Increased spacing between Sp1 and TATAA renders human immunodeficiency virus type 1 replication defective: implication for Tat function.

(1)

JOURNALOF

VIROLOGY,

Dec. 1993,p. 6937-6944 0022-538X/93/126937-08$02.00/0

Increased Spacing

between Spl and

TATAA

Renders

Human

Immunodeficiency Virus Type

1 Replication

Defective:

Implication

for Tat Function

LI-MIN HUANGAND KUAN-TEH JEANG*

Laboratory of Molecular Microbiology, Building

4, Room

306,

National

Institulte

of

Allergy and

Infectious

Diseases,

Bethesda,

Maryland

20892

Received 18 June 1993/Accepted23August 1993

Expression of the human immunodeficiency virus type 1 (HIV-1) is strongly activated byTat. The proper

action of Tat requires three elements: TATAA, TAR, andupstream motifs intheHIV-1 longterminal repeat. We show here that the correct spatial arrangement among Tat, Spl, and TATAA crucially influences HIV expression. Under conditions inwhich basal promoter activity is unperturbed, distancing

Spl

from TATAA markedly affected Tattrans activation. An increase in the Spl-TATAA distance from 18 to 101 nucleotides (depending on the inserted sequence) rendered HIV-1 either partially or wholly replication defective. This critical dependence onspacing suggests that Tat-, Spl-, and TATAA-binding factors mustcorrectly contact each other foroptimal expression and replication of HIV-1.

Human immunodeficiency virustype 1 (HIV-1) is the

etio-logicalagentofAIDS (11, 25). Like other retroviruses, HIV-1

reverse transcribes an RNA genome into double-stranded

DNA, which integrates into the host chromosome. Under normalconditions, the integrated provirus expressesits genes

and replicates productively (21, 44). All HIV-1 genes are

controlledbyasinglepromoterlocated in the 5' long terminal repeat (LTR). The transcriptional activity of this LTR is determined by cellular factors (13, 14) thatcooperatewith the viralTat protein (22, 23, 29, 34, 39). In the presence of Tat, transcription from the HIV-1 LTR is increased 100- to 1,000-fold. Without Tat, HIV-1 is defective for expression and replication (9, 30).

Although the precise mechanism of Tat function is being investigated, three elements in the LTRare required for Tat

action:TAR, TATAA, andupstream motifs (2, 4, 35, 37, 40). TARisanRNAelement found in Rwhich formsa

stem-bulge-loop configuration after it is transcribed nascently (3, 5, 32, 37). Tat attaches to TAR RNA and is thereby tethered to the vicinity of the LTR promoter (2, 38, 42). The other two elements, TATAA (12) andupstreammotifs (18, 24, 33),are

sites that Tat (once bound to TAR) interacts with in the

processoftransactivation.

In the HIV LTR, the three Spl binding sites and the two NF-KBbinding sites (18, 24, 33)arefunctionally important in

basaland intrans-activated expression (4). In the subgenomic context,NF-KB contributesmoretobasal expression, andSpl is more important for Tat responsiveness (4). Tat poorly activatesaminimal HIV-1 promoterthatdoesnothave either

Spl

orNF-KB motifs (2, 4, 41). Addition of enhancer-binding

sitescritically reconstitutes responsiveness toTat (2, 4). Two roles can be considered for enhancers in Tat trans

activation. First, enhancersmaysimply prime thepromoterto provide a basal level of transcription to make TAR RNA available for Tat attachment. Second, enhancer-binding

pro-teinscould be apartofamultiprotein complex which includes

cellular factors and Tat. Correct formation of this complex could thus leadto enhancedtranscription. In this setting, Tat

*Correspondingauthor.

trans activation occurs with the participation of cellular

tran-scription factors. One test of this is to determine whether spacing changes between enhancers and TATAA that donot affect basal activity canaffectTat-inducedtransactivation. We have therefore analyzed HIV-1 promoters (both in sub-genomic settings and in the context of infectious virus) with different distances between Spl and TATAA. We found spacings that maintained thebasalactivityof the promoterbut dramatically reduced Tat-inducedtransactivation.Thus, there is aspacing requirement for the Spl-TATAA-Tat interaction

which is distinct from that for the Spl-TATAA interaction. We also found that the correctorientation ofTat relative to Spl and TATAA is important. These observationsare compatible

withan adaptor-coactivator role for Tat in transcription

com-plex formation.

