

DNAs

(i.e.,

containing

atleasttwo

LTRs).

Acloned

deriv-ative ofthe

CD4+

T-cell line

A3.01,

designated 12D7,

was thentransfected withthe

ligated proviral

DNAsasdescribed

inMaterialsandMethods.

Forty-eight

hours

following

trans-fection,

the 12D7cellswerecocultivated with MT4cells and

progenyvirus

production

wasthen monitored

by

RTassay over a

51-day

period. Supernatants

were collected at the

peak

ofRT

activity

and served as virus stocks for subse-quent

infectivity

studies.

As

expected,

transfection-cocultivation with

wild-type

HIV

proviral

DNA resulted in

rapid

virus

production

(first

detected 4 to 5

days later),

whereas

pNL-ZERO

failed to

replicate.

As indicated in

Fig.

1, virus

production

was

observed

following

transfection-cocultivation of

proviral

DNAs

containing

one or two NF-KB

elements,

although

replication

oftheformer

(pNL-lKB)

was

markedly

delayed

(first

detectedon

day 27).

ClonedHIV

proviruses

containing

two orthree

Spl

sites

(pNL-2Spl

and

pNL-3Spl)

or a

single

pair

ofNF-KB and

Spl

sites

separated by

a mutated

inter-vening

NF-KB site

(pNL-KB/Spl-R)

all

replicated

following

transfection-cocultivation but with kinetics somewhat

slower than that of wild type.

Interestingly,

the

pNL-KB/

Spl-J

provirus

containing juxtaposed

NF-KB and

Spl

single

elements

replicated nearly

as

efficiently

as

wild-type

HIV

(data

not

shown).

Ofthe mutated HIV LTRs evaluated

by

transfection-cocultivation, only

the

pNL-lSpl

proviral

con-struction,

which contains the

single Spl

element

Spl(I),

failed to

replicate

in three

independent

experiments

lasting

51

days,

2.5

months,

and 4

months,

respectively.

Infection of PBLs and human T-cell lines with HIV-1

derivatives containing NF-KB or

Spl

motifs in their

recon-structed LTRs.

Although

the transfection-cocultivation

ex-periments

described above indicated which ofthe NF-KB/

Spl

insertions intothe

pNL-ZERO

LTRrestored

replicative

capacity,

how these elements influenced virus

infectivity

required

additional

investigation.

Infections weretherefore

performed

employing

virusstocks

generated

fromthe

trans-fection-cocultures referred to in

Fig.

1. The various T-cell lineswere infectedwith

equivalent

amountsof

wild-type

or LTRmutantvirus

(3

x 10532P-RTcpm,

approximately

equal

to a

multiplicity

of infection of0.001for

wild-type

virus),

and

subsequent

progeny virus

production

wasmonitored

by

RT

activity.

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| U3 |R |U5

NF-kB Spi TATA

-341 -186 -106 -56 -28 + 1 +19 +42

IZ7

Z7W1

*---- 11

1

NF-kB(II) NF-kB(I) Spi (III) Sp1 (II) SpI(I)

TACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTC

pNL-lSpl TAC

B pNL-2Spl TAC

pNL-3Spl TAC

TGGGGAGTGGCGAGCCCTC

C pNL-kB/Spl-J TAC

pNL-kB/Spl-R TACAAGGGACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGGCC

FIG. 1. Structure of reconstructed HIV LTRs associated with full-length proviral DNAs. The positions of several cis-acting regulatory elements within the HIV-1 LTRareindicated diagrammatically at thetop. The nucleotide sequencesofa72-bp region encompassingthe

NF-KB andSpl motifs of the wild-type LTR (pNL-WT)aswellaseight reconstructed LTRsareshown below. The nucleotide substitution

in pNL-KB/Spl-R, previously reported (28) to inactivate the NF-KB binding site, is underlined. Virus production following

transfection-cocultivation is indicated in the columnatthe right.

Inthefirstgroupof experiments, PBLs and different T-cell

lines (MT4, CEM [A3.01 clone], H9, and Jurkat [clone JE6.1])wereinfected withmutantHIVpreparations contain-ingone or twoNF-KB elements (andnoSpl motifs). Three

different replicative phenotypes were observed. The first, seenwithPBLsorMT4cells,wascharacterized by delayed

replication asthe number of NF-KB siteswasreduced from two to one (Fig. 2A). InH9 (Fig. 2B) and A3.01 (Fig. 2C)

cells, however, HIV with the single NF-KB(I) element failed to replicate; virus containing twoNF-KB sites in its

recon-structed LTR replicated slowly compared with wild-type HIV, generating virus progeny2.5to4weeks postinfection. Notably, infection ofA3.01 cells with HIVcontaining only twoNF-KBelements resulted in virusproductioninonlyone

ofthreeindependent experiments (the onedepicted in Fig.

