Functional analysis of human T-cell leukemia virus type I rex-response element: direct RNA binding of Rex protein correlates with in vivo activity.

JOURNAL OFVIROLOGY, Aug. 1991, p. 4408-4413 0022-538X/91/084408-06$02.00/0

CopyrightC 1991, American SocietyforMicrobiology

Functional

Analysis of Human T-Cell Leukemia Virus Type I

rex-Response Element: Direct

RNA

Binding of Rex

Protein

Correlates

with In

Vivo Activity

CLAUDIA BALLAUN,1G. KING FARRINGTON,2 MARIKADOBROVNIK,1JIMRUSCHE,2 JOACHIMHAUBER,1 AND ERNSTBOHNLEIN1*

SANDOZ Research Institute, Brunnerstrasse59, A-1235 Vienna, Austria,' andRepligen Corporation, One Kendall

Square, Cambridge,

Massachusetts 021392

Received 15February1991/Accepted 9May1991

The human T-cell leukemia virus type I rex gene product plays a critical role in the expression ofthe retroviral structuralproteins Gagand Envfromincompletely splicedmRNAs. Rexproteinactsthroughacis

element(rex-response element [RxRE]) whichislocated in theU3/R regionof the3'longterminal repeatand ispresentonallhumanT-cell leukemia virustypeI-specificmRNAs. Two domainsofthepredictedsecondary

structureofthe RxREarecruciallyimportantforRexactioninvivoasmeasuredbytwoassaysystems.Invitro studies using highly purified recombinant Rex protein revealed a specific and direct interaction with

radiolabeled RxREsequences.Thecorrelation betweenourin vivo results and thedirectbindingofRexprotein

to mutantandwild-typeRxREsequencessupportsboth theexistence of the predicted secondarystructureand theimportanceof this direct interaction with thecis-actingRNA sequenceforRexfunctioninvivo.

Human T-cell leukemia virus type I (HTLV-I), the etio-logic agentof adultT-cell leukemia andHTLV-I-associated myelopathy (8, 18-20, 22, 27), is provided with a complex genomeorganization. Inadditiontothe retroviral structural genes gag, pol, and env, HTLV-I encodes at least two

regulatory proteins (Tax and Rex) in the 3' region of the genome (7, 26). Rexactsposttranscriptionally, allowing the cytoplasmic accumulationofincompletely spliced retroviral mRNAsencodingthe structuralgenes gag,pol, and env(9, 11, 12). Function of the rex gene product depends on a cis-actingsequence(rex-response element [RxRE]) which is locatedinthe U3/R region of the 3' long terminalrepeat (1, 9, 23, 25) andseparates thepolyadenylation signal from the site of polyadenylation (1). The in vivo formation of a

predicted, stable, complex RNA secondarystructure, which wassupported by in vitro solution mapping analysis(25),not

only would allowaproperspacing of these signalsinvolved

in polyadenylation but also could benecessaryforrexgene

function.

In this report, we describe the in vivo mapping of cis-actingsequences,within theRxRE, whichare necessaryfor wild-type Rex-mediatedtransactivation.We have generated

aseriesof5', 3', 5'/3', and internalRxREdeletionmutants.

By employing two in vivo assay systems, we defined two

critically important substructures within the RxRE. The establishment of a Rex-specific RNA gel mobility shift assay, using highly purified recombinant wild-typeHTLV-I protein, enabledustodirectlyassaytheseRxREmutantsin vitro. The observed correlation between Rex activity and specificdirect binding ofRex proteintoradiolabeled RxRE RNA in vitro supports the importance of the predicted secondary structure of this cis element. Furthermore, our

results support the notion that a direct interaction of Rex

protein with RxRE sequences is required for HTLV-I rex

function invivo.

*Corresponding author.