MATERIALS AND METHODS

Plasmid constructions. All of the plasmids used were

de-rived from p-43CAT (4), which contains HIV-1 LTR se-quences from -43 to

+78,

positioned upstream of the cat gene.Threerestriction sites(from 5'to3', PstI,

Sall,

andXbaI)

are presentaroundposition -43. Anatural Spl triplet (from HIV-1 LAI)wasinserted into theSall sitetomakep-43Sp/26. Inournomenclature, the number following the slash indicates the distance (in base pairs) between the Spl binding site and TATAA, with the Spl-TATAA distance in HIV-1 clone pNL4-3 (1) being 18 bp. p4GSpTm/26 was modified from

p-43Sp/26

by insertion of four copiesofthe Gal4binding site (5'CGGAAGACTCTCCTCCG3' [15])atthe PstI site and has

a mutated TAR. TARwas mutated by changing the

nucleo-tidesfrom +24to +32 from TGAGCCTGGtoCCTCGGACC, eliminating the normal bulge and loop. pNL4G/101 was de-rived from HIV-1 molecular clonepNL4-3 (1), in which four copiesofthe Gal4binding site (total size, 78nucleotides)were

inserted at the -43 position into both LTRs. pNLUC/101 is similarto pNL4G/101 except that the insertedsequenceis 78 bpofpUC19sequence. Intact molecular clones of HIV-1were

prepared and propagated in Escherichia coli as described previously (25). Gal4-Tatwasobtained from MichaelR.Green (41). Gal4-VP16-expressing plasmid pCMVGal65 was a gift

6937

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6938 HUANG AND JEANG

A

TAR

T A A CAT

[image:2.612.66.562.77.449.2]

Sp x3 l -

.

---

JL

...'

...-.

-

.---Desig nation

p-43Sp/26

B4.0

3.6

3.2

r-as n/Ra

p-43Sp/47

0

-Fu_cw

p-43Sp/65 a

m

p-43Sp/84

_u

p-43Sp/1 04 c p-43Sp/1 86

2.8

2.4

2.0

1.6

1.2

0.8

0.4

0.0

3 2

103

= 3

o 2

CF 102

s

0

L) 2

cot

I-0 2

LL

I

~ 00

1 2 I0-1 26 36 47 65 84 104 186

0.5

A

%%

I.,

N

A 2 * 4 T

_ A

8 -;

! i

S1V

a

26 36 47 65 84 104 186

Distance between Spl bindingsites and TATA(bp)

p-43Sp/104 p-43Sp/l86

ToT(/Ig)

0 0.5 1 2 4 0 0.5 1 ₂ ₄ ₀ _0.5 1 2 4 0 0.5 1

i

_0

_

_io

_,

_.

AcCm _

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

FIG. 1. Increased spacingbetweenSplbinding sitesandTATAA decreases responsiveness ofapromoter toTat. (A) Plasmidconstructs.All

constructscontain HIV-1 (clone pNL4-3)promotersequence upto -43 (mRNAstart site is +1)and threecopiesofSpl binding sites(solid

circles).Sequences frompUC19 of different lengths (dotted lines)wereinserted between the Spl sitesand TATAA.(B)Basalactivities andtrans

activation responsiveness of different constructions. (Left) Relative basal activities of differentpromoters.The activity ofp-43Sp/26wasset at 1. The basal activities of otherpromoters wereexpressed relativetothat ofp-43Sp/26. (Right) Responsiveness of differentpromoters tovarious

amountsofTat,asindicated (inmicrograms). Themeansand standarderrors werederived from threetosix experiments. (C) Representative titration

seriescomparing the trans-activationresponsiveness of p-43Sp/26 (lanes 1to5),p-43Sp/84 (lanes6to10), p-43Sp/104(lanes 11to15),andp-43Sp/186 (lanes 16to20). Subconfluent HeLa cellsweretransfected with 1.5 jigofreporteraloneorwith 0.5, 1, 2,or4 jigof Tat-producing plasmid.Acetylation

of['4C]chloramphenicolwasresolvedby thin-layer chromatography. Cm, chloramphenicol;AcCm, acetylatedchloramphenicol. from PeterO'Hare

(8).