2C) monitored for periods of up to 42 days. The third replicative phenotype was observed in Jurkat cells and is

shown in Fig. 2D. Inthis case, virus withLTRs containing oneortwoNF-KBelements failedtoreplicate during nearly 4weeks of observation.

A similar approach was followed to monitor the role of Splmotifsonvirus infectivity. Sincenoviruswasgenerated

following transfection-cocultivation of pNL-lSpl, the infec-tivity studies were limited to virus stocks containing two

[Spl(I) plus Spl(II)] or three [Spl(I) plus Spl(II) plus

Spl(III)] Spl motifs as shown in Fig. 1. In all cell types

tested, the presence of a second Spl element restored

infectivity. In some cells, HIVs with reconstructed LTRs containing two or threeSpl binding sites had similar repli-cation kinetics (PBLs in Fig. 3A), while in others, virus harboring only two Spl elements (H9 cells in Fig. 3B) reproducibly exhibited retarded growth properties.

Infectivity of HIV preparations with reconstructed LTRs containing combinations of NF-KB and Spl elements. As

shown in Fig. 2 and 3, duplications of either the NF-KB or

Spl element frequently resulted in enhanced HIV replica-tion. Giventheproximityof theNF-KBandSplmotifs inthe wild-type LTR configuration, we wondered whether the observed augmented replicative capacity was limited to

combinations of the sameelement. Toassessthepossibility

that NF-KB andSpl elements mightfunction ina

combina-torial fashion,wegeneratedaprovirus containingLTRswith

single copiesof both motifs. In the five human T-cell systems evaluated,this virus

(HIVNL-KB,sp1_J)

replicatedasrapidlyor

more rapidly than HIV preparations with LTRs harboring duplications or triplications of the NF-KB or Spl elements (datanotshown).Thisphenomenonwasparticularly striking

inJurkat cellsinwhichHIV withtwoNF-KB elementsfailed to replicate during a 25-day observation period (Fig. 4A). Furthermore,theNL-KB/Spl-Jvirusreplicatedwith kinetics similar to that ofwild-type HIV in most of the cell lines tested(Fig. 4B).

We wondered whether the efficient viral replication di-rected by an LTR containing a pair of NF-KB and Spl

elements reflected theirjuxtaposition or simply their

pres-ence in the reconstructed LTR. Consequently, a different LTRcontainingthesinglefunctionalNF-KB(II)andSpl(III) elements spatially separated from one anotherby a

muta-genized (28) NF-KB(I)motif(seetheNL-KB/Spl-R provirus inFig.1)wasexamined for itsabilitytosupportaproductive

virus infection. In H9 cells, the replicative capacityof this viruswas similar tothat of virus containing thejuxtaposed NF-KB(I) andSpl(III) bindingsites(Fig. 5). Thus, an 11-bp

separationof functional NF-KBandSpl sites didnot

appre-ciably affectinfectivity; virions with this LTRconfiguration replicated more rapidly than those containing only two NF-KB ortwoSpl elements.

Replicative capacity of HIV proviral DNA containing a

PLASMID

pNL-WT

A

pNL-ZERO TAC

pNL-lkB TAC GCTGGGGACTTTCCAG pNL-2kB TACAAGGGACTTTCCGCTGGGGACTTTCCAG

VIRUS PRODUCED

CTC

-AGCCCTC +

CTC +

AGCCCTC

+

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Wild type

PBLs

IFkB only

10 20 30

DAYS POST INFECTION

U2

(0 I.

-E

I-5:

0

40

DAYS POST INFECTION

8000

go 0. U

E

0

F-C.)

'a

2

U!

3.

I-0

--

so

' - 0 10 20 30

0 10 20 30

DAYS POST INFECTION DAYS POST INFECTION

FIG. 2. Infections ofhuman T cells withwild-typeHIVor mutantHIVderivativeswith LTRscontainingone ortwoNF-KBandnoSpl elements. Activated PBLs orH9, CEM, or Jurkat cells were infected with approximately 3 x 105 32P-RT cpm ofthe indicated virus preparations obtained by transfection-cocultivation. VirusproductionwasmonitoredbyRTassay.

