Human immunodeficiency virus type 1 Rev activation can be achieved without Rev-responsive element RNA if Rev is directed to the target as a Rev/MS2 fusion protein which tethers the MS2 operator RNA.

(1)

0022-538X/92/127469-12$02.00/0

Human

Immunodeficiency Virus Type 1 Rev Activation Can

Be

Achieved without Rev-Responsive Element RNA if Rev Is

Directed

to

the Target as a Rev/MS2 Fusion Protein Which

Tethers

the MS2 Operator RNA

SUNDARARAJAN VENKATESAN,1* SUSAN M. GERSTBERGER,1HEESUNG

PARK,'

STEVEN M. HOLLAND,2AND

YONG-SUK

NAM'

Laboratoty of Molecular

Microbiology'

andLaboratory of Host Defenses,2 National Institute of

Allergy

and Infectious Diseases, Bethesda, Maryland 20892

Received16July1992/Accepted 9 September 1992

The posttranscriptionaltinnsactivation ofunspliced or partially spliced human immunodeficiency virus RNAs by the Rev regulatory proteiniscrucial forvirusreplication and is dependent on sequence-specific RNA binding by Rev. The cognate RNA target of Rev is contained within a

highly

structured, 244-nucleotide Rev-responsive element(RRE) RNA in the viral env gene. Here, we show thatspecific interaction with the RRE is not an absolute requirement for Revfunction.When the RRE is replaced by aheterologousMS2 phage operator sequence, Rev willfacilitatethe cytoplasmic expression of human immunodeficiency virus mRNAs containing this sequence if directedtotheMS2 operator via the RNA binding motif of the MS2 phage coat protein (MS-C) as a Rev/MS-C fusionprotein. Rev/MS-C

efficiently

activated both RRE and MS2 targets. A mutation in the MS2 operator that abolishedthecoatprotein binding in vitro rendered the mutant RNA nonresponsive to the fusion protein in vivo. Notwithstanding that Rev canbe tethered to the viral RNAs via another RNA binding motif, the structural

integrityof the N terminus of Revwasstillrequired for optimal trans activation. The life cycle of human immunodeficiency virus type 1

(HIV-1) includes several biochemical mechanisms that may also be general paradigms for eukaryotic gene regulation.

Sequence-specific interaction of viral RNAs with virus-encodedproteinsis acommon theme in manyfundamental

viral processes, such as RNA transcription and

posttran-scriptional activation and RNA encapsidation (13, 45).

HIV-1 transcription is activated by the viral Tat protein,

which recognizes a 22-nucleotide stem-loop nascent TAR RNA(7,9,21, 42,46).HIV Revactsposttranscriptionallyto modulate thesplicing (11, 30) and tofacilitate the extranu-clear transportand cytoplasmic utilization of viralmRNAs

by binding to a highly structured Rev-responsive element (RRE)sequencein theenvmRNA(15-17, 19, 20, 23,32, 35,

49). Rev isabasicprotein thatconcentrates inthe nucleoli (12, 14) and readily oligomerizes both in vitro and in vivo

(33, 36, 47, 50). Genetic studies have identified separate

nuclear localization-RNAbindingand activation domainsat the N- and C-terminalportions of the Revprotein,

respec-tively(31). Genetic analysesof theN-terminalregionof Rev have mapped a contiguous stretch of amino acid residues between positions 13 and 56 that confers RNA binding,

nucleolarlocalization,andoligomerization properties essen-tial forRevfunction

(5,

8, 27,33).

Interestinthe

specific

sites of functional contact of Rev

with the RRE has led to attempts to narrow the element down to a minimal

responsive

motif. The computer-pre-dictedsecondarystructureofRRE RNA

(32)

is

composed

of

fourstem-loops

(A, C,

D, arid

E)

andabranched

stem-loop

(B/B1/B2) surrounding a central loop sequence

(Fig.

1).

Largeportionsofthis RREstructure aredispensablefor the Rev response (17, 22, 23,

35),

and a 64-nucleotide subse-quence,predictedtofold into the branched

stem-loop B/B1/

*

Corresponding

author.

B2, retains the Rev response both in vitro and in vivo (23,

35). A 9-nucleotide 5'-CACUAUGGG-3' sequence of the RRE corresponding to the B/B1 regions represents the minimal Revrecognitionsequence,both in vitro and invivo, if it ispresentedas astem-bulge-stemstructureandcontains atleasttwo G's,one of whichmustbeunpaired (24).