MATERIALS AND METHODS

Plasmid construction. The wild-type RxREwas amplified by polymerase chain reaction by using the followingprimers: 5'-aaaaaagatctGGCTCAATAAACTAGCAGGAGTCTAT AAAAGCGT-3' and 5'-aaaaaaagcttGAGCAAGCAGGGT CAGGCAAAGCGTGGAGAGCCG-3'. The BglII-HindIII polymerase chain reaction fragment was ligated into the BamHI-HindIII sites of pKSII(-) (Stratagene) to generate pKS-RxRE. The RxRE polymerase chain reaction

frag-mentwasalsoinserted into the BglII-HindIII-treatedpgTat

vector (15), replacing the rev-response element (RRE) se-quences and generating pgTat-RxRE (2). A SalI-BamHI restriction fragment derived from pgTat-RxRE encompass-ing the RxRE sequence was subsequently cloned into a human immunodeficiency virus type 1 (HIV-1) HXB-2-derived infectious proviral DNA. The 5',3', and5'/3' RxRE deletion mutants were cloned by using the appropriate primers. The various mutants are listed in Table 1 and depicted in Fig. 1B. The internal deletion mutants were generated by oligonucleotide-directed mutagenesis (24) by

using the bacteriophage M13 mutagenesis system (Amer-sham). All mutantswere characterized by restriction analy-sis and dideoxy DNA sequencing (Sequenase 2.0; USB). Themutantfragments were ligatedasBamHI-HindIII

frag-ments into pKSII and pgTat and subsequently into the HXB-2-derived HIV-1 proviralplasmid.

Transfection and in vivoassays. ThepgTAT-RxRE DNAs (wild type and mutants) were cotransfected with a rex expression plasmid (2) into COS cells as described previ-ously (3). The immunoprecipitations and gel analyses were performed by the methods of Hauber et al. (10). HIV-1 proviral DNAs (1

jig)

were cotransfected with an HTLV-I

rexexpression plasmid (0.5 ,ug) (pLHRex; 2) into 5 x 106 COS cells. Supernatants were harvested 70 h posttransfec-tion and analyzed for HIV-1 p24 Gag expression by an enzyme-linkedimmunosorbentassay (ELISA) (Coulter).

Protein purification and in vitro assays. Expression and purification of Rex protein will be described in detail in futurepublications(5a, 6). Briefly,theHTLV-Irexgene was

4408

Vol.65,No. 8

on November 10, 2019 by guest

http://jvi.asm.org/

[image:2.612.51.553.89.274.2]

HTLV-I Rex/RxRE RNA BINDING IN VITRO 4409 TABLE 1. Descriptionof mutants

Invivoactivitiesa

Deletion HTLV-Icoordinates Free energy RNAmobility

Mutant type

(nucleotides)b

(kcal/mol)' Tat p24 shifta

shiftd (pg/ml)

Wild type 8600-8875 -111 +++ 365 +

RxRE1 5' 8633-8875 -89 +++ 178 +

RxRE2 5' 8646-8875 -76 +/- 16 +

RxRE3 3' 8600-8850 -96 +++ 104 +

RxRE4 3' 8600-8814 -75 - 0

-RxRE5 3' 8600-8768 -59 - 0

-RxRE6 5'/3' 8633-8850 -87 + + 115 +

RxRE7 5'/3' 8633-8814 -64 - 0

-RxRE8 5'/3' 8633-8768 -56 - 0

-RxREAI i.d.e A8653-8677 -108 +++ 406 +

RxREAII i.d. A8681-8696 -102 +++ 370 +

RxREAIII i.d. A8699-8744 -99 +/- 95 +

RxREAIV i.d. A8759-8841 -77 - 6

-RxREAIVc i.d. A9792-8815 -96 + + + 157 +

RxREAIVd i.d. A8773-8826 -81 +/- 44

-RxREAV i.d. A8653-8744 -84 - 12

-aThein vivoTatshift andp24(ELISA)activities and in vitro bindingresultsare

described.

bTheHTLV-Icoordinates (13) arelistedfor the end-pointdeletions.TheHTLV-1coordinates of thedeleted sequencesare also indicatedfortheinternal

deletionmutants.