All

plasmid

sequences were

confirmed

by

DNA

sequencing.

Cellculture andtransfection.

HeLa

cells

were

propagated in

Dulbecco's

modified

Eagle's medium with

10%

fetal

bovine

serum.The continuous humanT-leukemia cell lines C8166 and

12D7 were grown in RPMI 1640

medium

with 10% fetal

bovine serum. Human

peripheral blood lymphocytes (PBL)

were

stimulated

with

phytohemagglutinin (750

ng/ml)

for 4

days prior

to

infection

and

then maintained in

RPMI 1640with 10% fetal bovine serum

and 10%

interleukin-2

(Pharmacia).

Transfections

of

HeLa

cells

were

performed

with calcium

phosphate (17).

CAT and RT assays.

Chloramphenicol

acetyltransferase

(CAT)

assays were

performed

as

described previously (16).

After resolution

by thin-layer chromatography,

radioactivity

was

quantitated

with a

Fuji

phosphorimager.

For optimal

quantitations,

all

CAT activities

were reanalyzed

in

the linear

range of

acetylation.

All

transfections

and corresponding

as-sayswere donethree tosixtimes.

Reverse

transcriptase (RT)

assays were

performed

as

de-scribed

previously (36).

Each reaction mix contained 5 p.l of viralsupernatant in

50 ,ul of

RT

cocktail [60

mM Tris

(pH 8),

75 mM

KCl,

5 mM

MgCl2, 0.1%

Nonidet

P-40,

1 mM

EDTA,

5

,ug

of

poly(rA)

per

ml,

0.16

,ug of

oligo(dT)

per

ml]

andwas

incubatedat

37°C for

1h. From

each reaction,

5 p.lwas

spotted

ontoDEAE paper, whichwas

washed four

times

in 2x

SSC

(1

x SSC is

0.15

M

NaCl plus 0.015

M

sodium

citrate), and

radioactivity

was

counted

in

scintillant.

Western

(immunoblot)

analysis

and HIV

infection.

Three molecular

clones,

pNL4-3, pNL4G/101, and pNLUC/101 (7

jig

of

each),

weretransfected

separately

into HeLa cells. After 36 to 48

h,

cells and supernatants were harvested.

Cells

were

analyzed

forHIV-1

proteins, and the

supernatantswere used asvirus stocks forsubsequent infections.

For Western

analysis (6),

cellswerewashed with

phosphate-buffered saline and

resuspended

in sodium

dodecyl

sulfate

(SDS)

solubilization buffer

(50

mMTris

[pH 7],

2%

SDS,

5%

glycerol,

10%

2-mercaptoethanol). Proteins

were

resolved

ina

C

p-43Sp/26 p-43Sp/84

2 4

I

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CRITICAL SPACING FOR

Spl,

TATAA, AND Tat INTERACTION 6939

(A)

-.~~~~X

X-

4

.

. ._...

_.

...

(B)

30 25

a 20

cu

- 15

~0

.5

cU

U0

U-r

c

~.

-:'- -.-. . /- 5

0

F

_{F~ -.}/ ½.Ci

'7,rl

/

,A ₂₀ ₂₆ ₃₆ ₄₇ ₆₅ ₁₀₄

Distance betweenSpi and TATA (bp)

(C)

ApmiUp4Sprn/_O

p4CSp>|ri/6c4QCCSQJrr, T6 s I/2Tn.. t_ c r; ;-' 5;

1 2 3 4 5 6 77 8 9 C I 2

S.

.|

_

#*.

