Multiple positive and negative cis-acting elements that mediate transactivation by bel1 in the long terminal repeat of human foamy virus.

0022-538X/93/042317-10$02.00/0

CopyrightC 1993, AmericanSociety for Microbiology

Multiple Positive and Negative cis-Acting Elements

That Mediate

Transactivation by bell in the

Long

Terminal

Repeat of Human

Foamy

Virus

KIJEONGLEE, ANN HWEELEE,ANDYOUNGCHUL SUNG* Department of Life Science, Center for Biofunctional Molecules, Pohang Institute

of

Scienceand

Technology,

Pohang, Republic

of

Korea

Received 28 September 1992/Accepted 10January 1993

The bell protein of human foamyvirus (HFV), aretrovirus, regulates expression ofthegenelinkedtothe HFVlongterminalrepeat(LTR)and is essential for viralgeneexpression. The mechanism of actionof thebell

protein isunknown, but its action is mediated through the U3 region of the LTR. To determine which U3

sequences arecritical for transactivationby bell,aseries of hybridvectorsconsistingofamutantHFV LTR andthechloramphenicolacetyltransferasegenewereconstructed and tested for their responsivenesstothebell protein by using transientassays after transfection. The target sequences for transactivation by bell were

mappedtofive regions in the U3 domain ofthe LTR: nucleotides -559to-506, -454to-418, -360to-342, -327 to -284, and -116 to -89 (+1 represents the transcription initiation site). No significant sequence

similaritywasidentifiedamongthe fivetargetsites.The observation that the multiple distinct elements in the

HFV LTRarethetargets for bell transactivation is different fromobservations withother human retroviral systems.The regulation mechanism of HFV bell protein-mediated transactivationappearstobeanalogousto that ofsomeDNAvirustransactivators that increase transcription from numerousdifferent viralpromoters with little sequencesimilarity shared amongthem.Wedemonstratedthat multiplebell-responsiveelements (BRE) canactasbell-dependent enhancerelements, whilea singlecopyofoneBRE,BREe, can serve as an

upstream activating element in both orientations. In addition, the region between -466 and -498 was identified as responsible for the downregulation ofgene expression directed by BREa, which requires its

upstream sequenceelementtoactas abel1-dependent enhancer elementinaheterologouspromoter.

Humanfoamy virus (HFV) isamember of the subfamily Spumavirinae of the family Retroviridae. HFV has been detected in specimens from patients with nasopharyngeal carcinoma(1), chronic myeloid leukemia(42), toxic enceph-alopathy (5), de Quervain's thyroiditis (37, 40), and non-A, non-B hepatitis after blood transfusions (29). However, the role of HFV in thepathogenesis of these diseases remainsto

be determined (39). The full genome of HFV has been

molecularly cloned and sequenced (8, 24). Its genomic organization shows striking similarity to those of other humanretroviruses, especially human T-cell leukemia virus type I (HTLV-I)and humanimmunodeficiencyvirustype1 (HIV-1).

The nucleotide sequence analysis shows that the HFV

genomecontainsatleast threeopenreading frames,termed bell,bel2,andbel3, inadditiontogag,pol, andenv,which

arewell conservedamongretroviralgenomes.The bellgene

encodes a protein functioning as a transactivator for tran-scription directed bythe HFV long terminal repeat (LTR) (31).bell has been showntobe essentialfor virusreplication invitro (22). HIV-1 and HTLV-I also encode an essential

transactivator whichrequiresatargetelement in the LTR(3, 4, 32, 34, 36). HIV-1 tat stimulates LTR-directed gene expression by directly interacting with an RNA target

se-quence termed TAR located downstream of the promoter, positions +19 to +42 (7, 13, 15). Efficient transcription depends on the specific sequence, secondary structure, location, and orientation of the target element (33). In HTLV-I, thetax protein transactivates LTR-directed

tran-* Correspondingauthor.

scriptionviaa21-bpdirectrepeatsequencelocated between -252 and -75 in theU3regionof the LTR(4,34). However, thetaxproteinhasnotbeen showntobind DNAdirectlyand is instead believedtoactivate LTR-directedtranscription via currentlyunidentifiedcellularDNAbinding proteins (9, 23, 27). Previous reports revealed that the bell gene of HFV

encodes a transcriptional transactivator which stimulates

HFVLTR-directed gene expression. Also, the target sites for bell were roughly mapped to two regions in the U3 domain of the LTR (17, 38).

To determine theprecisetargetsequencesfor the action of the bell protein, wehavesystematically analyzedthe HFV LTRby mutagenesis. Furthermore, hybrid promoters con-taining the whole HFV LTR or a part thereof and the enhancerless thymidinekinase(tk) promoter ofherpes sim-plexvirus (HSV)or the enhancerless c-fospromoter were

made. By using transient expression assays, the target elements forbell,termed bell-responsive elements(BRE), weremappedtofiveregionsinthe U3 domain of theLTR. In addition, we have shown that the region consisting of nucleotides -498 to -466 (the -498 to -466 region)

con-tains a negative regulatory element which downregulates BREa-directedgeneexpressioninaheterologous promoter.

MATERIALS AND METHODS

Plasmid construction.Nucleic acid restriction and modify-ing enzymes were used according to the suppliers'

(Pro-mega, Boehringer Mannheim Biochemicals, and KOSCO) recommendations. Oligonucleotides were synthesizedwith an Applied Biosystems DNA synthesizer and purified by denaturing polyacrylamide gel electrophoresis. Plasmid

2317

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HFV-CAT was constructed

by

inserting

the

KpnI-NarI

DNA

fragment (-777

to

+351)

upstream of the bacterial

chloramphenicol acetyltransferase

(CAT)

gene,followed

by

the simian virus 40

poly(A)

sequence. HFV-CAT-M and

HFV-CAT-MI

(see Fig. 3)

wereconstructed

by generating

SmaI

and EcoRI sites at nucleotides -149 and -74 of HFV-CAT and a

HincII

site at nucleotide -119 of

HFV-CAT-M, respectively,

withthe Muta-Gene

phagemid

in vitro

mutagenesis

system

(primers

used: 5'-CAC

ClTl

CCC CCG GGC AAC T-3' for

SmaI,

5'-TTA CTA TAG AAT TCC TTA A-3' for

EcoRI,

and 5'-TCT GCG TTA ACG AAA GAT TCG-3' forHinclI

[Bio-Rad]) (20).