Thepredicted stability ofthe RxRE sequences is shown.

dActivity ranges from wildtype (+++) to undetectable (-).

ei.d.,internaldeletion.

expressed in Escherichia coli by translational coupling to the E. coli ,B-glucuronidase gene on a high-copy-number plas-mid, resulting in recombinant protein with the authentic HTLV-I amino acid sequence. The bacterial cells were harvested, lysed, and centrifuged, and the pellet was ex-tracted with 8 M urea in 50 mM MES [2-(N-morpholino) ethanesulfonicacid] (pH 6.5). The solubilized proteins were then subjected to S-Sepharose chromatography and gel filtration. Less-pure fractions were further purified by re-verse-phase chromatography on a C-18 column. Protein

purity was analyzed by sodium dodecyl

sulfate-polyacryla-midegel electrophoresis.Radiolabeled RNA (1.5 x 104 cpm) wasincubated with 10 U of RNasin, 150 gigof yeast tRNA

(Boehringer Mannheim), and 1% glycerol in

phosphate-buffered saline for10 minat30°C.The Rexproteinwasthen

added to the binding reaction mixture and incubated for

another 10 min (final volume, 10 ,ul) at 30°C. The reaction

mixtures were loaded onto 5% native polyacrylamide gels (29:1, wt/wt) andelectrophoresed at5 mA, driedon What-man 3MM paper, andexposedto Kodak XAR X-ray films. For the competition experiments, radiolabeled in vitro

tran-scripts and a molar excess ofnonradiolabeled RNA were added tothebinding reaction mixture and incubated for 10 minpriortotheadditionofRexprotein.

RESULTS

In vivo mapping of functional domains within the RxRE sequence. Todefine functional domains in theHTLV-IRxRE

element, wemolecularlycloned andmutagenizedthe RxRE sequence.Various5', 3', 5'/3', and internal deletionmutants werecreated(detaileddescriptioninFig. 1and Table 1) and inserted intoanRRE(5, 16)-deficientgenomicTatindicator

plasmid (15) to generate pgTat-RxRE plasmids (wild type and mutants). In the in vivo assay utilized, a functional RxRE sequence in the presenceofacotransfectedwild-type

rexgeneresults in the formationofthe72-amino-acid(72-aa)

form of the HIV-1 Tat protein (9, 15) as detected by

immunoprecipitation(Fig. 2, lane 2). The 5'deletion mutant RxRE1 (Fig. 2, lane 3), the 3' deletionmutant RxRE3 (Fig.

2, lane 5), the 5'/3' mutant RxRE6 (Fig. 2, lane 8), and the

internal deletionmutants RxREAI, -All, and -AIVc (Fig. 2, lanes 11, 12, and 15) displayed a phenotype comparable to thatofwild-typeactivity (Fig.2, lane2) in this assay system. Transfection of pgTat-RxRE2 and the internal deletion mu-tants pgTat-RxREAIII and pgTat-RxREAIVd only poorly supportedRexfunction(Fig.2, lanes 4, 13, and 16). All other mutants failed to yield detectable levels of the indicator 72-aa Tatprotein(Fig.2 andTable 1). Toanalyze the various RxRE mutants more quantitatively, we constructed

infec-tious HIV-1 proviral clones in which the HIV-1 RRE was

replacedby the variousRxREsequences. With these

provi-ral constructs, thesynthesis ofthe HIV-1p24Gag polypep-tiderequiredthe presenceoffunctionalHTLV-IRexprotein (data not shown) and was quantitated by using an HIV-1

Gag-specific ELISA (Coulter). The results of this assay

(Table 1) supported the mapping data obtained in the Tat shift assay (Fig. 2).

PurifiedRexproteinspecifically binds RxRERNA in vitro. The interaction of HIV-1 Rev protein with the RRE

sug-gested a similar direct interaction of Rex protein with its

HTLV-I cis-acting element which, like the RRE, has the

potentialtoformastablesecondarystructure(Fig. 1A) (1, 9,

25, 28). To test this hypothesis directly, we expressed

HTLV-I Rexprotein in E. coli(6). Thepurity ofthe

protein

was analyzed in denaturing polyacrylamide gels (data not

shown) andWestern blots(immunoblots) (Fig. 3B, lane 1).

Incubation ofRexprotein withradiolabeled HTLV-I RxRE

transcripts resulted in a clearly retarded mobility of the

complex(Fig. 3A,lane2)ascomparedtofree RNA

(Fig. 3A,

lane 1). Immunodepletion of the protein

preparation

with a

Rex-specific rabbit antiserum (2)resulted in the loss of the

ability to form complexes with radiolabeled RxRE RNA

(Fig. 3A, lane 3). The addition of

protein

A agarose alone

(Affi-Gel; Bio-Rad) had noeffecton the complex formation

VOL.65, 1991

on November 10, 2019 by guest

http://jvi.asm.org/

4410 BALLAUN ET AL.

A

1I4tli..4114I

S"14.t11 144441|}11 4

f

t

.