Wild type PBLs

0

-2-Spl

1000 p

Mock

0 I-10 20 30

0 10 2003

B

to

E

a

3.- I-0

ci

8000

DAYS POST INFECTION _{DAYS POST INFECTION}10 20 30

FIG. 3. Infections of human T cells withwild-typeHIVormutantHIV derivatives with LTRscontainingtwoorthreeSplandnoNF-KB elements. Activated PBLsor H9 cells were infected withthe indicated virus preparationsasdescribed in Materials and Methods. Virus

productionwasmonitoredbyRTassay.

I0

a

E

C.

a

3..

5:

I-0

2000-

1000-0

A

to

E

C)

S

0. a

I-0

A

0

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B

a.

0

20 30 10 20

DAYS; POST IN.FECTION

[image:5.612.109.534.72.243.2]

DAYS POST INFECTION

FIG. 4. Infections of human T cells with wild-type HIV or mutant HIV derivatives with LTRs containing pairs of homologous or

heterologous cis-acting elements. Jurkat orCEM cells were infected with the indicated virus preparations as described in Materials and

Methods.Virus productionwasmonitoredby RTassay.

single enhancer-activator element.The results shown in Fig. 4 and 5 suggest that paired NF-KB and Spl elements are

acting in a combinatorial manner since HIV preparations

withduplications of thesameelements exhibitedanimpaired

replication phenotype in some cell lines. Moreover, the

transfection-cocultivation results presented in Fig. 1 indi-cated that the single Spl motif Spl(I) failed to restore replication competence to pNL-ZERO, suggesting that a

single Splelement couldnotdirect virus production unless it

was associated with either a second Spl element or an

NF-KB element. These conclusions all assume that the

biological activity of each of the single NF-KB or Spl

elements canbe predicted fromthe data showninFig. 1.

However, since the sequences of the differentNF-KB or

Spl motifsin the HIV-1LTRarenotidentical,weundertook a series of experiments to evaluate more critically the

functionalcontribution of individual enhancer-activator ele-mentsin thereconstructed LTRs. Toward this end, proviral DNAs containing only single NF-KB or Spl elements were

Wild type H9

Juxtaposed

6000~- y NFkB/Spl

E Separated

CL ~~~~~NFkB/Spl

>'4000

o- , ... .~~- k

0 10 20 30

DAYS POST INFECTION

FIG. 5. Infectionof H9 cells with HIV containingan LTRwith

single separated NF-KB and Spl elements. H9cells wereinfected

with theindicated viruspreparations containing pairs of homologous or heterologous motifs. Virus production was monitored by RT

assay.

assayed for virus production by transfection-cocultivation. AsshowninFig. 6,and inagreementwith the datapresented in Fig. 1, a proviral clone containing only the Splelement Spl(I) failed toreplicate in threeindependent experiments. In contrast, the presence of the single Spl motif Spl(III) restored the capacitytoproducevirus. Splelement Spl(II) had an intermediate effect in these assays: in one

experi-ment, progeny virus was detected by day 25, while in a

second experiment, none was produced after 77 days. A

similar disparity was also observed when the two NF-KB elementswereexamined individually. NF-KB(I)was associ-ated with virus production in three separate experiments, whereas NF-KB(II) restored replicationcompetenceinonly

one oftwo independent experiments. In view of the very

long time(61 days) beforeprogenyvirions weredetected in

this latter experiment, the replication of NF-KB(II) most likely reflects a reversion event similarto that observed in the LTR sequences of other late-arising HIV variants (see nextsection). It isourcurrentbelief thatNF-KB(II) byitself cannot restore replicative capacitytothepNL-ZEROLTR.

Second-site reversions whichappearinreconstructed LTRs following virus infection of human PBLsor T-cell lines. We previously reported that second-site revertants of replica-tion-defective HIV mutants may emerge during long-term tissue culture infections(46). Consequently,theappearance

ofprogenyvirionsatverylatetimesfollowingHIV infection inthisstudyraised thepossibilitythatacompensatoryLTR alteration was responsible for the infectivity observed. We therefore focused attention onthegroup ofproviruses

con-taining only NF-KB elements in their reconstructed LTRs since they exhibited a spectrum of replicative capacities related to the cell type infected. As indicated in Fig. 2, a

hierarchy of cellular permissiveness was observed when HIVharboring onlyone ortwoNF-KB elementswas

exam-ined. PBLs and MT4cellswerethemostpermissive, allow-ingHIV containing onlya single NF-KB motifto replicate, whereas constructs containing one or twoNF-KB elements completely failed to replicate in Jurkat cells. H9 and CEM cells exhibited an intermediate phenotype: replication of virus witha single NF-KB sequence wasrestrictedin these

cells, but HIV containing both NF-KB elements was infec-tious intwooftwoexperiments inH9 cellsandone(shown

in Fig. 2C) of threeexperiments in CEM cells.