Underlined nucle-otidesweremutatedtogenerate therestrictionenzymesites. The 5' and 3' deletion mutants wereconstructed

by

first

cleaving plasmids

HFV-CAT and HFV-CAT-M withXhoI and

EcoRI,

respectively,

andthen

incubating

the linearized DNA with BAL 31 exonuclease.

Digested

DNAs were removed at various

times,

and the

protruding

ends were filled

by using

the Klenow

fragment

of DNA

polymerase

I.

The end-filled DNAs were

ligated

with

KpnI

linker for 5'

deletion mutants and with EcoRI linker for 3' deletion mutants.

Next,

the DNAswere double

digested

with

KpnI

and

ClaI

for 5' deletion mutants orXhoI and EcoRI for 3'

deletionmutants.Deleted

fragments

ofthe HFV LTRwere

gel

purified and

replaced by

the

corresponding

fragments

of HFV-CAT and HFV-CAT-M

digested with

the same en-zymes. Each deletion mutant was

analyzed by

restriction

digestions

and double-stranded DNA

sequencing

with

prim-ers

complementary

to either the R

region

ofthe HFV LTR

(+54

to

+72;

5'-CAA TAT AAA ATA

CTT-3')

or the M13 forward

primer (Promega).

Plasmid

-11/+351

was con-structed

by deleting

the

XbaI-ClaI

fragment (nucleotides

-11 to

+351)

of HFV-CAT.

Internal deletion mutants were constructed

by

treatment

with

appropriate

restriction

and/or

modifying

enzymes and

ligation

with T4 DNA

ligase. HFV-IC(Ac)

and

HFV-IICE(Ac,e)

were constructed

by digesting plasmids

HFV-CAT and

HFV-IE(Ae), respectively,

withHindIIIand

treat-ing

the

resulting digests

with BAL31 exonuclease. Deleted

plasmids

weremade flushwiththe Klenow

fragment

of DNA

polymerase

I and

religated

with T4 DNA

ligase

in the presence of SmaI linker.

HFV-IA(Aa*)

and

HFV-IE(Ae)

were obtained

by deleting

theBstEII-SalI

(-570

to

-506)

and SmaI-EcoRI

(-149

to

-74)

DNA

fragments

from

HFVAp-1MII

(see below)

and

HFV-CAT-M, respectively.

HFV-IIBC(Ab,c)

was constructed

by inserting

the

KpnI-AvaII

(-777

to

-434) fragment

ofHFV-CAT into theKpnI site of

plasmid

-341/+351.

To obtain

plasmid

HFV-III(Aa,c,e),

aSall site was

generated

at

position

-512 with respect tothe

wild-type

LTR

transcription

initiation siteof

HFV-IICE(Ac,e) by oligonucleotide-directed

invitro

muta-genesis (5'-CTA

ATT TCATCC

TGTLCGLACTC

TCTGTC AAT

G-3')

followed

by

deletion of the

BstEII-SalI

DNA

fragment (-570

to

-506).

HFV-IV(Aa,b,c,e)

and

HFV-V(Aa,b,c,d,e)

were constructed from HFV-IICE(Ac,e) and HFV-CAT-M

by deleting

theBstEII-SmaI (-570 to -302) and BstEII-EcoRI

(-570

to -74) fragments, respectively.

HFV-ANRE was constructed by deleting the Sall-SmaI

(-508

to

-454)

DNA

fragment

ofHFVAp-1MII.

The AP-1 mutants

HFVAp-1MI

and

HFVAp-1MII

were constructed

by oligonucleotide-directed

in vitro mutagene-sis. The

primers

used were 5'-CTA ATT TCA TCC

T(I

CGA

CTC TCT GTC AAT G-3'

(HFVAp-lMII)

and5'-GGG TCC ATC TCG AGC ACT TCC CCG GGC AAT GAA

GGG-3'

(HFVAp-lMI

and

HFVAp-1MII).

Hybrids

of HFV LTR

regions

with the truncated tk

promoterof HSV and with the c-fos promoter were gener-ated by placing the HFV LTR either upstream or down-stream of the enhancerless tk promoter or the c-fos pro-moter. The configuration of LTR along the tk and c-fos promoter in each hybrid plasmid is schematically drawn in Fig. 6A. LTR-tk-U, LTR-tk-U-R, and LTR-tk-D were con-structedbyinserting the entire HFV LTR (nucleotides -777

to +351)into theSall site in both orientations and the SmaI

site of -37tk CAT (6), respectively. fos-U and LTR-fos-Dwereconstructedby inserting the HFV LTR (-777to

+351)into theSalI site andXhoIsite of A-71c-fos CAT (11,

28), respectively.