,

A

S1i.

x

-t (

('1\11II i BB

'%It-11114,4 )1

/ \

%f.is \ FA I

ts-0

{,-\

I-- e.

1,-,

\_

w...t_ \ t

{,l

\

{,_

_

t, 91tsill \ t,=

4.-I

_t

s.s.5

!e

\

1l\

Xs

I

\-l

I

l,<|

4

\I

t|

%#.|q: %,-:

5'- RxRE deletion mutants

Mlutant RxRE 1

SL II ,!

Mutant RxRE 2

SL II

SL IV

SL I

3'-. RxRE deletion mutants

Mutant RxRE 3

SL I I s

Mutant RxRE 4 SL II

SL III

SL IV

SL I

Mutant RxRE 5 SL III

5'/3'- RxRE deletion mutants

Mutant RxRE 6 Mutant RxRE 7

SL II

SL III SL III

SLI

IV

SL

Mutant RXRE 8

SL II

°

ISL

III

SLL I

B

J. VIROL.

I

on November 10, 2019 by guest

http://jvi.asm.org/

HTLV-I Rex/RxRE RNA BINDING IN VITRO 4411 FIG. 1. (A) Schematic outline of the predicted secondary structure ofHTLV-I RxRE. The depictedHTLV-I sequence extends from nucleotide 8600 to nucleotide 8879 (14). The cap and splice donor sites are indicated, as are the various predicted substructures (28) (stems AandB; stem-loops I through IV). The internal deletion mutants(RxREAI, RxREAII, RxREAIII, RxREAIV, RxREAIVc,RxREAIVd,and RxREAV) are indicated by different shades of gray. (B) Schematic outline of the RxRE mutants. The5',3', and5'/3'RxRE deletion mutants areoutlined. Thedeleted sequenceshave been omitted from the wild-type structure (see panel A). The displayed structures of the RxRE mutantsdo not correspond with the most stable predicted secondary configurations (28) but were depicted in this manner to highlight the substructures whichareaffected by the specific deletions.

(Fig. 3A, lane 4). The untreated protein preparation con-tained a Rex-immunoreactive species in Western blot anal-yses (Fig. 3B, lane 1). However, the immunodepleted prep-aration which failed to complex radiolabeled RxRE RNA contained no detectable amounts of HTLV-I Rex (Fig. 3B, lane 2), while the pellet of the immunoprecipitation and the

Affi-Gel-treatedpreparation clearly yielded an immunoreac-tive protein of the correct molecular weight (Fig. 3B, lanes 3 and4).

The observed Rex-RxRE complex formation was depen-dent on the protein concentration (Fig. 4, lanes 2 to 5). In contrast, purified HIV-1 Rev protein, previously shown to specifically interact with HIV-1 RRE sequences (5, 17), did not bind to the heterologous HTLV-I RxRE sequences under identical experimental conditions (Fig. 4, lanes 7 to 10). Furthermore, increasing amounts of nonradiolabeled RxRE in vitro transcripts specifically competed with the

complex formation (Fig. 5, lanes 3 to 5). Additionof equal amounts of nonspecific HIV-1 RNA, which could not sup-port Rexfunctioninvivo (datanotshown),didnotaffect the RxRE RNA-protein complex (Fig. 5, lanes 6 to 8).

Direct binding of Rex protein to mutant RxRE in vitro transcripts. The Rex-specific RxRE-RNA gelmobility shift

assay permitted us to analyze our series of RxRE mutants

for direct binding in vitro. The synthesized RNAof RxRE mutants 1, 2, 3, and 6 formed complexes with the purified

Rex protein comparable to those of the wild-type RxRE

(Table 1). However, the RxRE mutants 4, 5, 7, and 8failed

to yielddetectable RNA-protein complexes under the same

experimental conditions (Table 1), which correlateswith our invivoresults. Invitro binding studiesof the RxRE internal deletion mutants also correlated with the observed activity in vivo (Table 1). Mutants which had failed to form com-plexes with purified Rex protein also did not compete for the binding of Rex protein to wild-type RxRE RNA (data not shown).