A

7 6000 E 5000'

a.

400

4-

I.-2000

C 1000'

JURKAT JE6.1

Wild type

2-Spl only A\/,\Juxtaposed

d

VCS \ ^/NFkB/SpI

IL L @ 2-NFkB only

-- L -6 -.

10

-..Iir. . . -A

aA

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RECONSTRUCTED HIV LTRs 4355

NF-kB Spl TATA

+1

II

xs~~~~~~IU

I 1111 I

xDATE(S)Ox

VIRUS DATE(S)0OF

PRODUCTION HARVEST

0/3

1/2 d25

2/2 d17,26

3/3 d17,26,27

[image:6.612.141.474.72.251.2]

1/2 d61

FIG. 6. Replication of HIV-1 proviruses withreconstructed LTRs containing single NF-KBorSplbindingmotifs following

transfection-cocultivation. Thestructure of theHIV-1 LTRisshown diagrammatically atthetop. Inthe reconstructed LTRs,afilledsquareorcircle

indicates thepresenceofanindividualSplorNF-KB motif, respectively. NRE denotesthe negative regulatoryelement. The frequencyof

virus production(numberofRT-positivetransfections-cocultivations pernumber ofindependent experiments)and the day of virusharvest

areindicated in the columns ontheright.

Toassesswhetherthe LTRs might have undergone alter-ation during these virus infections, we performed "RNA

PCR" on thegenomic RNAs isolated from particular

sam-ples ofprogeny virions. These samples included (i)H9 cells

infected with

HIVNL-2KB

(twoNF-KBandnoSplelements),

(ii) CEM cells infected with

HIVNL-2KB

(twoNF-KBand no

Spl elements), and(iii) PBLs infected withHIVNLlKB(one

NF-KB andnoSpl elements).Theresultantamplified cDNA

wascloned, andthe 5' 445bpofthe U3 portionof the LTR were sequenced. In all cases, the original deletions

gener-atedin vitro remained intact.Furthermore, asshown in

Fig.

7, none of the eight cDNA clones derived from H9 cells infected with pNL-2KB contained additional changes in a

regionof the LTRextendingfromtheNF-KB elementstothe TATAA site. In contrast, 8 of 10 PCR clones of virus recovered from CEM cells infected with

HIVNL-2KB

con-NF-KB(II) NF-KcB(Q) Spl(il) Spl(II) Spl(l) TATAA

pNL4-3 ACAAGGGACT TTCCGCTGGG GACTTTCCAG GGAGGCGTGGCCTGGGCGGG ACTGGGGAGTGGCGAGCCCTCAGATGCTGC ATATAAG H9 2tNFkB ...

.... .. ... ...

CEM 2NFkBN ...

...

.-.-PB1N**skB*s

...----..-.--*-

---PBNk3.--- ----...

*. e----...

...

.T .T

.T

...

.T .T .T

.T

.T .A.T

...T

.A.T .A.T

.A.T

..T

...T

.A.T

A.T

.-.A....

...

...A.. .

...

....

*... *... ...

...e

...

...A....

... ...

FIG. 7. PCRanalysis ofaportion of the U3 region of HIV LTRmutantslacking Spl bindingmotifs. The RNA associated with the cell-free

virus emerging from H9orCEM cellsinfected withHIVNL-2,B(Fig.1)orPBLsinfected with HIVNL1KB (Fig. 1)was reversetranscribedand amplifiedbyPCRasdescribedinMaterialsandMethods. The nucleotidesequencesof individual clonesweredetermined andcomparedwith thatoftheparental wild-typeHIVNL4-3shownatthetop.Identical nucleotidesareindicatedbythedots;dashes denote deletednucleotides. Nucleotide substitutionsareindicated.

A-; VOL.65, 1991

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tainedaC-to-T

change

atnucleotide-30

(27),

2

bp

upstream from the TATAA site. In

addition,

this same substitution wasobservedin all 10 clonesarisingfrom the PBLinfection with HIVNL 1KB virus and was

accompanied

by a second

change (T

to A) at nucleotide -32 in 8 of these 10 clones. Some of the

amplified

LTRclones hadnoalterations in other

regions

of the LTR; in the others

examined,

a maximum of twoadditional

changes

perLTRwere

noted,

noneof which were clustered in a

specific region.