*

*S

-

-AcCm

[image:3.612.96.521.82.505.2]

-Cm

FIG. 2. Targeting of Tat to promoter via DNA tethering is less efficient and is sensitive to the distance separating Spl and TATAA. (A) Constructions usedtoassayactivationbyGal4-TatwithGal4sitespositioned upstream of Spl binding motifs. These constructs are derivatives of thep-43Sp plasmids shown in Fig. 1 except that in the starting plasmid, the distance separating Spl and TATAA is 20 instead of 26 nucleotides. Thereare twoothermodifications: (i) four copies of theGal4binding site (open squares) were inserted upstream of Spl and (ii) the TAR sequence wasmutated,rendering it Tat nonresponsive. (B) Behavior of plasmids in response toGal4-Tatfusion protein. Each construction wastransfected aloneorwithasecondplasmid expressingGal4-Tat.Ineach experiment, 1, 2, or 4 ,ug ofGal4-Tat-producingplasmid was used, and the foldtrans activationfor themostoptimalratio of reporter to effector was used. A total of three to six experiments were done. The means and standard errors are shown. (C) Typical thin-layer chromatogram showingtransactivation in HeLacells. Odd-numbered lanes, basal activities; even-numbered lanes, cotransfections withGal4-Tat. Cm, chloramphenicol; AcCm, acetylated chloramphenicol.

10% polyacrylamide-SDS gel

and

transferred

toImmobilon-P

(Millipore), which

was

then blocked with

TEN

buffer (10

mM Tris

[pH 8],

1 mMEDTA, 50 mM

NaCI)

containing

2%

nonfat

dried

milk.

The filters

werethen

incubated with

hyperimmune

patient

serum

overnight, washed,

and

subsequently

reacted with

'25I-labeled

protein

Ato

visualize

antibody-antigen

com-plexes. Autoradiography

and

quantitation

with a

Fuji

phosphor-imager

were

performed.

For HIV-1

infections,

viral supernatants were passed

through

0.45-p.m

filters and normalized for RT

activity.

Cells

(4

x

106) of

C8166 or

12D7

or PBL(3 x

106)

in a

1-ml

volume wereabsorbed with virus supernatant which contained various

amounts of RT at

37°C. After

I

h,

3.5 ml of

medium

was

added. Cell

supernatants were

sampled

every 2 or3

days for

RT

determination.

Forthe PBL

infections,

1.5 x

106

newcells were added every 10

days.

RESULTS

Increased spacing between

Spl

and TATAA

dramatically

reduces Tat transactivation without

affecting

basalpromoter

activity.

Spi

motifs are found

proximal

to TATAA in the HIV-1 promoter. Since Tat trans

activation

is crucial for the

expression

of HIV-1 genes, this

spatial

arrangementsuggestsa

-~~~~~~.mdmm

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(A)

0

-.

ctI c)

4-0 CZ ct

-o

11

2

1 03

2

102

2

1 01

1

*

Gal4-VP1

6 Gal4-Tat

*

Tat

20 26 36 47 65 104

Distance between Spl and TATA

(bp)

.'4G'3pTmn/;".3 5'Ip rn/I. p4'33pTr%''D '--b4.3.1, 4i ?'_!rn' ',' p4.SSpTn/l 10

, 3 4 45 1T07 8 9 1 1 1 2

0 _

_

.

0

_

_VI

.* *

AcCrr

-Cm

FIG. 3. tranis-activationabilityofGal4-VP16isnotdistance sensitive.(A)Schematic comparisonof tr-ans-activation responsivenessof HIV-1

promoters todifferentversions of Tat. Aplasmid expressingGal4-VP16wascotransfectedwithplasmids containingdifferentspacingbetweenSpl

and TATAA, asshown in Fig. 2A. In each set ofexperiments, 1, 2,or4 pLgofGal4-VPI6-producing plasmidwasused to derive the optimal conditions for transfection.Thegraphshows themeansandstandarderrorsfor threetosixsetsofexperiments.TocompareGal4-VP16withTat andGal4-Tat,the trans-activation resultsfromFig. lBand 2BaresuperimposedontheGal4-VPl6 data.(B) RepresentativeCATassay,showing trans-activation responsiveness toGal4-VPI6. The reporterplasmids areindicated. Odd-numbered lanes,basalactivities; even-numberedlanes, cotransfections with Gal4-VP16. For thevalueusedinpanel A,theassayswererepeatedtoobtain results in the linear range ofacetylation. Cm, chloramphenicol; AcCm,acetylatedchloramphenicol.