DISCUSSION

Although the trans regulatory proteins of HTLV-I and HIV-1 share no obvious amino acid homology, therexgene productcanfunctionally substitute for Rev action (21). This

observation suggests thatbothtrans-acting proteins sharea common functional mechanism mediated by cis-acting se-quences(4);the HTLV-I Rexactivity dependsonthe RxRE sequence and the HIV-1 Rev functiondepends on the RRE sequence (9, 16). We generated a series of site-directed RxRE mutants todefine importantdomains in the predicted secondary structure of the HTLV-I RNA element. Our in vivo experiments defined two independent domains within the RxRE. Our data (Fig. 1A) and the results recently

reported by Toyoshima et al. (25) map one essential cis elementto a structureonstem-loop IV, whichalso coincides with the sequence requirements determined by Ahmed and

colleagues (1). An additional domain is located on the

cloverleaf structure comprising stem-loops I, II, and III (mutant RxREAV), which is in agreement with a previous

report(25). Intriguingly, deletion oftheindividual

substruc-- 43

1 2 3 4 5 6 7 8 9 10 1 l 12 13

___R__S.

wFw

=

*

Xa

...

r.. ,_wt_ _S? _ _ _

.

E __

-L _t

tffi_w_+__56w7<;

. ._ .7_

- - sZ s^

86aa Tat

72aa

- 29

- 18,4

- W

4_

-

_b

- m - 14,3

-P -P

:k, ;k'

;k~

!k

I

~--k

-k

"

k

k~

Op

1k

k

K=K

I(C)

K;

K

.,

K K K K KK K

K

KK K K K

C $+,

7 V

+Rex

FIG. 2. In vivo analysis ofthe RxRE mutants by immunoprecipitation. Thevarious RxRE constructs (indicated atthe bottom) were

cotransfected withachloramphenicol acetyltransferase expression plasmidorpLHRex(2). Thepositions ofthe86-and 72-aaformsof the HIV-1 Tatprotein and molecularmassstandards(in kilodaltons) areindicated.

lbs. wli.ld&.-

I--W-wr ijlllllllll. .411M

VOL. 65,1991

V1-14 1 5 1 6 17 ,*.,A

1%

on November 10, 2019 by guest

http://jvi.asm.org/

[image:4.612.104.494.476.697.2]

4412 BALLAUN ET AL.

1 2 3

4

5

6

7 8

2 3 4

46-30 4ow

__

_10 Rex

21-

14-FIG. 3. (A) HTLV-I Rex protein specifically interacts with RxRE RNA. Rex protein (1 ,uM; 10-,u reactionvolume) causes a

distinct mobility shift (lane 2) compared to free RNA (lane 1) in nondenaturing 5% polyacrylamide gels. Rex-depleted protein

prep-arations failtoform specific complexes(lane 3). Pretreatment with Affi-Gel has noeffect onthe complex formation (lane 4). Arrows

indicate the positions of free and complexed RNA. (B) Western blot analysis of the Rex proteinpreparation. Rex-immunoreactive poly-peptidescanbedetected in lanes 1 (untreated Rex protein), 3 (pellet of the Rex-depleted preparation), and 4 (Affi-Gel-treated protein preparation). Noimmunoreactive proteincanbedetected in lane 2

(anti-Reximmunoprecipitatedprotein preparation). Molecularmass

standards (inkilodaltons) areindicatedontheleft.

ture stem-loop IorII displayed wild-type activity in both in

vivo testsandthe RNAgelmobility shift assay. Compared

to these mutants, RxREAIII yielded little 72-aaTatprotein andsignificantly reduced p24 Gag values, suggesting that the functional domainmustpartly overlap with stem-loop III. Of

note, mutation of the conserved bulge sequence (CUCAG GUCGAG; nucleotides 8783to8793; seeFig. 1), whichwas crucially important for HTLV-I Rex function (1), also abro-gated HTLV-II Rex responsiveness in vivo (13). Intrigu-ingly, stem-loop III contains an identical bulge sequence (GUCG; nucleotides 8715 to 8718; Fig. 1), which might

represent a functional site of interaction in this region. Experimental results obtained with mutant RxRE2 further indicate that thepresentation of the bulgesequencemight be important. Although all necessary nucleotides are present

(Fig.1andTable1), themoststableconfiguration of RxRE2 isnotpredictedtoform the cloverleafstructure(28;datanot

1 2 3 4 5 6 7 8 910

FIG. 4. Protein titration of the Rex-RxRE complex formation.