In spite of the accumulated evidence of the role of the RREas ananchor for Rev, thepossibilitythatthe RREalso recruits cellular factors that may be agonistsorantagonists of Rev function cannot be excluded. For instance, Rev cannotactivate the 5'-most132-nucleotide RREfragment in

vivo, despite having a greater in vitro affinity for this

fragment than the full RRE (23). It is possible that this

132-nucleotidefragmentlacksanessentialrecognitionmotif

for a putative cellular helper

factor(s).

In fact, a 56-kDa

RRE-bindingcellular factor that mayassist in Rev function has been demonstrated previously (44). Also, both stably

and transiently expressed Rev functions poorly in murine cells, implying a requirementfor tissue- or

species-specific

Revhelperfactors(43).

To explore the role of the RRE in the trans-activation

processes other than Revbinding and toexamine the con-tributions of thepeptidemotifs of Revtoactivation,wehave deviseda functional assayfor Revwithheterologous RNA targets. We show that the RRE may be

replaced by

the

heterologous MS2 phage operator RNAwithout

losing

the Revresponse invivo if Rev is directedtothe operatoras a Rev/MS2coatprotein fusion.

Using

bivalent targets recog-nized byboth Rev and the MS2 coat

protein,

we demon-stratethatthe in vivo responses of the targets

parallel

their in vitro affinities for the

respective

RNA

binding

proteins.

These studies indicate that Rev

binding

maybethe

primary

function of the RRE and that

RRE-binding

cellularfactors maynotbeanabsolute

requirement

for thetrans-activation effect of Rev.

7469

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7470 VENKATESAN ET AL.

MATERIALS AND METHODS

gag expression plasmids. All of the recombinants were constructed

by

site-directed

mutagenesis

of RRE DNA with a commercial M13-based protocol (Mutagene kit; Bio-Rad

Laboratories,

Richmond, Calif.), and the respectivemutant DNAs were

exchanged

for the RRE in the HIV-1 long terminal repeat

(LTR)-linked

gag

expression

vector

contain-ing

the

wild-type

(wt)

RRE

(23).

RREZ-MSwasconstructed

by

first

deleting

the

Rev-responsive B/B1/B2

subdomain(23) in the RRE

(RRE

coordinates49 to113)andtheninsertinga modified version of

phage

MS2 translational operator se-quence

(6).

RREZ-MS in M13 was further mutagenized to generateRREZ-MSAA,

RREZ-MS(U/A),

and RREZ-MS(A/

C),

which refer to the mutants which deleted the bulgedA residue

(underlined

in the -GAGG-sequence) orsubstituted A for

U(-AUIJA-)

orC for A

(-AUUA-)

in theloopsequence of MS. In

addition,

the RREwas alsocompletely exchanged

foramonomeric MS sequence in thesense(MS)orantisense

(SM)

orientation and forahead-to-tail [(MS)(MS)] or head-to-head

[(MS)(SM)]

dimer of the modified operator. (MS) wasconstructed

by

inserting

themodified operatorflanked

by

twoAresidues at the Asp-718

(KpnI)

site ofM13mpl8,

andtheMS monomerwas subsequently exchanged for the RRE in the wtgag RRE

plasmid.

To generate [(MS)(SM)]

and

[(MS)(SM)],

the MS monomerwas recovered from the recombinant M13

replicative

formby Asp-718 digestionand

self-ligated.

TheMSdimer from this reactionwasrecovered

by polyacrylamide gel

electrophoresis and insertedinto the above gag

expression

plasmidinplaceof the RRE. (MS)+A wasconstructed

by inserting

the RRE sequence

correspond-ing

to

positions

31 to49andpositions204to221, upstream and

downstream, respectively,

of the MS sequence in the M13 recombinant. In

(MS)+a,

RRE sequence between po-sitions 39to49and 204to213flankedthe MS sequence. The

following

two chimeras

incorporating

the targets for both Rev and the MS coat

protein

were also engineered. (i) To construct

(MS)+B/B1/B2,

the

B/B1/B2

subdomain(positions

49to

115)

of the RREwas

initially

clonedintoM13mp18.MS sequenceflanked

by

three A'swastheninserted upstream of the

B/B1/B2

motif.

(ii)

M13 containing

B/B1/B2

was also

mutagenized

to

replace

the

Bi

stem-loop(positions59to82) with the MS2 sequence. The resulting recombinant B/3G/

MS2/B2

preserved the B stem and contained the RRE

recognition

sequence -CACUAUGGG- upstream of the op-erator. Each ofthe bivalent chimeraswasindividually

mu-tagenized

tosubstitute the three G's in the RREBstemwith three

A's,

todelete thebulgedAresidue intheMS operator,

ortodo both. All ofthe chimerasweretheninserted into the gag

expression plasmid

pRMK4RREinplace of the RRE.