BRE(a)-tk and BRE(a)-tk-R were constructed by subclon-ing the BstEII-EcoRI fragment (-570 to -506) ofplasmid

-777/-506into theXbaI site of -37tk CAT in the same and

oppositeorientations with respect tothe promoter,

respec-tively. BRE(a')-tkwasconstructedby placing the

KpnI-SalI

DNA fragment (-777 to -509) of HFVAp-1MII into the XbaI site of -37tk CAT in the same orientation. BRE(a)-tk-D andBRE(a2)-tk-Dwereobtainedby inserting thesame BstEII-EcoRIfragment into theSmaI site of -37tk CAT as a monomer and a dimer, respectively. BRE(b)-tkwas ob-tained by inserting the SmaI-HindIII fragment (-453 to

-360)ofplasmidHFVAp-1MIIinto theXbaI site of -37tk

CAT. To construct BRE(c)-tk and BRE(c3)-tk, synthetic

oligonucleotides (5'-AGC Tfl-T CAC ATA CTC AGT AGC

TGT TT-3' and its partially complementary sequence

5'-CTA GAA ACA GCT ACT GAG TAT GTGAA-3')

contain-ing a portion of the HFV LTR (-360 to -335) were

prepared.Thecomplementary oligonucleotidesweretreated

with T4polynucleotide kinase, hybridized,andinserted into the XbaI site of -37tk CAT. BRE(d)-tk, BRE(dl)-tk,

BRE(d2)-tk, and BRE(d3)-tk contain the

Kp4nI-Hinfl

DNA

fragments of plasmids -342/+351 (-342 to -127), -284/

+351

(-284

to

-127),

-249/+351 (-249 to -127), and

-182/+351 (-182to -127), respectively, attheXbaI site of

-37tk CAT in the orientation opposite to that of the pro-moter. BRE(d')-tk was constructed by inserting the DNA

fragment containing the -327 to -276 region, which was

prepared by polymerasechainreaction,into theXbaI site of

-37tk CAT in the opposite orientation. BRE(e)-tk and

BRE(e)-tk-Rwereconstructedby insertingthe SmaI-EcoRI

fragment (-149to-74)of HFV-CAT-MintotheXbaI site of

-37tk CAT in the same and opposite orientations,

respec-tively,

with respect to the promoter. BRE(e'1)-tk and

BRE(e'2)-tk

wereconstructed byinsertingtheSmaI-HincII

(-149 to -117) and HincII-EcoRI (-116 to -74) DNA

fragmentsinto the XbaI site of-37tkCAT in the same and

opposite orientations, respectively. BRE(e)-tk-D,

BRE(e)-tk-DR,andBRE(e2)-tk-DcontaintheSmaI-EcoRIfragment

at the SmaI site of -37tk CAT in the same and opposite

orientations and as a dimer with respect to the promoter.

Theorientation of eachfragmentin allhybrid constructs was

confirmed by double-stranded DNA sequencing with the

CAT gene

primer

(5'-GGC CGT AAT ATC CAG G-3') or reverse

primer (Promega).

BREa/NRE-tkwas obtained by

inserting

the BstEII-EcoRI fragment (-570 to -466) of

plasmid-777/-466 into the XbaI site of -37tk CAT. BREe/

NRE-tk-R, BREe/NRE-tk-R', and NRE/RSV-CAT were

constructed by inserting the SalI-SmaI fragment (-512 to

-435)

of

HFVAp-1MII

into the SalI site ofBRE(e)-tk-R in

thesameandoppositeorientations and into theNdeIsite of

pRSV-CAT, respectively.

Cell culture and transfection assay. BHK-21 cells were grown in Dulbecco's modified Eagle's medium supple-mented with 10% fetal calf serum. Plasmid transfections

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x

U3 RU CAT Poly(A) |

~~b ~TATA

777 7 a

+11

m

I

BFV-CAT -561/+351 -506/+351

i I

i I

CAT Activity % conversion bol-A bol-S <0.1 275 <0.1 182 <0.1 107 <0.1 165 <0.1 65.2 <0.1 87.5 <0.1 85.8 <0.1 10.7 <0.1 8.7 <0.1 7.5 <0.1 6.8 <0.1

<0.1 <0.1

I I <0.1

8.1 7.3 7.4

2.5

i <0.1 0.2

i -I <0.1 <0.1

I-I <0.1 <0.1

FIG. 1. Schematicdiagramof thewild-typeHFV LTRand the 5' deletionmutantsof theHFV LTRand theirresponsestobell.Sequential

5' deletionmutantsweregenerated by controlled BAL 31 exonuclease digestion from parental plasmid HFV-CAT, and the deleted fragments usedtoconstructCAT fusion plasmidsaredepicted with their 5' endpoints extending from nucleotides -561to-13 (+1 correspondstothe transcription initiation site) and theircommon 3' endpoint at +351. The effectorplasmids pSbell-S (bel-S) and pSBell-A (bel-A)were

constructedby insertinga1,048-bp SspI fragmentofpHSRV comprisingtheentire bellopenreading frame downstream of the simian virus

40earlypromoterin thesenseand antisenseorientations, respectively (31). Regulatory regions defined by 5' sequential deletion analysisare

indicatedby theopenboxes (BREc, -360to-342; BREe, -127to-89). The solid vertical bars and the solid ovalrepresentthe AP-1 binding

sites and TATAbox, respectively. Deletion mutantsweretransfected into BHK-21 cells and levels of CATactivitywere determinedas

described in Materials and Methods. Restrictionenzymesites:K,KpnI;X, XhoI; N,Narl; C, ClaI.

were carried out by the DEAE-dextran method (30). Cells (106) were plated in a 100-mm-diameter dish a day before

transfection and transfected with 2 p,geach of effector and reporterplasmidsin thepresenceof80,M chloroquine.The cellsweretreated with10%dimethylsulfoxide for 150safter

exposuretothetransfection cocktail.Forty-eighthours after transfection, the cells were harvested and assayed for the expression level of CAT as previouslydescribed (12). The cell extractswere prepared bythree cyclesoffreezingand thawing. The level ofacetylated [14C]chloramphenicolwas

measuredby liquidscintillationcountingof the spotscutout from the plate after the acetylated and unacetylated forms

were separated by thin-layer chromatography. A 30-min time point and 50 ,ug of protein lysates were used for all reactions. In cases in which the level of acetylation of chloramphenicol was more than 80%, the protein lysates were diluted for the reaction and the CAT values were

correctedbythe dilution factor.AllCATassaydatareported inthis articlearefrompointsin the linearrangeof theassay.