DISCUSSION

In our

previous studies,

a series of deletion and point

mutationswere introduced into the HIV LTR to

study

the rolesof the NF-KB and

Spl

binding

sites and the TAR

region

in virus

production

(23).Deletion of thetwoNF-KBsites did not render the

resulting

proviral

DNA

replication

incompe-tent. The removal of the three

Spl binding

sites with

preservation

ofthe NF-KB

binding

sites resulted in avirus

capable

of

replicating

in humanPBLsbutnotincells

lacking

NF-KB

binding activity

(30).

Furthermore,

thesimultaneous

excision of both NF-KB and all three

Spl binding

sites

eliminatedthe

capacity

of HIVto

replicate

inhumanTcells. These observations suggested that discrete

binding

sites in the LTRfunctionasmodular

units,

called enhansons(10, 29,

43),

which can

functionally

substitute for one another and whose

importance

toviralreplicationvaries in differentcell

types

depending

on the nature of cellular

transcriptional

factors present in those cells. In the present

studies,

we

attempted

to define minimal sequences

required

to support viral

replication

in a variety of human T lymphocytes and furthercharacterizedthenatureofthe enhansons present in the HIV

promoter-enhancer

sequences.

Our efforts to determine minimal

promoter-enhancer

se-quences

required

forviral

replication

wereundertakenusing

areconstructive

approach.

Theinsertion ofthe

single

NF-KB element

NF-KB(I)

into

pNL-ZERO

was sufficientto restore HIV

replication

in CEM-MT4

cocultures;

in contrast,

NF-KB(II)

didnot restore

replication

competence. Inanalogous

LTR reconstruction

experiments involving

individual

Spi

motifs,

acontinuum of

replicative capacities

was observed:

Spl(III)

>

Spl(II)

>

Spl(I).

This latter result is consistent

with reports

showing

that the

Spl(I)

element has lower

affinity

for

Spl protein (20)

andfor factorspresentin crude nuclearextractsof human

lymphocytes

(35)than doeseither

element IIorIII. The

ability

of HIV

preparations

containing

only

Spl(III)

to infect human T cells is currently under evaluation.

Asneitherthe NF-KBnor

Spl binding

sitesareabsolutely

required

forvirus

replication

in PBLs(aslongasthe otheris presentin the LTR), the marked conservation ofthe pres-ence and arrangement of these two types of

cis-acting

sequences in all known HIV-1 isolates is puzzling. The

experiments

shown in

Fig.

4 and 5 suggest that the

arrange-mentand

spacing

of

adjacent

NF-KBandSpl bindingsites in thenative

configuration

ofthe HIV-1 LTR contribute more

significantly

to virus replicationthan duplications or

tripli-cations of either element. In this regard, we previously reported that point mutations affecting theadjacent NF-KB and

Spl

elements resulted inasignificantreductionof virus

replicative

capacity

(23). Thus, two independent studies suggest the existence ofafunctional interaction(s) between these two different cis-acting elements and the proteins whosefunctions theymediate.Notably, NF-KB binding sites

are

frequently

situated adjacent to binding sites for

consti-tutive cellular

transcriptional

factors. Onestriking example

is in the promoter of the cytomegalovirus immediate-early gene, in which a series of NF-KB binding sites are inter-spersed with the binding sites for NF-1 (12, 16). When these elementswereplacedadjacenttoeachother, 5' tothewhey acidic protein promoter, a synergistic enhancement of pro-moteractivitywasobserved, suggestingthat in thiscontext, NF-KB wasabletointeract with factorsbindingtothe NF-1 sitetoaugmenttranscription (17). Likewise, similar studies have been performed to study the interactions between

adjacentbindingsites forSpland OTF-1. AnSplelement in combination withahigh-affinityOTF-1 siteaugmented

tran-scription

2-fold; however,whenplacednext to alow-affinity

OTFsite, the Spl site increased transcription 20-fold (19). Thus, as both NF-KB and Spl caneach interact with other

transcription factors to augment transcription, it is not

surprising

thattheymayinteract with each other in the HIV LTRto promoteviralreplication.

An interesting aspect of this analysis is the apparent

hierarchyof cellularpermissivenessfor HIV infectionseen best with proviruses containing only one or two NF-KB elements. The order observed(PBLs = MT4> H9> CEM > Jurkat cells) is consistent with our previous findings (PBLs= MT4>CEM)withanHIV LTRmutantcontaining a wild-type arrangement of NF-KB motifs but lacking all three Spl sites and presumably reflects various levels of

cellular factors

capable

of

binding

toNF-KB

(30).

Asshown in Fig. 2 of this study, virus containing a

single

NF-KB elementwascapableofinfectingPBLs and MT4 cells (data notshown)

exclusively.