Expression plasmidsforRev/MS-C fusion proteins and their derivatives. HIV-1 Revwasexpressed either from the

Tat-responsive

HIV-1 LTR or from the constitutive Rous

sar-coma virus

(RSV)

LTR (29). MS-C and MS-N were

ex-pressed

from the

pSRa

promoter (6). Rev/MS-C denotes a tandem fusion of Rev and MS-C open reading frames

(ORFs).

The fused gene was constructed by a two-step

polymerase

chain reaction(PCR)procedure. In the first step, the Rev ORF was PCR amplified with anXbaI restriction

site-tagged

5'oligonucleotideprimer and a 3' primer that was

tagged

with sequencescorresponding to codons 2 through 7 of

MS-C,

MS-D,orMS-N. TheMS2 coat protein expression

plasmids

were generously provided by Benjamin Berkhout of the

University

of Amsterdam, The Netherlands. The ORFs for the respective MS2 coat proteins were PCR

amplified

with a 5' primer tagged with a sequence

corre-spondingtotheC-terminal 6codons of Rev and a3'

primer

tagged with anXbaI site. The PCR-amplified Rev and MS

DNAs were gel purified, annealed together, and

amplified

further with XbaI restrictionsite-tagged 5' primers

comple-mentary to the codons at the N terminus of Rev and the C terminus ofMS,respectively. The resulting

Rev/MS

fusion genes werepurifiedfromgels, cleavedwithXbaI, and cloned into M13mp18. After the DNA sequence of the

Rev/MS-mpl8 recombinants was verified, the fusion genewas PCR

amplified byprimerpairs tagged with T7

phage

RNA

poly-merase promoter and terminatorsequences at the 5' and 3' ends, respectively. The resulting T7 template was tran-scribed in vitrowith T7 RNApolymerase, and theRNAwas translated in a rabbit reticulocyte translation system with [35S]methionine. The translation yielded a fusion

protein

with the expected molecular mass that was readily im-munoprecipitable with anti-Rev antiserum. All other Rev/MS-Cderivativescarryingthe indicated mutationswere constructed by site-directed mutagenesis of Rev/MS-C

M13mpl8 phage DNA according to a commercial protocol

(Mutagene kit, Bio-Rad Laboratories). All of the Rev/MS

chimeraswereclonedattheXbaI site ofanRSV LTR-linked

eukaryotic expression plasmid, pRSV.5 (29).

Escherichia coli expression of Rev/MS-C fusion proteins. Rev/MS-C fusion protein and the indicated mutants were expressed in E. coli as fusion proteins linked to the C

terminus of E. coli maltose-binding protein (MBP) from an isopropyl-,-D-thiogalactopyranoside (IPTG)-inducible

,-ga-lactosidase promoter with a commercial kit (New England Biolabs, Beverly,Mass.). Rev/MS-C andRev(LE/PE)/MS-C mutant ORFs were recovered from the respective RSV

LTR-linked eukaryotic expression plasmids byXbaI diges-tion and cloned downstreamof the lone XbaI sitein the MBP vector. As a negative control, an Rev/MS-C coding sequence was alsoclonedin the antisense polarity with respect to the MBP ORF. Biosynthesis of the MBP-fused proteins was inducedby IPTG(1 mM) at an optical density at 600 nmof 0.3 followedby 2 hof incubation in at 37°C. Bacterialcells were harvested, rinsed with phosphate-buffered saline (PBS), and resuspended in lysis buffer containing 50 mM Tris-HCl (pH 7.8), 50 mM NaCl, 1 mM EDTA, 1 mM ethylene glycol-bis(,B-aminoethyl ether)-N,N,N',N'-tetra-acetic acid(EGTA), 1 mM dithiothreitol, and 1 mM phenyl-methylsulfonyl fluoride. Cells were disrupted with a French pressat 10,000lb/in2 and centrifuged at 25,000 x g for 15min

to collect the pellet containing the inclusion bodies. The pellet wascompletely dissolved in Tris buffer containing 50 mMTris-HCl, 50 mMKCI,and 5 mM EDTA supplemented with 6 M guanidine HCI and dialyzed successively against 200 volumes of the above buffer containing 1, 0.5, and 0.05 M guanidine HCI for 4 h at each step. Samples were centrifuged at 10,000 x g, and the supernatant was stored at -70°C or directly bound to amylose resin under conditions recommended by the manufacturer. The fusion protein was eluted with 20 mM maltose, concentrated by ultrafiltration, and used for binding studies.