The results shown are the averages of a minimum of four separate experiments for each deletion mutant, with stan-dard deviations withinarangeof 20%.Theprotein

concen-trationwasdetermined withaBio-Radproteinassaykit with bovineserum albuminas a standard.

RESULTS

5' and 3' progressive deletion analysis. Previous reports revealed that the bellgeneof HFV encodesatranscriptional transactivator that acts on the U3 region of the LTR. To identify the functional regions responsible for bell action,

we constructed plasmid HFV-CAT, which contains the entire HFV LTR(nucleotides-777to+351)upstreamof the CATgene.A series of deletionsstartingat the 5' end of the LTRwere carried outby controlled digestion of the HFV-CAT DNA withBAL31exonuclease. The structure of each deletion is illustrated in Fig. 1. The activities of individual LTR deletion mutants were determined by measuring the levels of CATactivityafter cotransfectioninto BHK-21 cells either with the bell expression plasmid (pSbell-S) orwith

the controlplasmid (pSbell-A).Theparental plasmid, HFV-CAT,showedmorethan2,750-fold inductionbybell.

Dele-tion of the R andU5 regionsin -11/+351did not haveany effect on the bell response, as expected on the basis of a -443/+351

-412/+351 -380/+351 -360/+351 -342/+351 -284/+351 -249/+351 -182/+351 -174/+351 -169/+351 -133/+351 -130/+351 -127/+351 -106/+351 -89/+351 -53/+351 -13/+351

i i <0.i 11.0

0 s

<0.1 11.0

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[image:3.612.158.470.71.391.2]

U3 RU5 CAT I Pol(A)

/.tI.~..4-b1J1IIIIIIIIIIIII2 zj

CAT

Activity

-777 %-i

;, i'W

1 t conversion

I

| F

+1

bel-A be1-S

HWV-CAT I--11/+351 1--777/-1491 -777/-186

1--777/-270

--777/-276

--777/-304

--777/-320

--777/-328 -_-, I,IJ3 v.

-777/-357I i

-777/-366I i

-777/-4181 l

-4 <0.1 I <0.1 I I.-I <0.1 I a-I <0.1

---14 I <0.1

----I <0.1

- I-I <0.1

i I <0.1

a I <0.1

I I <0.1 i- <0.1

I.-.--I<0.1

I - <0.1 275 292 248 244 270 260 221 216 242 262 229 168 153

-777/-4401 1 ' <0.1 34.1

-777/-4541 1 1 <0.1 6.5

-777/-4661 -1 l <0.1 6.1

-777/-4981 1 l- <0.1 102

-777/-5061 1

1-1

<0.1 140

-777/-5591

-

1 <0.1 <0.1

-777/-587i- <0.1 <0.1

FIG. 2. Localization oftwoBREandoneNREby transient-expressionanalysisof 3' deletionmutants.Deletionmutants weregenerated

byBAL31exonucleasedigestionfromthe EcoRIsite(nucleotide-74)toward the 5'direction,with theircommon5' and3'endpointsat-777

to +351,respectively. Each deletionmutantcontainedadeletionwith 5'endpoint varyingfrom -587to-149 anda common3'endpointat -74(+1 denotesthetranscription initiationsite). -11/+351wasconstructedbydeletingtheXbaI-ClaIfragmentofHFV-CAT.Regulatory regions defined by3'serial deletionanalysisareindicatedbythe open boxesand the hatched box:BREa,-559to-506; BREb,-454to-418; NRE, -498to-466. The solidvertical bars and solid ovalaredescribedinthelegendtoFig.1.The deletionmutants weretransfectedinto BHK-21 cells, andlevels of CATactivityweredeterminedasdescribedinMaterials andMethods. Restrictionenzymesites: K,KpnI;X, XhoI;XI,XbaI;C,ClaI; SI,SmaI;E,EcoRI. bel-A andbel-S indicatepSbell-AandpSbell-S, respectively.

previousreport(Fig. 2)(17).Deletions from the 5' end of the

LTRtopositions -561, -506, -443, -412, -380,and -360

resulted in slightly progressive reductions of the bell re-sponsebyuptothreefold(Fig. 1). These reductions may be due to the removal of the target sites for bell or other

regulatory sequences such as AP-1 binding sites that are

required for basal promoter activity(14, 25).In contrast, an

eightfolddecrease in thebell responsewasevident after the

deletion of an additional 18 nucleotides (deletion -342/

+351), butaresidual bell response remained. The level of

CAT activity remained relatively constant from this point

until the deletionwas extended up to -127 from the tran-scription start site, indicating that the DNA sequence 3' downstream toposition -127 still contains sufficient infor-mationtomediatebell transactivation. However, deletion of a further 38 nucleotides, resulting in plasmid -89/+351, almostcompletely abolished the bell response. The results from the 5' deletion analysis demonstrate that the ability of the HFV LTRtorespond tobell is dependent on at least two

specific sequences located at positions -360 to -342 and

-127 to -89. We propose that these elements be termed BREc and BREe, respectively.

To confirm the two regions, BREc and BREe, identified by the 5' deletion analysis, we generated an EcoRI site at

position -74 by oligonucleotide-directed mutagenesis

(HFV-CAT-M) and introduced a series of deletions toward

the 5' side starting atthe EcoRI site (Fig. 2). Interestingly,

deletion of positions -74 to -418 (deletion -777/-418),

includingboth BREc andBREe, didnotshow anysignificant

effectonthebellresponse,suggestingthat neither BREcnor

BREeisabsolutelyrequiredfor thebellresponse. Afurther

36-bp deletion (-777/-454) resulted in a considerable de-crease in CAT activity, with some of the bell response retained. Surprisingly, deletion ofpositions -466 to -498 resulted in an increase in bell-mediated transactivation of about16-fold, suggestingthat thisregioncontainsanegative

regulatorysequencewhichdownregulates the bellresponse.