At theother extreme, HIV withtwo NF-KB elements failed to replicate in Jurkat cells. This continuum of cellularsusceptibilitywasalso reflected in the number and typesofsecond-siterevertants whichappeared

during

infection. H9 cellsconsistently supported replication

of HIV with two NF-KB elements; progeny virions from suchaninfection containedanunaltered LTR

(Fig.

7).This same virus inoculum produced progenyvirions in onlyone of three

independent

infections ofCEM (A3.01) cells. PCR

analysis

revealedaconsistent second-site mutationin 8 of 10 cDNA clones

arising

from this single positive experiment

thatwas localizedtothe TATAregion ofthe LTR.Finally,

the HIV construction with a single NF-KB binding site

(NL-1KB) replicated slowly in PBLs but, in the process,

acquired

two

changes

also situated

immediately

upstreamof the TATA box. Thus, a selection for virions

containing

a similar TATAmutationis operativeinbothCEMcells and

PBLs, possibly

reflecting

the 40-bp deletion

encompassing

the three Spl sites which effectively places the NF-KB(I)

motif 13

bp

upstreamofthe TATAbox. This TATA altera-tion is consistent with reports describing the restricted interaction between a cell-specific enhancer and discrete

VIRUS

UILD TYPE

CE11REUERTRNT

POLREUERTRNT

TATR BOX

TGCTGCF|iG

TGCTG ATATAG

TGCRG TATAG

FIG. 8. TATAboxrevertants which appearfollowing infection of CEM and activated PBL cells with HIV containing only an

NF-KBmotif(s)in theNF-KB/Spl domain.

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[image:7.612.373.520.588.687.2]

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TATA sequences (39, 42, 44). Perhaps the alteration of the wild-type HIV TATAA box to TATATAA is necessary for functionalinteraction with the dislocated NF-KB element(s). It is worth noting that the base change common to both second-site revertant viruses extended the TATA box two nucleotides in the 5' direction relative to that of the parental virus pNL4-3 (Fig. 8). Since the TATA element positions where RNA polymerase II initiates RNA synthesis, this change could shift the transcriptional start site from its normal location at the U3/R border. Experiments to ascer-tain whether the transcriptional start site has been altered in the LTR revertant viruses and to assess the effects of such TATA changes on HIV replication are presently in progress.

ACKNOWLEDGMENT

The following reagent was obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases: Jurkat clone E6-1 from the Amer-ican Type Culture Collection.

REFERENCES

1. Adachi, A., H. E. Gendelman, S. Koenig, T. Folks, R. Willey, A. Rabson, and M. A. Martin. 1986. Production of acquired immu-nodeficiency syndrome-associated retrovirus in human and non-human cells transfected with an infectious molecular clone. J. Virol. 59:284-291.

2. Ahmad, N., and S. Venkatesan. 1988. Nef protein of HIV-1 is a transcriptional repressor of HIV-1 LTR. Science 241:1481-1485.

3. Arya, S. K., C. Guo, S. F. Josephs, and F. Wong-Staal. 1985. Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science 229:69-73.

4. Berkhout, B., and K.-T. Jeang. 1989. trans activation of human immunodeficiency virus type 1 is sequence specific for both the single-stranded bulge and loop of the trans-acting-responsive hairpin: a quantitative analysis. J. Virol. 63:5501-5504. 5. Berkhout, B., R. H. Silverman, and K.-T. Jeang. 1989. Tat

trans-activates the human immunodeficiency virus through a nascent RNA target. Cell 59:273-282.

6. Durand, D. B., J.-P. Shaw, M. R. Bush, R. E. Replogle, R. Belagaje, and G. R. Crabtree. 1988. Characterization of antigen receptor response elements within the interleukin-2 enhancer. Mol. Cell. Biol. 8:1715-1724.

7. Dynan, W. S., and R. Tjian. 1983. The promoter-specific tran-scription factor Spl binds to upstream sequences in the SV40 early promoter. Cell 35:79-87.

8. Feng, S., and E. C. Holland. 1988. HIV-1 tat trans-activation requires the loop sequence within tar. Nature (London) 334: 165-167.

9. Folks, T., S. Benn, A. Rabson, T. Theodore, M. D. Hoggan, M. Martin, M. Lightfoote, and K. Sell. 1985. Characterization of a continuous T-cell line susceptible to the cytopathic effects of the acquired immunodeficiency syndrome (AIDS)-associated retro-virus. Proc. Natl. Acad. Sci. USA 82:4539-4543.