RNA synthesis and protein binding. DNA templates, tagged attheir 5' ends with theT7promoter, were generated by PCR with primer pairs corresponding to the ends of the desired RNA (23). T7 promoter-tagged, PCR-amplified DNA fragments (0.5 pmol) were used as templates to generate unlabeled or uniformly labeled ([a-32P]UMP) RRE tran-scripts with a commercial kit from Promega Biotec (Milwau-kee, Wis.). All of the RNAs were purified by gel filtration on Sephadex G-100 columns and then electrophoresed on urea-acrylamide gel (10%), if necessary. RNA-protein binding J. VIROL.

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was evaluated by RNA gel mobility shift experiments. A reaction mixture containing protein samples and heparin (5 ,ug per reaction mixture) in HEPES binding buffer (20 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]-KOH, pH 7.9, 62 mM KCI, 0.15 mM dithiothreitol, 6% glycerol) was preincubated at 30°C for 10 min before the labeled RNA (5 x

103

cpm) was added and further incubated for 10 min at 30°C. Samples were loaded onto 5% native polyacrylamide gel in 0.5x Tris-borate-EDTA and

electro-phoresed at30 mA at 4°C. Radioactivity was visualized by

autoradiography of dried gels. Rev used in these experi-ments was expressed in E. coli and purified to near

homo-geneity (47).

Transient expression assay.Although the results presented in this article were obtained with HeLa cells, all

transfec-tions were repeated four times with Cos-7 cells. Approxi-mately 2 x 106 HeLa orCos-7 cells were electroporated at 300 V and 250 ,iF in a Bio-Rad electroporator with the individual gag plasmids (5

jig)

and 2jigof pHIV-Tat(pSV40 TatforCos-7 cells) and with or without the indicated Rev or theRev/MScoatprotein fusion protein expression plasmids. For the expression of Rev, 2.5 or 5 ,ug of an HIV-1 LTR-linked (23) or an RSV LTR-linked plasmid was used. The Rev/MS fusion protein plasmids (5 jigperexperiment) were expressed from the RSV LTR. All transfections also included HIV-1 LTR-linkedchloramphenicol acetyltransfer-ase (CAT) (2 ,ug) plasmid to monitor the transfection effi-ciency. Aliquotsof transfected cellswereroutinely removed at 48 hand used forimmunodetection of Rev and Rev/MS

fusion proteins (see below). Alternatively, aliquots of cells from each transfection were plated on coverslips

immedi-ately after electroporation and processed for immunofluo-rescenceof Rev and Rev/MSfusionproteins.Transfections were routinely terminated at 48 to 60 h, the cells were disrupted bythree freeze-thawcycles,and theextracts were clarified by low-speed centrifugation.gag

expression

was quantitated by a p24 enzyme-linked immunosorbent assay

(ELISA) of cell extracts (Coulter Diagnostics).

p249a&

expression levels were normalized to constant values of CAT activity

expressed

from HIV LTR-linked CAT

(23).

Each gag ELISA value from HeLa cell transfections repre-senteda meanfromatleast eight independenttransfections withmultiplepreparationsofplasmidDNA.CAT

expression

was quantitated by a commercial CAT ELISA kit

(Boehr-ingerMannheim).

Immunoblotting. After electroporation, cells were

plated

on60-mm-diameter dishes and harvested 48to72 h later. A one-thirdequivalentof cells from each dishwascollected

by

centrifugation, rinsed twice in

PBS,

and

lysed by

two freeze-thaw cycles in 50 ,ul of a buffer

containing

50 mM

Tris-HCl, pH 7.4, 0.5% Triton X-100, and 0.5%

3-[(3-cholamidopropyl)

-

dimethyl

-

ammoniol

-1-

propanesulfonate.

Thecelllysateswere then

adjusted

to 100 jiM

phenylmeth-ylsulfonyl fluoride-1

jig

of

aprotinin

perml-1

jig

of

leupeptin

per ml and digested at

37°C

for 15 min with a mixture of

pancreatic

RNase A

(2

jig/ml)

and 10 U of

RQ

DNase

(Promega

Biotec).

The

digestion

was

stopped by

theaddition

ofanequalvolume ofabuffer

containing

50mM

Tris-HCl,

pH 7.4, 4% sodium

dodecyl

sulfate

(SDS),

12%

(wt/vol)

glycerol, and 5%

(vol/vol)

2-mercaptoethanol

which was

heated at95°Cfor 5 min.