Successive deletions to -506 resulted in only a slight in-crease in the bell response. In contrast, a deletion of a further53 nucleotides to -559completely shut off

transac-tivation by bell. These unexpected findings suggest that

there are twomore targetsites forbell (positions -454 to -418 and -559to -506) as well as one negative regulatory elementatpositions -498to -466. We propose that the two additional targetsites forbell,positions -454 to -418 and

-559 to -506, be termed BREb and BREa, respectively,

and that thenegative regulatory element, positions -498 to -466, be termed NRE.

Internal deletion and point mutation

analysis.

Taken

to-gether, the results obtainedby sequential deletion analysis

identified four positive elements and one negative element

responsible for the regulation of bell transactivation.

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J

UJ3

RUJ5

1I CAT 1I

POlY

(A) I

A CAT Activity

-777 "

HZ

w +1 +351% conversion

MM MM i

I

bel-A bel-S

NFV-CAT NFV-CAT-K HNV-CAT-KI

Y-TIA(Aa*)

BV-IC(Ac)

NFV-IZ(A.) NFV-IIBC(Ab, c)

UvIICZ(Ac,.)

EV-III (a,c, ) WFv-Iv (Aa,b,c,a)

W3V-V

(A,b,c,d,

*)

BUV-ANRE

NFVAp-lMII

1 B A 1

i

III

,

~~~~~~~~~

T1

SI_~MC

i I - i

8I

I.-4I I I

I~~~~F- i . F

I8o

--<0.1 275 <0.1 281 <0.1 279 <0.1 106 <0.1 135 <0.1 270 <0.1 119 <0.1 147 <0.1 256 <0.1 59.0 <0.1 <0.1

<0.1 178 <0.1 145 <0.1 138

FIG. 3. Effects ofdeletionsof putative BRE and NRE on HFV LTR promoter activity. Internaldeletion mutants were constructed by

treatmentwithappropriate restrictionand/or modifying enzymes. The deleted regions of each deletion mutant are as follows:

HFV-IA(A&a*),

nucleotide-570 to -506; HFV-IC(Ac), -365 to -322;HFV-IE(Ae), -149 to -74; HFV-IIBC(Ab,c), -432 to -342; HFV-IICE(Ac,e), -403 to-302 and -149 to-74;HFV-III(Aa,c,e), -570 to -506, -403 to -302, and -149 to -74;HFV-IV(Aa,b,c,e),-570 to -302 and -149 to -74; HFV-V(Aa,b,c,d,e), -570 to -74; HFV-ANRE, -508 to -454. The point mutants containing the mutated AP-1 binding sites, HFVAp-1MI andHFVAp-IMII, were generated by oligonucleotide-directed in vitro mutagenesis. Point mutations are indicated as solid vertical bars in themapsof the mutants. Open boxes represent the putative BRE. All of the BRE except BREd are described in the legends toFig.1and2, and theBREd maps to the region from -302 to -149. These deletion mutants were transfected into BHK-21 cells, and levels of CATactivityweredeterminedasdescribed inMaterials and Methods. Restrictionenzymesites: K,

KpnI;

B, BstEII; A,AvaII;H,HindIII;

Hc,HincII; SI,SmaI;E, EcoRI; X, XhoI;

S,

SalI.bel-A and bel-S refer to pSbell-A and pSbell-S, respectively.

quential deletion mutagenesis could notidentify redundant

elements in whichasinglecopyis sufficient to activate gene

expression. In addition, 3' sequential deletion mutagenesis

mighthave resulted in thejuxtaposition of distal sequences,

resultingin artifactualactivationorinactivation of

transcrip-tion. To investigate whether there are unidentified target

sites forbell,wegeneratedinternaldeletion mutantswhich

havedeletions of one, two,three,orallof the BREidentified

by progressive deletion. Combinatorial deletions of one,

two,orthree of the BRE didnotshowanysignificant effect

on thebell response (Fig. 3). Interestingly, plasmid

HFV-IV(Aa,b,c,e)

from which all four BRE are absent, retained

bell-mediated transactivation, albeit at a lower level than

that of thewild-typeHFV-CATplasmid. Bycontrast,

addi-tional deletion of a region from positions -302 to -149,

resulting inplasmid

HFV-V(Aa,b,c,d,e),

resulted ina

com-plete lossof transactivationbybell (Fig. 3).These

experi-ments indicate that the region between -302 and -149 contains thefifth target site forbelltransactivation (BREd).

To investigate the role of NRE in the HFV LTR as a

whole, we constructed HFV-ANRE by deleting the

SalI-SmaIDNAfragmentofHFVAp-lMIIandassayed it for the

bellresponse(Fig. 3).Deletion of NRE appears to have little influenceonthebell-mediatedtransactivation of the whole LTR.

Since the deletion from the 5' end to position -360,

including three AP-1 binding sites, showed a moderate

decrease in thebellresponse (Fig. 1),wetested the role of these AP-1binding sites in the bell response. Mutationsin

these AP-1 binding sites (HFVAp-lMI and HFVAp-lMII)

led to anapproximately twofold decrease in bell-mediated

transactivation, suggesting that three AP-1 binding sites

contribute to the optimal activity of the HFV promoter. These resultsareingoodagreementwith those ofa

previous

report(25).

Effect of BRE and NRE in a heterologous promoter. To determine whether the sequences

mapped

asthe targetsites forbell were themselves sufficient for the bell response in the context ofa

heterologous

promoter, we subcloned the

DNA fragments containing each BRE upstream of the

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EIFV LTR 4 JA II

-777 Id IQ U w +1 +351

H

m

CAT activity

(% conversion)

-37tk

CAT

ll-IFikjIIIATII---777 -509

/|mz CAT ,>

q/

ERE(a')-tk

A

S

A

S

-570 -506 A