role forSpl in Tat function. Indeed,manystudies have shown that

Spl

cooperateswith Tat(4, 27, 41).Tobetter understand the Spl-Tat interaction, we determined whether there is a

spatial constraintonSpl withrespecttoTATAA.We

progres-sively increased the distance separatingSpl from TATAA (see constructs in Fig. IA). In this manner, we hoped to differen-tiate between the contribution of Spl to basal transcription

versus Tattransactivation (i.e., whethera distance separating

Spl

from TATAA that affects the latter but not the former could be achieved).

Basal activities from our constructions did not change uniformly as the distance separating Spl and TATAA was

increased (Fig. 1B,left). In fact,the highest basal activitywas

seenwhen the distance between the twoelements was65bp. Overall, fluctuations in basal activities from all of the con-structs were less than threefold different from that of the starting construct, p-43Sp/26. Basal activity actually rose

slightly as the distance increased from 104 to 186 bp. These results suggest that the effect of Spl on the basic HIV-1 TATAA promoterremains relatively constant overthe range

of distances tested. However, when the same promoters were

cotransfected with aTat-producing plasmid,asteep declinein Tat-mediatedtransactivationwas seen asthe distance between

Spland TATAAincreased (Fig. IB, right [note log scale]; Fig. IC).Therewas a 103-fold difference inactivityforspacingsof 26 and 186 nucleotides (Fig. lB, right; Fig. IC). Thus, these

(B)

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(5)

CRITICAL SPACING FOR Spl, TATAA, AND Tat INTERACTION 6941

(A)

e 4

O" 7L:- 4

(B) pSp4GTm/1 04 pSp2GTm/65 pSpl GTm/47

1 2 3 4 5 6

]AcCm

FIG. 4. Ga14-Tat is not functionalwhen placed between Spl and TATAA. (A) Constructions used toassayGal4-Tatactivitywhen the Gal4site(s)wasinserted between SplandTATAA.These constructs

are similar to those shown in Fig. 2A except that one, two, orfour

copies of the Gal4binding site (O) were inserted between Spl and TATAA. (B) Representative thin-layer chromatogram, showinglack

oftrans-activation responsiveness. Odd-numbered lanes,basal activi-ties;even-numberedlanes, cotransfectionswith Gal4-Tat.Cm,

chlor-amphenicol; AcCm, acetylated chloramphenicol.

constructionsclearly segregated the effectsofspacingbetween

Spl

and TATAAfor basalversus Tat-inducedtranscription.

Distancebetween

Spl

and TATAAis alsocritical when Tat

is targeted to the promoter via DNA binding. In the above experiments,Tatwasintroduced to thepromoterviatethering

to TARRNA. In suchanapproach,whenwemoved

Spl

away

fromTATAA,wealsodistanced

Spl

from Tat. Tocontrol for

the latter variable, we used a second approach that keptthe

distance between Tat and

Spl

constant while varying the

distance between

Spl

and TATAA. We positioned Gal4

bindingsitesnext to the

Spl

motifs anddirectedGal4-Tat(41)

tothe promoter by usingthe Gal4bindingsites (Fig. 2A).To

make sure that Gal4-Tat functioned only throughGal4 sites,

TAR was mutated so that it would be nonfunctional (see

Materials andMethods).We hadobserved thatwhen Gal4-Tat

was targetedto thepromotervia TARRNA,itwas asefficient

as Tat in trans activation (data not shown). However, when

Gal4-Tat was targeted by using DNA, the efficiency oftrans

activation was decreased 10'- to 102-fold (Fig. 2). This is in

agreementwithourprevious findingswithanalternative DNA

tether (2). Nevertheless, Gal4-Tat did maintain a moderate

level of trans activation (Fig. 2B; Fig. 2C, lanes 1 and 2) that

could be tested for

spatial sensitivity.