Aliquots

were

electrophoresed

on

12.5%polyacrylamide gelsinSDS. The

proteins

were blot-ted to

0.2-jim-pore-size

Immobilon filters

(Millipore

Corp.)

and reacted first with rabbit

polyclonal

anti-Revantiserumor

mousemonoclonal antibodies

against

Revand thenwith the

appropriatesecond

antibody

tagged

with horseradish

perox-idase.Theimmunoreactive bandswerethen visualizedbya commercial chemiluminescence

protocol

(Amersham Corp.)

and

quantified by scanning.

Immunoprecipitation. HeLaorCos-7 cellswereroutinely

labeled 48 h posttransfection. For labeling, a one-third

equivalent

ofcells from each confluent 60-mm-diameter dish wasstarved for 15 min in RPMI mediumlackingmethionine and

cysteine

and

containing

2.5%

dialyzed

fetal bovine serum. The cells were then

metabolically

labeled with

[35S]methionine

and

[35S]cysteine (2 mCi/ml)

inatotal vol-umeof 0.1 ml for 1 h at37°C.The cellswereharvested and

lysed as described above for immunoblotting, except that RNaseand DNasetreatments werein atotal volume of 0.2 ml.Followingthedigestion,thesamplesweredilutedwith 5 volumes of a buffer

containing

50 mM

Tris-HCl,

pH 7.4, 0.25% Triton X-100, 0.25%

3-[(3-cholamidopropyl)-dimeth-yl-ammonio]-1-propanesulfonate,

0.15%bovine serum

albu-min,

60 mM

NaCl,

100

jiM

bound

phenylmethylsulfonyl

fluoride,

1

jig

of

aprotinin

per

ml,

and 1

jig

of

leupeptin

per ml. The

lysates

werethen

precleared by being

boundat

4°C

for 1 h to

Staphylococcus protein

A beads that had been

preloadedwith nonimmune

immunoglobulin

G. The super-natantsfrom this stepwere then bound to

protein

Abeads coated with rabbit

polyclonal

or mousemonoclonal anti-Rev antibodies for 2 hat

4°C.

The

protein

Abeadswerecollected by

centrifugation, rinsed,

and

processed

for

electrophoresis

through

10%

acrylamide gels

inSDS.

Immunofluorescence microscopy.

Following

electropora-tion,

2 x

104

cellswere

plated

on7-mm-diameter

coverslips

in 24-well

plates.

At48 to 72 h

posttransfection,

the

mono-layers

wererinsed twice with PBS andfixedfor 15 min in4%

paraformaldehyde

in PBS. The cellswererinsed three times with PBS and

permeabilized

with 1% Triton X-100 in PBS for 3 min.

They

wererinsed four times in PBS and incubated witheitherrabbit

polyclonal

anti-Rev antiserum

(1:200

dilu-tion)

oranti-Revmousemonoclonal

antibody

(1:500 dilution)

in 0.2% bovine serum albumin in PBS for 2 to 4 h. After

being

washed

extensively,

the

monolayers

were reacted for 1 h with fluorescein-labeled goat anti-rabbit Fab

fragment

(1:500)

or

donkey

anti-mouse antiserum

(1:200)

in 0.2% bovineserumalbumin in PBS. After several washes in

PBS,

the

coverslips

wererinsed in deionized waterand mounted in MOWIOL

(Calbiochem

Corp.)

in 15%

glycerol

supple-mented with5%

(wtlvol)

DABCO

(Sigma

Chemical

Co.)

to prevent

fading.

The cellswereexamined

by

regular

interfer-ence or Nomarski

optics

and

by

dark-field immunofluores-cencewith aZeiss

Axiophot

microscope.

RESULTS

Rev/MS2

coat

protein

fusion activatesHIV-1 gag

expression

from a

hybrid

RNA

lacking

the

Rev-responsive

domain. To

investigate

whether the RRE can be

replaced by

another

RNA, the RRE sequence in the gag

expression

vector

pRMK4RRE

(23)

wasmutatedto

exchange

the64-nucleotide

Rev-responsive

domain for a modified

MS2

phage

transla-tion operator sequence of 19nucleotides

(Fig. 1,

RREZ-MS).

TheMS2 operator hadbeen modifiedtointroduce twoG-C base

pairs

to stabilize the stem

region

without

overtly

affecting

the MS2 coat

protein

binding

(6).