HRE

(a) -tk

/-

f j

i

Z-Cff

S

-453 -360 A

BRE(b)-tk II t/ CT

-360 -335 A

BRE (c)-tk /

/-S

-360 -335 A

ERE(c3) -tk CAT

S

-127 -342 A

U~~~~~~~

BRE(d)-tk

//MSWtk

A

-74-149 A

BRE(e) -tk-R k

-149 -117 A

BRE (e'1)-t k //

-74-116 A

BRE(e'2) -tk / /

-127 -342

BRE(d)-tk /CAT

2.1

* 3.5

* 2.0

*4S

25.2

* 21.5

*

* 31.3

*

4.5

*

*

80.6

* 2.2

5.6

* 2.1

*

*

42.3

O 4.5

*

B85.8

.

5.2

*

p

52.0

* 2.3

*

4

4.5

* 3.6

* z

~45.7

CAT activity

(% conversion)

bell-A bell-S

-I1

4.5 85.8

-127 -284

BRE(dl) -till CAT >

-127 -249

BRE(d2) -tk

/j

-tkJI7CII----127 -182

HRE(d3) -tk/ic CAT >--K

-276 -327

BRE(d') -tk / t/ /

2.1

1.8

1.7

4.5

4.2

3.9

4.1 75.9

FIG. 4. Transactivation of the enhancerless HSVtkpromotercontaining the putative BRE by bell. Hybridconstructswereconstructed by inserting the fragment containing the putativeBREupstreamofthe enhancerlesstkpromoterofHSV. Numbersrepresentthepositions of the

HFV LTR withrespect toitstranscription initiation site. Arrows indicate the orientation of theDNAfragment relativetothat of theenhancerless

tkpromoter.(A)Schematicdiagram of theHFVLTR-containingmultiple regulatory regionsandhybridconstructs, includingeachputative

BRE,and theirresponsetobell.Aand SrepresentpSbell-A and pSbell-S, respectively.The BREaredescribed in thelegendstoFig. 1, 2,

and 3. CATactivitywasmeasuredaspreviouslydescribed (12)and is indicated beside each lane of theautoradiogram. (B)Finemappingof

BREdbyserial deletionanalysis.The construction of eachplasmidand the transfectionassayaredescribed in Materials and Methods.

2322 B)

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[image:6.612.143.467.56.667.2]

hancerless HSV tk promoter andassayed them for thebell response(Fig. 4A).Thetruncated tk promoter contains only 37bpupstreamfrom thetranscriptionstartsitewith only one

transcription element, aTATA box(6).BREainBRE(a)-tk

significantly elevated the basal promoter activity in the

absence of bell. However, no significant induction bybell

wasdetected, indicating that BREa acted as a bell-indepen-dent element in a heterologous promoter. This result sug-geststhat BREarequiresanyotherelement within the LTR in ordertoconfer thebellresponse.To testthispossibility,

we constructed BRE(a')-tk, which contains BREa and its

upstream sequence, and assayed it for the bell response. BREa in BRE(a')-tkfunctionsas a bell-dependentelement

(Fig. 4A), indicatingthat BREa requires itsupstream DNA

sequencein ordertoact as abell-dependentelementinthe context of the heterologous promoter as well as the HFV promoter. By contrast, the plasmids containing BREb,

BREd, and BREe upstream of the enhancerless promoter

showed significant increases in CAT activity bybell,

sug-gestingthattheyaresufficienttoconferthebell

responsive-ness tothetk promoter.

To determine the 5' border of BREd, we subcloned the

K,pnI-Hinfl

DNA fragment of the plasmids -342/+351,

-284/+351, -249/+351,

and -182/+351 in the opposite

orientationupstreamof theenhancerlesstk promoter,

result-inginBRE(d)-tk, BRE(dl)-tk, BRE(d2)-tk, and BRE(d3)-tk,

respectively, and assayed them for the bell response.

Plas-mid BRE(d)-tk was transactivated by bell, but plasmids

BRE(dl)-tk,BRE(d2)-tk,andBRE(d3)-tk completely lacked

thebell response

(Fig. 4B).

Todetermine theprecisetarget sequence of BREd for bell transactivation, we placed the -327to -276 DNAfragmentupstreamof theenhancerless tk promoterto generate BRE(d')-tk and assayed it forthe bell response. Plasmid BRE(d')-tk was transactivated by

bell,

and the level oftransactivationwascomparabletothat of

BRE(d)-tk.

By combining these observations with the results ofthe internal deletion analysis, we concluded that the fifth target site for bell (BREd)is located in the region between -327 and -284. Inaddition,we subdivided BREe

into twofragments andpositionedthem upstream of the tk

promoter.PlasmidBRE(e'2)-tk, containingthe -116to-74

fragment,wastransactivatedbybell,butplasmid

BRE(e'1)-tk, containing

the-149to-117fragment,was not.Thus,we couldultimatelynarrowBREetotheregion between

nucle-otides -116and -89.

Asinglecopyof BREc inBRE(c)-tkhad littleeffectonthe

level of the CATactivityin thepresenceof bell (Fig. 4A).

However, when three copies of BREc were inserted

up-stream ofthe promoter at the same position, the resulting

plasmid, BRE(c3)-tk, showed a marked increase in CAT

activityinthe presence ofbell (Fig. 4A).

To examine theeffect of NREon twootherBRE, BREa

and BREe, and a

heterologous

viral promoter, the Rous

sarcomavirus

(RSV) LTR,

weconstructed

BREa/NRE-tk,

BREe/NRE-tk-R,

BREe/NRE-tk-R',

and

NRE/RSV-CAT

and then

assayed

them for the bell response. NRE in

BREa/NRE-tk

completely

abolished the increase in basal

promoter activity directed by BREa in the presence or absence of bell

(Fig. 5). However,

NRE in

BREe/NRE-tk-R,

BREe/NRE-tk-R',

and

NRE/RSV-CAT

did not show

anyeffecton the

ability

oftransactivation of BREe andon

the promoter

activity

of the RSV LTR

(Fig. 5).