In this

instance,

when we

increased the

distance between Spl and TATAA,

we

did

not

affect the distance between Tat and Spl. This lengthening of

distance did

compromise

Tat trans

activation

(Fig. 2B and C),

and this result

supports the idea that correct

spacing

between

Spl and TATAA is important for optimal Tat function.

However,

because there is

no

clear linear

relationship between

distance and loss of

trans

activation, it is possible that the

relative

positioning of Spl and

TATAA on

the

DNA

helix is

also

important.

To

control

for the possibility that the Gal4 binding sites

might be

inherently malpositioned,

we

checked for the ability

of Gal4-VP16

to

activate the

plasmids shown

in

Fig. 2A. Potent

trans

activation by Gal4-VP16

was

observed, and this activation

was not

sensitive

to

the distance between

Spi

and

TATAA

(Fig. 3). This result

suggests

that

Gal4-Tat and Gal4-VP16

differ in their

spatial

restrictions

(Fig. 3A)

and

is consistent

with observations from others that both

Tat

and VP16

can

initiate

transcription (but in

nonidentical manners) (43).

Orientation

of Tat

relative

to

Spl

is

important.

The above

experiments

address the issue of the influence of spacing

between

Spl and

TATAA on Tat tranis activation. We

won-dered whether

the orientation of Tat relative

to

Spl and

TATAA

could be

similarly important.

We

have demonstrated

previously that

a

specific orientation of

Tat

when

presented

from

TAR RNA

is

crucial for function

(2).

To

further

explore

this from

a

DNA-binding perspective, instead of positioning

Gal4-Tat

upstream

of

Spl,

we

interposed

Gal4

binding

sites

between

Spl

and TATAA

(Fig. 4A). Interestingly,

we

found

that

in this

configuration, Gal4-Tat

was

completely inactive

(Fig. 4B, lanes I and 2).

In

the

pSp4GTm/104 plasmid, the

distance between

Spl and

TATAA

is

104

bp.

We

decreased the

Spl-TATAA

distance

to

47 and 65 nucleotides

(Fig.

4A,

pSplGTm/47 and pSp2GTm/65,

respectively). Gal4-Tat still

did

not

activate these

two constructs

(Fig. 4B, lanes

3 to

6).

Control

experiments with

a

Gal4-VP16

plasmid

demonstrated

that

pSp4GTm/104, pSplGTm/47,

and

pSp2GTm/65

were all

fully

activated

by

VP16

(data

not

shown). Thus,

Tat

activation

of

the HIV-1 promoter

is

orientation

specific for Spl

and

TATAA, whereas

VP16

activation is

not.

Spacing

between

Spl

and TATAA is critical for the virus.

The

distance between Spl and

TATAA

profoundly influences

Tat

responsiveness in the subgenomic

HIV-1 context (Fig. 1 and

2).

To

extrapolate this

finding

to

the

virus,

we

engineered

two

molecular clones of

HIV-1

clone

pNL4-3,

in

which the

distance between Spl and

TATAAin

both the 5' and 3'

LTRs was

increased

to 101

bp (Fig. 5A).

Two

different

DNAs were

inserted

asspacers.

pNL4G/101 contained

four

copies of

Gal4

binding sites positioned between

Spl and TATAA, while

pNLUC/101 contained

a

randomly selected

78-bp sequence

from

pUC19.

Other than

this

difference,

the two

clones

were

isogenic.

pNLUC/101, pNL4G/101,

and

pNL4-3

were

independently

transfected into

HeLa

cells.

Viral

protein synthesis,

superna-tant

viral

RT

production,

and

the

ability

to propagate a

spreading infection

were

assessed

for each of the three

molec-ular

clones

(Fig.

5and

6).