To direct the

binding

of Rev to RREZ-MS

RNA,

MS2

coat

protein

was

appended

to the C-terminal end of the Rev. A

single-nucleotide deletion atthe seventh codon of the MS-C

ORF,

which introduced a frameshift and

early

termination of the coat

protein

(Rev/MS-D),

served as a

negative

control. Anothermutantthatdeleted 48 residuesatthe Nterminus of

on November 9, 2019 by guest

http://jvi.asm.org/

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Rev

ex

1 Nef

H Bs SpH BgHc K

I I

lIII

I.

gYag

I

pol

I

RRE

A

B c a cu URU

a1uCa C G e u RA I 13

c

C RRE

1 244

50

RREZ-MS ...I-ZZ

31

MS+A

El

MS

+a

B/3G/MS/B2

MS

+

B/B1/B2

39

El

[image:4.612.62.555.71.418.2]

113

..._......

... ...

...

204 222

213 El

50 113

.... ...

....

50 113

m..zzi

MS

-(MS) -(MS)

FIG. 1. Schematicdiagram ofHIV-1 LTR-linkedgag expressionplasmids containing RRE and RRE-MSchimeric inserts. The basic

structureofthepHIVLTR-gag-RRE plasmid (previouslyreferredtoaspRMK4RRE)has been described before(23) and isdrawntoscale

under the linear genomicmapof the HIV-1provirusNL4-3(1).ThepolORF in theexpression plasmidwastruncatedattheIjpnI(Asp-718)

site and is denotedby pol'.Theletter codes refertoafew of the relevantrestriction sites. The 244-nucleotide RREsequencein thisvector,

indicatedby the lightly shaded rectangle,hadbeeninserted between the

YpnI

(Asp-718)site inthepolORF and theKpnIsite in thenefORF. Thepredicted secondarystructureof the RREis shown in the boxatleft, and theindividualstem-loopsarelabeled. The MSsequenceis

denotedby aheavilyshaded block with the modified MS RNAsequence (6)illustrated as astem-loopstructure in the boxat right. The

asterisksontheMSRNAstructurerefertothenucleotides critical for MS2phagecoatproteinbinding. TheMSmonomeranddimerare

denotedby singleand dualheavilyshaded boxes. Thestructuresof the different chimerasareindicatedbycombinations oflightlyandheavily

shadedboxes. The numbersbythese boxes refertothe RRE coordinates. These chimerasare morefullydescribed inMaterials and Methods.

Revnecessaryfornuclear-nucleolartargeting,RNAbinding, andoligomerizationwasalso constructed(A3-5ORev/MS-C).

Toensurethe nucleartargetingofA3-5ORev/MS-C, a-GRK

KRR- sequence, corresponding to the nuclear localization signalof Tatprotein,wasinsertedatthethirdpositionin the MS-C ORF of the above deletion mutant to generate

A3-5ORev/MS-N(Fig. 3).

HeLa cellsweretransiently cotransfected with Revorthe

respective fusion protein expression plasmids and the indi-catedgag expression vector. Aliquots of transfected cells

were taken formetabolic labeling, and the labeledextracts

wereimmunoprecipitated with anti-Rev antiserum.

Alterna-tively, total cellular extractswere electrophoresed through

SDS-polyacrylamide gels, and the fusion proteins were

detected byimmunoblotting. As shown in Fig. 2A and B, fusionproteins of the predictedsizeswereeasilydetected by immunoblotting or immunoprecipitation with the anti-Rev

antiserum. Thesubcellular distribution ofthe various fusion proteinswasinvestigated by indirectimmunofluorescence of

fixed, permeabilized cells with anti-Rev antiserum. As

ex-pected, Revwasconcentrated inthenucleoli oftransfected

cells (Fig. 2C). Although the Rev/MS-C fusion protein

ap-peared concentrated in the nuclei, the protein was also

uniformly expressed throughout the cytoplasm andthe

nu-clei ofmanytransfectants. A3-5ORev/MS-C,whichlackeda

nuclearhoming signal, accumulated exclusively in the cyto-plasm.A3-5ORev/MS-N,withtheTatnucleartargetingsignal at the third codon ofMS-C, had adiffuse nuclear

accumu-lation with some hint of condensation, as well as diffuse

cytoplasmicstaining (Fig. 2C).

gagexpressionfrom plasmids containing thewtRREwas

compared with that from plasmids containing RREZ-MS following transfection of HeLa or Cos-7 cells. In thegag

ELISA,all of the cellextractswereserially dilutedtoobtain opticaldensityvalues of between 1 and 2 for RREactivation by Rev. Under these conditions, the ELISA values for a

positiveRevresponsevariedbetween 100 and500 timesthat ofgagexpression in the absence of Rev. The cutoffvalue

B2

* U U

*A

A

G-C G-C

* A G-C U-A U-A G-C G-C

5'

3'

MS

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0 1 2 3 4

---Gomm

B 4 3 2 tO M

97

66

45

31 _

22 -

-IMMUNOPRECIPITATION

[image:5.612.118.514.72.302.2]

REV 3-50REVMS-C 3-50REViMS-N REVMS-C

FIG. 2. Eukaryotic expression and subcellular localization of Rev and selected Rev/MSfusion proteins. HeLacellsweretransfected with

therespectiveplasmids in thecontextoftransientgag expression fromanHIV-1LTR-linked, RREZ-MS-containinggag expressionplasmid.