10. Fromental, C., M. Kanno, H. Nomiyama, and P. Chambon. 1988. Cooperativity and hierarchical levels of functional orga-nization in theSV40 enhancer. Cell 54:943-953.

11. Garcia, J. A., D. Harrich, E. Soultanakis, F. Wu, R. Mitsuyasu, and R. B. Gaynor. 1989. Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcrip-tional regulation. EMBO J. 8:765-778.

12. Ghazal, P., H. Lubon, B. Fleckenstein, and L. Hennighausen. 1987. Binding of transcription factors and creation of a large nucleoprotein complex on the human cytomegalovirus en-hancer. Proc. Natl. Acad. Sci. USA84:3658-3662.

13. Guyader, M., M. Emerman, P. Sonigo, F. Clavel, L. Montagnier, and M. Alizon. 1987. Genome organization and transactivation of the human immunodeficiency virus type 2. Nature (London) 326:662-669.

14. Harrich, D., J. A. Garcia, R. Mitsuyasu, and R. B. Gaynor. 1990. TAR independent activation of the human

immunodefi-ciency virus in phorbolesterstimulatedTlymphocytes. EMBO J. 9:4417-4423.

15. Harrich, D., J.A.Garcia, F. K. Wu, R. Mitsuyasu, J.Gonzales, and R. B. Gaynor. 1989.Role ofSpl-bindingdomains in in vivo transcriptionalregulation of the humanimmunodeficiencyvirus type 1 long terminalrepeat. J.Virol. 63:2585-2591.

16. Hennighausen, L., and B. Fleckenstein. 1986. Nuclear factor 1 interacts withfiveDNAelements in the promoterregion ofthe humancytomegalovirus major immediateearlygene.EMBO J. 5:1367-1371.

17. Hennighausen, L., P. A. Furth, and C. W. Pittius. 1989. KB elements strongly activate gene expression in non-lymphoid cells andfunction synergistically withNF1 elements. Nucleic AcidsRes. 17:8197-8206.

18. Jakobovits,A., D. H.Smith, E. B.Jakobovits,and D.J.Capon. 1988. Adiscrete element 3'ofhumanimmunodeficiencyvirus 1 (HIV-1) and HIV-2 mRNA initiation sites mediates

transcrip-tional activation by an HIV trans activator. Mol. Cell. Biol. 8:2555-2561.

19. Janson, L., and U. Pettersson. 1990. Cooperative interactions between transcription factorsSplandOTF-1.Proc.Natl.Acad. Sci. USA87:4732-4736.

20. Jones, J. A., J. T.Kadonga, P. A. Luciw, andR. Tijan. 1986. Activation of the AIDS retrovirus promoter by the cellular transcriptionfactor, Spl. Science 232:755-759.

21. Kawakami, K., C. Schneidereit, and R.G. Roeder. 1988. Iden-tification andpurificationofahumanimmunoglobulin enhancer-binding protein (NF-KB) that activates transcription from a

humanimmunodeficiency virus type 1 promoter in vitro. Proc. Natl. Acad. Sci. USA 85:4700-4704.

22. Kunkel, T. A. 1985.Rapid andefficientsite-specificmutagenesis without phenotype selection. Proc. Natl. Acad. Sci. USA 82:488-492.

23. Leonard, J., C.Parrott, A. J. Buckler-White, W.Turner,E. K.

Ross, M. A.Martin, and A. B. Rabson. 1989. The NF-KBbinding

site in the human immunodeficiency virus type 1 long terminal repeat are not required for virus infectivity. J. Virol. 63:4919-4924.

24. Lu, Y., M. Stenzel, J. G. Sodroski, and W. A. Haseltine. 1989. Effects of longterminalrepeatmutationsonhuman immunode-ficiency virus type 1 replication. J. Virol.63:4115-4119. 25. Miyoshi, I., H.Taguchi, I. Kubonishi, S.Yoshimoto, Y.Ohtsuki,

F. Shiraishi, and T. Akagi. 1982. Type c virus-producing cell linesderived from adult T-cell leukemia. GannMonogr. 28:219-228.

26. Morgan, R. A., K. Cornetta, and W. F. Anderson. 1990.

Appli-cations of thepolymerase chain reactionin retroviral-mediated gene transfer and theanalysis ofgene-markedhuman TILcells. Hum.Gene Ther. 1:135-149.

27. Myers, G., A. B. Rabson, J. A. Berzofsky, T. F.Smith, and F.

Wong-Staal(ed.). 1990. Human retroviruses and AIDS 1990, p. I-A-64. Theoretical Biology and Biophysics, Los Alamos Na-tionalLaboratory, Los Alamos, N.Mex.