At48 h

after transfection into

HeLa

cells,

we

found

(on

aper

microgram

of

input

DNA

basis)

less

production

of viral

proteins from pNLUC/101

and

pNL4G/101

than from

pNL4-3. Supernatant

RT

production

wasfound at relative

ratios

of

1,

3.5,

and 14

for

pNLUC/101, pNL4G/101,

and

pNL4-3,

respectively (Fig. 5B);

relative

protein (p24

and

p55)

ratios

were

1,

3.5,

and

7.2,

respectively (Fig.

5B).

We next

assayed

the

infectivity

of the three viruses. Three

different cell lines

(PBL,

12D7

[CEM],

and

C8166)

were

used

(Fig. 6). Despite normalization of input virus

at the start

of

VOL.67. 1993

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[image:5.612.63.299.84.409.2]

(6)

W%

.Q Q C5 K

I.\

..v I

?,

,q

B

!.

ir

A

Designation U3 RU5

pNL4-3 U3 RU5

puc 19

sequence

seq_IMNdIuI _l|I pNlUC//101

69.8-No_

oI

<--p55

43.3-

28.3-*-p24

Gal4 binding

sites

"i

!

pNL4G/101

18.1-RT--

p

[image:6.612.104.513.80.318.2]

1 2 3

FIG. 5. HIVswith increased Spl-TATAA intervals show poor expression. (A) Schematics of threeHIV-1molecular clones. In bothpNL4G/101 and

pNLUC/101,

the distance between Spl and TATAA was increased to 101 bp. The spacer was either four copies of the Gal4binding site

(pNL4G/101)

or 78bpof pUC19 sequence (pNLUC/101). (B) Increased Spl-TATAA spacing reduces viral expression. A typical Western analysis is shown. Positions of molecular size markers are indicated to the left of the gel (in kilodaltons). The supernatant RT produced for each transfection is shownunder the lanes. Thepositionsofp55and p24 proteins are indicated. Equal amounts of totalproteins were loaded in each lane.

infection in all three cell lines,

pNLUC/101

was severely

delayed in peak RTproduction (no peak RT detected during the course of the experiment), while pNL4G/101 was

moder-ately delayed (3 to 15 days) (Fig. 6). Reinfection of cells with virus harvested at peak RTproduction (for

pNL4G/101)

and with medium supernatant at the end of 6 weeks of infection (forpNLUC/101) reproduced the same kineticpatterns (data notshown). Polymerase chain reaction analysis of thepNL4G/ 101 virus alsodemonstrated that the insertedspacersequence was retainedstably (19a).

DISCUSSION

Although it is well accepted that Tat is a transcriptional

activator, the mechanism of Tatactionremainsunclear(20-22, 31, 34, 39). One way to appreciate the mechanism is to understand the immediate interactive sites for Tat in the promoter(4). For example, it has been shown thatTatpoorly activates an HIV-1 minimal promoter which has intact TATAAand TAR elements (2, 4). Providing enhancer bind-ing-sites conferred optimal Tat responsiveness on the pro-moter (4, 27, 41). Inparticular, Spl sites have been shown to most effectively augment Tat function (4, 27). Our current study extends these observations by characterizing the spacing and relative orientation requirements for

Spl,

TATAA, and Tat for function. We also show that correct spacing between SpIandTATAAin theproviral LTR isveryimportant for viral

infectivity.

How do Spl, TATAA, and Tat interact? One possibility is that these three componentscoalesce to form a multiprotein

transcription complex. The fact that Spl and TATAA interact functionally (4, 18) isnotsurprising, since Spl motifsarefound

ubiquitously upstream ofmanyTATAA elements (26). How Tat interplays with this process is intriguing. In our

experi-ments, we

found

a

dramatically

steep

drop in

Tat-mediated

trans

activation when

we

progressively separated

Spl farther

from

TATAA

(Fig.

1B, right).

At

the

same

time, the effect of

Spl

on

the basal

activity of

the promoter

remained

relatively

unimpaired

(Fig.

IB, left).