Increasing amountsofrecombinant Rexprotein (0.3, 0.6, 1.2,and

1.8 p.M; 10-p.I reaction volume) result in Rex-RxRE complexes

(lanes 2 to5). Addition of identical amounts of recombinant Rev

protein (5) failed to yield any detectable complexes with

radiola-beled RxRE RNA(lanes7to10). Lanes 1 and 6 show themigration

of RxRE RNA in the absence of protein. The positions of the

complexesandfree RNA areindicatedbyarrows.

I

RXRE

unrelated

RNA

FIG. 5. RNAgel mobility shiftcompetition experiment. Nonra-diolabeled RxRE RNA(lanes3to5)andanunrelatedHIV-1 RNA (HIV-1HXB-3 nucleotides 6619to7202; 5) (lanes6to8)wereadded

tothebindingreaction mixtureattheindicated molarexcess.The positionsof freeRxRE RNA(lane 1)andRex-complexedRxRE RNA (lane2) in the native 5% polyacrylamide gelareindicated byarrows.

shown), which could explain the failure of this RxREmutant tosupportRex actionin vivo. The structural stability of the various RxREmutantsmight also influence theirphenotype

in vivo. However, the observed difference in biological

activity between RxREAIII andmutantRxREAV isunlikely

tobetotally accounted forby the loss of stability (Table 1).

Other RxREmutants(e.g., RxRE1 and RxRE6), which form the cloverleaf structure and have predicted free-energy

valuesverysimilartothat ofRxREAV,supportRex-mediated

transactivationsignificantly better than RxREAV (Table 1).

Toassessthecapability of the various RxREsequencesto interact withRex protein directly, weestablished an RNA gel mobility shift assay using purified recombinant Rex protein(6). Several lines of evidence supportthespecificity of the observed Rex-RxRE complex formation. (i) Rex-specific depletion of the protein preparation abrogated the Rex-RxRE complex formation (Fig. 3A) which was inter-nally controlled by Western blot analysis (Fig. 3B). (ii) Rex-RxRE complex formationoccurredinadose-dependent manner, whereas purified recombinant HIV-1 Rev protein (5) failedtoform specific complexes under identical experi-mental conditions(Fig. 4). (iii) Nonradiolabeled RxRE RNA specifically reduced theRex-RxREcomplexes(Fig. 5), while nonrelevant HIV-1 RNA did not abrogate the Rex-RxRE complex formation (Fig. 5). Of note, all Rex function-supportingRxRE mutantsspecifically interacted with wild-type Rex protein in vitro (Table 1). Although it is still possible that Rexproteinisinvolved inRNA3'processing,our experiments clearly indicate that binding of Rex protein to RxRE RNAoccurs intheabsenceofpolyadenylation signals. The correlation betweenourin vivoexperimentsandour in vitrobindingstudiesindicates thatthe interactionofRex protein with RxRERNA isaprerequisitefor Rex-mediated

trans activation in vivo. Itfurthersuggeststhat the putative

A B

1 2 3 4

.I.

zk.

J. VIROL.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.612.76.305.58.173.2] [image:5.612.361.529.75.301.2] [image:5.612.100.276.516.645.2]

HTLV-I Rex/RxRE RNA BINDING IN VITRO 4413 cellular factors(s) involved in theRev- and Rex-dependent

transport of

incompletely

spliced RNA to the cytoplasm

might

interact with the RNA-protein complex. Only the

resultsobtained withmutantRxRE2werenotconsistent. As

already discussed,

thismutantisunlikelytoformthe clover-leafstructure(28; datanotshown)andisrelativelyunstable

(free

energy,-76

kcal/mol;

Table1). However,itispossible

that our in vitro reaction conditions partly support the cloverleaf

configuration

while the probability thatthis

sec-ondary

structure is formed in vivo is less likely.

Alterna-tively,

a

secondary

event

following

Rex-RxREcomplexation

is

required

to support Rexaction in vivo.

In

conclusion,

ourin vivo studies definedtwofunctionally

important

substructures in the RxREcis-actingelement. The

precise

localization and the sequence

requirements

of these domainsare

currently

under

investigation.