(A) Immunoblotting. After electroporation, HeLa cellswereplatedon60-mm-diameter dishes and harvested 48to72 h later. Aone-third equivalent of cells from each dishwasused forimmunoblotting with polyclonal rabbit anti-Rev antiserum asdescribed in Materials and Methods. The results fromimmunoblotting of 12.5%polyacrylamidegelsareillustrated. Lanes: 0, notransactivator; 1, Rev; 2, Rev/MS-D;

3, A3-5ORev/MS-N; 4,Rev/MS-C. Molecular sizes (in kilodaltons)areindicatedatthe left. (B) Immunoprecipitation. Total cell lysateswere

prepared and processedforradioimmunoprecipitation with polyclonal rabbit anti-Rev antiserum. The immunoprecipitateswereresolved by SDS-polyacrylamide gel electrophoresis with 10% gels, and the radioactive proteinswerevisualized byfluorography. Lanes: M, mock transfection; 0, transfection without RevorRev/MS expression plasmids; 1, Rev; 2, A3-5ORev/MS-C; 3, A3-5ORev/MS-N; 4, Rev/MS-C.

Molecular sizes (in kilodaltons) are indicated at the left. (C) Indirect immunofluorescence assay. After DNA was electroporated, approximately2x 104HeLacellswereplatedoncoverslips in 12-or24-well plates and harvested 48to72 h later forimmunofluorescence

assay.The resultsobtained with four differentproteinsareillustrated.

(recommended by the manufacturer) for the negative

re-sponse was at ELISA readings that fell below 10% of RRE-Revvalues afterdilution. Valuesthatrangedbetween 10to 20% of those with the RRE and Revwere scored as

marginal responders. gag expression from the RREZ-MS

plasmidwasinducedbytheRev/MS-Cfusionproteinbutnot by Revorthe MS-C protein, alone orincombination (Fig. 3). TheRev/MS-Cfusionwas equallyefficacious with both

theRRE and theRREZ-MSplasmids (Fig. 3,row5).When

theMS ORF inRev/MS-Cwasinterrupted byasingle-base

deletion, causinga frameshift, theresultingfusion protein, Rev/MS-D, lost all activity with the RREZ-MS chimera while itpreservedthe activation of the RREplasmid (Fig.3,

row6). Thedeletion of 48 residuesnear the Nterminus of Revgenerated a fusion protein (A3-5ORev/MS-C) thatwas

predominantlylocalizedtothecytoplasmandwasinert with both the RRE and the RREZ-MS targets (Fig. 3, row 7). Insertion of the Tat nucleartargeting signal into A3-5ORev/ MS-C allowed the resulting mutant, A3-5ORev/MS-N, to accumulate in thenucleus but did not restore the activation for the RRE.A3-5ORev/MS-Nwasmarginallyactive with the RREZ-MS target (Fig. 3, row 8), which suggested that although A3-50Rev/MS-N most likelybound RREZ-MSvia the MS2 coat protein end, the N terminus of Rev was

requiredfor the activation oftheheterologous target. MS operator mutants with reduced in vitro coat protein affinities have corrsponding decreases in the in vivo Rev

response. To examine whether trans activation of the RREZ-MS chimerabythefusionproteinwasdictatedbythe

binding of the MS coat protein, the MS2 operator was

mutagenized to introduce changes known to reduce coat protein bindinginvitro. Deletion of the entireMSsequence

(RREZ) resulted in the lossof the Revresponse (Table 1).

Removalof thesinglebulgedAresiduein thestem structure of theMS2 operator has been showntoreduce the in vitro bindingaffinityof thecoatprotein bygreaterthan 2 orders of magnitude (10, 40).AnRREZ-MS chimera with this deletion (RREZ-MSAA) was defective for trans activation by Rev/ MS-C (Table 1). Changes at two other positions in the -AUUA-loopsequence [RREZ-MS(U/A) andRREZ-MS(A/

C)]knowntodiminish in vitrobindingbyatleast 10-fold(10,

40) ledto amoderate decrease or no change in the in vivo

responsetothe fusionprotein (Table 1).