28. Nabel, G., and D. Baltimore. 1987. An inducible transcription

factoractivates expression ofhumanimmunodeficiencyvirus in Tcells. Nature(London) 326:711-713.

29. Ondek, B., L. Gloss, and W. Herr. 1988. The SV40 enhancer contains two distinct levels oforganization. Nature (London) 333:40-45.

30. Parrott, C., T. Seidner, E. Duh, J.Leonard, T. S.Theodore,A.

Buckler-White, M.A.Martin, andA. B.Rabson. 1991. Variable role of the long terminal repeat Spl-binding sites in human immunodeficiency virus replicationin Tlymphocytes. J. Virol. 65:1414-1419.

31. Popovic, M., M. G. Sarnagadharan, E.Read, andR. C. Gallo. 1984. Detection, isolation, and continuous production of cyto-pathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224:497-500.

32. Rosen, C. A., J. G. Sodroski, and W. A. Haseltine. 1985. The location ofcis-acting regulatory sequences in the human T cell lymphotrophic virus type III (HTLV-III/LAV) long terminal repeat. Cell 41:813-823.

33. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular

on November 10, 2019 by guest

http://jvi.asm.org/

(9)

cloning: alaboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

34. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc-ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA74:5463-5467.

35. Seidner, T. J., and A. B. Rabson. Unpublished data.

36. Selby, M. J., E. S. Bain, P. A. Luciw, and B. M. Peterlin. 1989. Structure,sequence,and position ofthe stem-loopintar deter-minetranscriptional elongation by tat through the HIV-1 long terminalrepeat. Genes Dev. 3:547-558.

37. Shaw, J.-P., P. J. Utz, D. B. Durand, J. J. Toole, E. A.Emmel, andG. R.Crabtree. 1988. Identificationofaputativeregulator of earlyTcellactivation genes. Science 241:202-205.

38. Siekevitz, M., S. F. Josephs, M. Dukovich, N. Peffer, F. Wong-Staal, and W. C. Greene. 1987.Activationof the HIV LTR by T cellmitogens and the trans-activator protein of HTLV-1. Sci-ence238:1575-1578.

39. Simon, M. C., T. M.Fisch, B. J. Benecke, J. R. Nevins, and N. Heintz. 1988.Definition ofmultiple, functionally distinct TATA elements, one of whichisa target in the hsp7Opromoter for ElA regulation. Cell 52:723-729.

40. Sodroski, J., C. Rosen, F. Wong-Staal, S. Z. Salahuddin, M. Popovic, S. Arya, R. C. Gallo, and W. A. Haseltine. 1985. Trans-acting transcriptional regulation of human T-cell leuke-mia virustype III longterminal repeat. Science 227:171-173. 41. Sodroski, J. G., C. A. Rosen, F. Wong-Staal, R. Patarca, and

W. A. Haseltine. 1985. Location of the trans-activating region on the genome of human T-cell lymphotropic virus type III. Science 229:74-77.

42. Struhl, K. 1986. Constitutive and inducible Saccharomyces cerevisiae promoters:evidencefor two distinct molecular mech-anisms. Mol. Cell. Biol. 6:3847-3853.

43. Tanaka, M., U.Grossniklaus, W. Herr, and N. Hernandez.1988. Activation ofthe U2 snRNA promoter by the octamer motif definesanewclassofRNApolymeraseIIenhancer elements. GenesDev.2:1764-1788.

44. Wefald, F. C., B. H. Devlin, and R. S. Williams. 1990. Func-tional heterogeneity of mammalian TATA-box sequences re-vealed byinteraction withcell-specificenhancer. Nature (Lon-don) 344:260-262.

45. Weiss, A. L., R. L. Wiskocil, and J. D. Stobo. 1984. Therole of T3 surface molecules in the activation of human T cells: a two-stimulus requirementfor IL 2 production reflects events occurringat apre-translational level.J. Immunol. 133:123-128. 46. Willey, R., D. H. Smith, L. A. Lasky, T. S. Theodore, P. L. Earl, B. Moss, D. J. Capon, and M. A. Martin. 1988. In vitro mutagenesis identifiesaregion within the envelopegeneofthe humanimmunodeficiency virus that is criticalforinfectivity. J. Virol. 62:139-147.

47. Zoller, M. J., and M. Smith. 1983. Oligonucleotide-directed mutagenesis: asimple methodusing twooligonucleotide prim-ersandasingle-stranded DNAtemplate. DNA 3:479-488.