Furthermore, we haveestablisheda

specific

Rex-RxREinvitrobindingassay. For the first

time, purified

recombinant HTLV-I Rexprotein

was shown to

specifically

bind

directly

to RxRE RNA in vitro. The observed correlation between the in vivo and in vitro

experiments

supports the notion that binding of Rex

protein

to the RxRE RNA is necessaryfor Rex action. In

addition,

the

mapping

of

functionally important

domains to

substructures in the RxRE

(RxREAIV

and RxREAV)

indi-catesthe

importance

ofthe

predicted secondary

structurein

vivo.

Hence, overexpression

of

optimized

RxRE RNA or chimeric RxRE

transcripts

could be used in somatic gene

therapy

approaches

directed

against

HTLV-I.

ACKNOWLEDGMENTS

Wethank Jan-MarcusSeifertand FranzAmbergerfor oligonucle-otidesynthesis,BryanCullenandMichaelMalimfortherevgene, and WarnerC. Greene fortherexgene. Wealso thankourcolleague

Helmut Bachmayer for critical comments and Barbara Falle and Martina Ruhlfor excellent secretarial assistance.

REFERENCES

1. Ahmed, Y.F., S. M. Hanly,M. H. Malim,B. R. Cullen, and W.C. Greene.1990.Structure-functionanalysesof the HTLV-I Rex and HIV-1 Rev RNA responseelements: insightsinto the mechanism ofRex and Rev action. GenesDev.4:1014-1022. 2. Bohnlein, S., F. P. Pirker, L. Hofer, K. Zimmermann, H.

Bachmayer, E. Bohnlein, and J.Hauber. 1991. Transdominant repressors for human T-cell leukemia virus type I Rex and human immunodeficiency virus type 1 Rev function. J. Virol. 65:81-88.

3. Cullen, B. R. 1986. Trans-activation of human

immunodefi-ciencyvirus occursviaabimodalmechanism. Cell46:973-982. 4. Cullen, B. R., and W. C. Greene. 1989. Regulatory pathways

governingHIV-1replication. Cell 58:423-426.

5. Daly, T. J., K. S. Cook, G. S. Gray, T. E. Maione,and J. R. Rusche. 1989. HIV-1recombinantRevproteinbindsspecificallyto

the Rev responseelementin vitro.Nature(London)342:816-819.

Sa.Farrington,G.K.,R.Doten,C.

Bailaum,

E.Bohnlein,andJ.R. Rusche. Unpublisheddata.

6. Farrington, G.K.,P. Lynch,A.Jensen,E.Bohnlein,R.Doten,

T.Maione,T.Daly,andJ.R. Rusche. 1991. InImmunobiology

of

proteins

andpeptides VI,inpress.

7. Gallo,R.C.,F.Wong-Staal,L.Montagnier,and M. Yoshida.1988.

HIVMHTLV-I

genenomenclature. Nature(London)333:504. 8. Gessain, A., F. Barin,J. C. Vernant, 0. Gout, L. Maurs, A.

Calender,andG. deThe.1985.Antibodiestohuman T-lympho-tropicvirustype-1in patients withtropical spastical parapare-sis. Lancet ii:407-410.

9. Hanly,S.M.,L. T.Rimsky,M. H.Malim,J.H.Kim,J.Hauber,

M. DucDodon,S.-Y.Le,J.V. Maizel,B. R.Cullen,and W.C. Greene. 1989. Comparative analysis of the HTLV-I Rex and HIV-1 Rev trans-regulatory proteinsand their RNA response elements. GenesDev. 3:1534-1544.

10. Hauber, J., A. Perkins, E. P. Heimer, and B. R. Cullen. 1987. Trans-activation ofhuman immunodeficiencyvirusgene expres-sion ismediatedbynuclearevents.Proc.Natl.Acad.Sci. USA 84:6364-6368.

11. Hidaka, M., J. Inoue, M. Yoshida, and M. Seiki. 1988. Post-transcriptionalregulator (rex)of HTLV-I initiates expressionof viral structural proteins but suppresses expression of regulatory proteins. EMBO J. 7:519-523.