These

observations

suggest

that

Tat

"bridges" SpI and

TATAA

(similar

to

the

function of

an

adaptor-coactivator [10]). The fact that

a

functional

Spl-Tat-TATAA

interaction is orientation sensitive

(Fig.

4) is also

consistent with

a

bridging

role

for

Tat.

Biochemical evidence

that

Tat

physically binds Spl

(22a)

and that

Tat

also

binds

TFIID

(Sa)

supports

this

bridging

hypothesis.

The bridging

hypothesis

also

explains why

Tatand VP16 can

both initiate

transcription

but in

different

ways

(43).

We suggest

that while

VP16

is

a

direct activator

of

a

minimal

TATAA promoter, Tat does not

activate

directly

but

instead

"adapts"

the

effect

of

an upstream factor

(e.g.,

Spl)

to

the

TATAA promoter.

Thus,

Tat

requires Spl (4, 27,

41),

a

specific

spacing between Spl and

TATAA

(Fig.

1),

and

a

specific

orientation relative

to

Spl

(Fig.

4). VP16, in

contrast,

needs

none

of

these.

Finally,

we

verified

the

spacing

requirements

in the context of HIV-1. We

transferred

our

Spl-TATAA moieties into

infectious molecular clones of

HIV-1

(Fig.

6) and found that,

in

the

setting of virus

replication,

a distance of 101 bases between

Spl

and TATAA

dramatically

changed

virus

growth.

In three

T-lymphocyte

cell types

(PBL,

12D7,

and

C8166),

the

replication

of

viruses

with an

Spl-TATAA distance of

101

bp

was

moderately

(pNL4G/101)

or

severely

(pNLUC/101)

de-layed (no

replication detected by

supernatant RT at 6weeks

postinfection).

The reasons

for

the

differences

seenwhen the spacer was four

copies

of the Gal4

binding

site or 78

bp

of

pUC19

sequence are not

clear.

However, both results

are

clearly consistent

with the

interpretation that increased

spacing

J. VIROL.

"--.4 = ..I

on November 9, 2019 by guest

http://jvi.asm.org/

(7)

CRITICAL SPACING FOR Spl, TATAA, AND Tat INTERACTION 6943

~~~~~~~~~~~~p

NL4-3

LO

so

pNL4G/1

01 020-E

10 +<

pNLUC/lO0

-~mock

1 i

C 8

co

0 ~~~~~~~~~~~~~5

0

12D7 PBL

I-=

4 3

Days After

Infection

FIG. 6. Viruseswith increased Spl-TATAA spacing in U3 replicate poorly. Replication kinetics of thethreeviruses fromFig. 5Aareshown.

Cellularsupernatantscontaining5 x 105cpmof RTactivitywereusedtoinfect 12D7and C8166cells.For PBLinfection,supernatantscontaining 106cpmof RTwereused. Althoughnotshown, allinfectionswerecarriedoutfor 6weeksbefore termination. The growthcurvesfor pNL4-3 and

pNLUC/101inC8166 and 12D7 cellswererepeatedtwice.

between

Spl

and

TATAA

negatively affects virus replication.

Thecrucial

consideration

of

spacing for

HIV-1 LTR

function

is

important

in

light

ofrecent

controversies

over

the need for

NF-KB

and/or Spl

elements

for

virus viability (28,

30,

36).

Differences in

competing studies

are

likely

explainable by the

factthat

mutations

in

the

respective

NF-KB

and

Spl sites did

notconserve

spacing relative

toTATAA

in the

provirus clones.

Itis

important

tonote

that insertions of

sequences

into

the

HIV-1 U3

could affect

polyadenylation.

We

have

examined

transcripts

produced from

the

pNL4G/101 virus and found that

they

were

polyadenylated

at the

expected position (data

not

shown).

In

addition,

recent

studies by

Valsamakis

et al.

(45)

and

Cherrington

and

Ganem (7)

suggest

that

ourparticular

insertions in

U3

wouldnot

have

a

major

effecton

polyadenyl-ation.

ACKNOWLEDGMENTS

WethankAnneGatignol, Oliver Semmes, and Lung-Ji Chang for readingsof themanuscript.

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