These results suggestthat NRE canfunction

only

on

adjacent

BREa in a

heterologouspromoter.

Multiple BREfunction as a

bell-dependent

enhancer.

En-hancer elements have beendefinedas

regulatory

sequences

CAT activity (% oonvrsion)

bell-S

-37tk CALT

-570 -506

ER

(a)-tk

/okc

7 R

BRZa/NRX-tk

BRZ(o)-tk-R

BRZo/NRZ-tk-R

-512

Be/NR-tk-R

' //1IkI M

RSV-CLT

2.1

21.5

2.4

3.5

31.3

3.5

45.8

//T

, 90.1 90.5

-512-453

[image:7.612.326.565.80.358.2]

NRN/RSV-CAT CA 88.7 91.5

FIG. 5. Effects of NREonBRE andonRSV LTR-directedgene

expression.Theregionbetween nucleotides-570and-466,

includ-ingBREa andNRE,wasplaced upstreamofanenhancerless HSV tk promoter. TheSalI-SmaI fragment (-512 to -453) containing

NREwasplaced upstreamof BREe and the RSV LTR. Numbers above the maps represent the positions of the HFV LTR with

respect to its transcription initiation site. Arrows indicate the orientation of the DNAfragmentrelative to that of the enhancerless

tk promoter. Hatched boxes, hatched ovals, and stippled ovals

representNRE, BREa,andBREe, respectively.Cellswere cotrans-fected with each hybrid construct and abell expression plasmid (pSbell-S) or the control plasmid (pSbell-A). CAT assays were

performed48 h laterasdescribed in Materials and Methods.

that activate transcription of a linked gene at a distance, regardless of their orientation. To determine whether an HFV LTRcontainingfive BRE andoneNREcould actas an enhancerelement,the entire LTRsequence(-777to+351) was inserted upstream or downstream of the promoter in both orientations into two testplasmids lackinganenhancer, -37tk CAT and A-71c-fos CAT (Fig. 6A). The resulting plasmidsweretransfected intoBHK-21cells withorwithout the bellexpression plasmid, and transientexpressionof the CAT enzymewas usedas a measure of thetranscriptional activity.Asexpected,two controltestplasmidsexhibiteda

basal level of CATactivityand did notrespondto bell(Fig.

6A). In the absence of the bell expression plasmid,all five

recombinantplasmidsgavebackgroundlevels of CAT activ-ity that were about the same as those observed for test plasmids.When cellswerecotransfected withabell expres-sionplasmid, pSbell-S,and the recombinant plasmids

con-tainingthe HFVLTR,CAT activitieswere increased 10- to

50-fold. It is noteworthythat the level of the CATactivity

mediatedbybellwas somewhathigherwhenthe LTR was

placed upstream than downstream of each promoter (Fig.

6A).These data indicate that the HFV LTRcontainingthe

-570-466

A'

i

tk

-74 -149

-512 -453

1

1=

t

I k

I

Cwr-::..

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A) CAT activity (% Converslon) bell-A bell-S

-37tk CAT 2.1

LTR-tk-U | CAT 5.3

LTR-tk-U-R9^^T5.1

LTR-tk-D CAT/ 2.4

A-71c-fos CAT 1.4

LTR-foa-U CATfoQ 1.4

LTR-fos-D/

fo|

CAT

/O

1.3

B)

3.5 86.1 83.2 23.6 1.5 64.0 39.7

21.5 31.3

BRN

(a)

-tk

/

[

Z

BRZ(a)-tk-R

/

CALT/

BRZ(a)-tk-D

BRN(a2)-tk-D/I-

tklIQT /

BRN(0)-tk

/I|@CZIT>-ff

BRN(0)-tk-R

/

[/

URN(e)-tk-D

I-4Ei

LiQS>-BRN

()-tk-D

IH

JL

QT

-3 '-IR

BRN(e2)-tk-D

10.3 10.5 16.9

3.5 5.2 2.1 2.0 3.1

13.0 10.1 16.0

43.5 52.0 4.5 4.6 15.4

FIG. 6. Characterization of the enhancer element of the HFVLTR.(A)Theentire HFVLTRwasplacedupstreamordownstreamofthe enhancerless HSVtk promoterorc-fospromoterfusedtothe CATgene.Arrowsdenote theorientation of the HFV LTRrelativetothat of theenhancerlesspromoter. (B)The sequencebetweennucleotides -570 and -506containingBREaand theregionbetween-149 and -74 containingBREewereplacedupstreamordownstreamofanenhancerless HSVtk promoterin both orientationswith eitherone ortwocopies

of therespectiveBRE.Cellswerecotransfected with eachhybridconstructandabellexpression plasmid(pSbell-S)orthecontrolplasmid (pSbell-A). CATassayswereperformed 48hlaterasdescribed in Materials and Methods.

five BRE and NRE does function as a bell-dependent enhancer.

Todetermine whether asingle BREcould independently

act as an enhancer element, two of the BRE, BREa and

BREe,wereplaced in both orientations either upstream or

downstreamofthe tk promoter(Fig. 6B). BRE(a)-tk,which

hasasinglecopyofBREa,showed elevated basal promoter

activity, not responding to bell. Also, BRE(a)-tk-R and

BRE(a)-tk-Dshowedelevated basal promoter activity, albeit

at alowerlevelthanBRE(a)-tk.

Interestingly,

twocopies of

BREainBRE(a2)-tk-D conferredmore elevated basal

pro-moter

activity

thanthesinglecopyinBRE(a)-tk-D,

suggest-ing that twocopiesof BREaseem tohave acooperative or

additive effect. Plasmids containing one copy of BREe upstreamofthepromoterineither orientationshowed about a10-foldincreaseinCATactivitywhen cotransfectedwitha

bellexpression plasmid (Fig. 6B). Bycontrast,theplasmids

containing a single copy of BREe downstream of the

pro-moterineither orientation didnotshowany

significant

bell response

(Fig. 6B).

The observation that both BREa and BREe downstream of the promoter showed a lower bell responseis reminiscent of theCATactivityfound in LTR-tk-D and LTR-fos-D (Fig. 6A). Whentwo copies of BREe were inserted downstream of the promoter at the same