12. Inoue, J.-I., M. Yoshida, and M. Seiki. 1987. Transcriptional (p4Ox) and post-transcriptional (p27x-III) regulators are re-quired for the genes.Proc. Natl. Acad.Sci. USA 84:3653-3657. 13. Kim, J. H., P. A. Kaufman, S. M. Hanly, L. T. Rimsky, and W. C. Greene. 1991. Rex transregulation of human T-cell leukemia virus type II geneexpression. J. Virol. 65:405-414. 14. Malik, K. T. A., J. Even, and A. Karpas. 1988. Molecular

cloning and complete nucleotide sequence of an adult T cell leukaemiavirus/human t cell leukaemia virus type I (ATLV/ HTLV-I) isolate of Caribbean origin: relationship to other members of the ATLV/HTLV-I subgroup. J. Gen. Virol. 69: 1695-1710.

15. Malim, M. H., J. Hauber, R. Fenrick, and B. R. Cullen. 1988. Immunodeficiencyvirus rev trans-activator modulates the activity of the viralregulatory genes. Nature (London)335:181-183. 16. Malim, M. H., J. Hauber, S.-Y. Le, J. V. Maizel, and B. R.

Cullen. 1989. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of

un-splicedviral mRNA. Nature(London) 338:254-257.

17. Malim, M. H., L. S. Tiley, D. F. McCarn, F. R. Rusche, J. Hauber, and B. R. Cullen.1990. HIV-1 structural gene expres-sion requires binding of the Rev trans-activator to its RNA target sequence.Cell 60:675-683.

18. Osame, M., K. Usuku, N. Uichi, H. Amitani, A. Igata, M. Matsumoto, and H. Tara.1986. HTLV-I associated myelopathy, a newclinicalentity. Lancet i:1031-1032.

19. Poiesz, B. J., F. W. Ruscetti, A. F. Gazdar, P. A. Bunn, J. D. Mihna, and R. C. Gallo. 1980. Detection and isolation of type C retrovirusparticlesfrom fresh cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc. Natl. Acad. Sci. USA 77:7415-7419.

20. Poiesz, B. J., F.W. Ruscetti, M. S. Reitz, V. A. Kalyanamaran, and R. C. Gallo. 1981. Isolation of a new type C retrovirus (HTLV) in primary uncultured cells of apatient with Sezary T-cell leukemia. Nature(London) 294:268-271.

21. Rimsky, L. T., J. Hauber, M. Dukovich, M. H. Malim, A. Langlois, B. R. Cullen, and W. C. Greene. 1988. Functional replacement of the HIV-1 Rev protein by the HTLV-I Rex protein. Nature(London)335:738-740.

22. Seiki, M., S. Hattori, Y. Hirayama, and M. Yoshida. 1983. Human adult T-cell leukemia virus: complete nucleotide se-quenceof theprovirusgenomeintegratedin leukemia cell DNA. Proc. Natl. Acad. Sci. USA80:3618-3622.

23. Seiki, M., J.-I. Inoue, M.Hidaka, and M. Yoshida. 1988. Two cis-acting sequencesresponsible for post-transcriptional

trans-regulation ofgene expression ofhumanT-cell leukemia virus type I. Proc. Natl. Acad. Sci. USA85:7124-7128.

24. Taylor, J. W., J.Ott,and F. Eckstein. 1985. Therapid genera-tion of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 13:8765-8785.

25. Toyoshima, H., M.Itoh, J.-I. Inoue, M. Seiki,F. Takaku, and M. Yoshida. 1990. Secondary structure of the human T-cell leukemia virustype1rex-responsiveelementis essential forrex

regulationof RNAprocessingand transportofunsplicedRNAs. J. Virol. 64:2825-2832.

26. Wong-Staal, F.,and R. C. Gallo. 1985. HumanT-lymphotropic retroviruses. Nature(London)317:395-403.

27. Yoshida, M., I. Miyoshi, and Y. Hanuma. 1982. Isolation and characterization of retrovirus from cell lines of human adult T-cell leukemiaandits implication in the disease. Proc. Natl. Acad. Sci. USA 79:2031-2035.

28. Zuker,M., and P. Stiegler. 1981. Optimal computerfoldingof largeRNA sequencesusing thermodynamicandauxiliary infor-mation. Nucleic Acids Res. 9:133-148.

VOL. 65,1991

on November 10, 2019 by guest

http://jvi.asm.org/