position, the resulting plasmid showed about a fivefold

increase in CATactivity by bell (Fig. 6B).These observa-tions suggest thattwocopiesofBREe haveacooperativeor

synergistic effect.

DISCUSSION

The results presented heredemonstrate that as manyas

fivedifferentcis-actingelements needed forbellfunctionare

presentin the U3regionof the LTR:positions -559to-506

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for BREa, -454 to -418 for BREb, -360 to -342 for BREc, -327 to-284 for BREd, and -116 to -89 for BREe. One of the five BRE, BREa, required its upstream DNA sequence element for bell-dependent transcriptional activation, whereas each of the other four BRE functioned in a bell-dependent manner by itself in a heterologous promoter. Our results arepartially consistent with those of previous work showing two target regions for bell in the U3 domain of the LTR (17, 38). Furthermore, we showed that the -498 to -466region downregulates the gene expression directed by BREain aheterologous promoter.

The HFV LTRcontaining at least five BRE and one NRE as a whole appears to act as a bell-dependent enhancer element. A single copy of BREe activated bell-dependent

transcriptionwhen positionedupstream of the enhancerless

tk promoter but had little detectable function at 1.65-kb downstreamof thetranscription start site,suggesting that a single copy of BREe by itself is sufficient for action only as an upstream activating element. A dimer of BREe was

sufficient to activate the enhancerless tk promoter

down-streamof the CAT geneatthe same position. In addition, a singlesyntheticcopyofBREchad little effect on thelevel of

CATactivitymediatedbybell,whilethreesyntheticcopies

of BREc showed a markedincrease in CAT activity in the presence of bell. The increase in CAT activity was much greater than would be expected if the effects of the two copies of BREa and three copies of BREc were additive,

suggestingthat adjacentBRE sites cooperateto produce a

synergistic effectin the bell transactivation. Since a single

targetsite for bell appearstobesufficientforbell-mediated

transactivation, it will be interesting to determine why an

arrayofBREexists in the U3 regionofthe HFV LTR. The presence of multiple copies ofBRE could be expected to

giveanoptimal level ofLTR-directed geneexpressionandto

have a strong evolutionary advantage; mutations arising in

onesite would becompensatedforby other sitesinthe LTR. The fact that five distinct BRE which have no apparent sequence similarity are the targets forbell transactivation suggests that the mechanism of action of the HFV bell

proteinissimilartothat of some DNA virus transactivators

which increase transcription from numerous differentviral

promoters that havelittle sequencesimilarity. The

adenovi-rus ElA protein, for example, activates transcription from

manydifferent adenovirusearly promoters(16, 26), implying

thattransactivation isnotmediated via direct interactionof

the ElA protein with a specific ElA-responsive promoter

element. In some other promoters, ElA increases the

activ-ity ofafactor that interacts with the TATA box(21, 41).The

generality

ofElA

protein

canbefurther emphasized by its

abilitytostimulatetranscription byRNApolymeraseIII

(18,

19,35). Therefore,it is clear that further studiesare

required

in order to determine the mechanism by which HFVbell

regulates LTR-directed gene expression via at least five

different DNA target sites

(BRE)

and one negative

regula-torysequence

(NRE).

There are a number of

putative

DNA binding sites for

various cellulartranscription factorsintheU3regionof the HFV LTR. Most notable is the established consensus se-quence for AP-1 binding, which occurs three times. The

AP-1 bindingsites arecritical for the action ofsome other

retroviraltransactivators, suchasthevisna-maedi virus Tat

protein (10, 14). In contrast, point mutations in the three

AP-1bindingsites of the HFV LTR resulted in

only

a

slight

decrease (about

twofold)

in response to bell (Fig. 3),

indi-catingthat themechanism ofbell-mediated transactivation

differs from that of visnavirus,inwhichanAP-1 site closeto

the TATA boxseems tobe one

major

targetfor the

trans-activator. In

addition,

a

plasmid containing

five

copies

of

synthetic

AP-1

binding

sites from the

collagenase

gene promoter

(5'-AGC

TTG ATG AGT

CAG CAG-3')

upstream of the HSV tk promoter

(TRE-CAT

[2])

results inanincrease

of CAT

activity only by

twofold in the presence of bell

(unpublished results), suggesting

that the roleof the

putative

AP-1 site inbell-mediated

transactivation,

if any, is minor.

ACKNOWLEDGMENTS

Wethank V.terMeulen for his kindgiftofplasmids pSbell-Sand

pSbell-A,

W. A. Haseltine forplasmid pRSV-CAT, and Yo Han Choifortechnical assistance. WeacknowledgeH.S.Shin and C.B. Chae forcritical reviews of themanuscript.

Thisworkwassupported by Research Institute of Science and Technology grantR92061 and Center forBiofunctional Molecules grantCBM-92-06.

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