To demonstrate directly that the Rev/MS-C fusion

re-tained the authenticbinding properties of the Revand MS coat proteins, the Rev/MS-C protein wasoverexpressed in

E. coli as a fusion protein tagged to the C-terminal end of the MBP in the sense (MBP/Rev/MS-C) or the antisense (MBP/Rev/MS-9 orientation. Additionally, a derivative of

Rev/MS-C carrying a defective interfering mutation in theactivation domain of Rev[MBP/Rev(LE/PE)/MS-C]was

alsoexpressed andpurified. TheMBP fusionproteinswere

induced with IPTG and purified from inclusion bodies by affinity chromatography on amylose resin. Asingle major band of ca. 68 kDa was visualized in the affinity-purified fractions from the MBP/Rev/MS-C and the MBP/Rev(LE/ PE)/MS-C recombinants (Fig. 4A). The (MBP/Rev/MS-C) recombinantsynthesized anelongatedMBPtaggedattheC A

97 66 45 31 22 14

C

IMMUNOBLOT

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REV,

MS-C

and Fusion

proteins

p24 GAG Production (SD)

l

RRE RREZ-MS MS+BI3AIB1/B2 (MS) (MS)

1 |

695sREV

3 s 100 2.4 (0.6)

1.9

(1.3) 6.8 (2-6)

2

MS-N ( MS-N 1.8(0.4) 4.2(0.9) 3.7(1.9) 2.8(1.4)

35 50 6985 116

R=EV12

3

REV+MS-C ( MS-C ND

5.1 (1.8)

2.7

(1.2)

8.2

(2.3)

35 so 6985 116

4 |REV+ MS-N

(

REV)

M28

ND

4.7 (2.1)

5.1 (2.8)

6.7 (2.7)

35 50 685N 11612

5

REV/MS-C

111 (17.8)

143(19.7)

113 (12.2) 42.7 (8.7)

6 REV/MS-D

C

MS-D

54.3 (9.3)

5.7 (2.8)

6.1 (2.8)

4.3 (1.3)

7 |A3I5OREVIMS-C |Z7chZZE

ieXZ

s-ZZ 4.1

(2.3)

1.7

(0.8)

3.2

(1.8)

5.7

(2.3)

8

A3/5OREV/MS-N

|

9ZZ

lie

6.3 (2.8)

|1..a

(4.1)

27.Z (5.8)

9.1 (3.8)

FIG. 3. trans activation of HIV-1gag mRNAs containing RRE, RREZIMS, (MS)+B/3A/MS/B2, and

[(MS)(MS)]

targets to Revand

selected Rev/MS fusion proteins under transient conditions in HeLa cells. The ORFs of the various transactivators are schematically illustrated. In eachcase, the shaded square nearthe Nterminus ofRev representstheArg-rich domain requiredfor RNAbinding and nuclear-nucleolarlocalization, and therounded, shadedrectanglecorresponds totheleucine-rich activation domain. The hatchedregions flanking the RNAbinding motif representthe putative oligomerization domains. InMS-N,sixamino acids from thenuclear localization domain ofTathavebeeninsertedatthe thirdpositionofMS-C(6).MS-D refersto asingle-nucleotidedeletionatthe sixth codon of MS-C which resultsinaframeshift andearly termination(6).gagexpression values,normalizedfor transfectionefficiency,aretabulated with the respective standard deviations(SD) derived fromatleasteightindependentexperiments.Thepositivevaluesaredenotedbyboldtypeface, andintermediatevaluesareunderlined.Rev-dependentgagexpressionfroma wtRRE-containing plasmidisarbitrarily assignedavalue of 100. Theresults of thisandall othersubsequentexperimentswerealsoduplicatedwith Cos-7 cells. ND,notdone.

terminusby 12residuesencodedbytheantisense strandof

the Rev/MS-Cgene. Themajor 68-kDa band inthe lysates

from theMBP/Rev/MS-CandMBP/Rev(LE/PE)/MS-C cells

[image:6.612.63.560.75.363.2]

immunoreacted with murine monoclonal anti-Rev antibody

TABLE 1. Invivoresponsesof mutationsinthe MSsequenceof RREZ-MStoRevandRev/MS-C

p24019production(± SD) relativetoRREand

RRE orRRE-MS Rev in the presence of:

RNAchimerae

No transactivator Rev Rev/MS-C

wtRRE 2.2(1.5) 100 111(17.8)

ERR(reverse RRE) 1.8(1.1) 3(1.3) 2(1.4)

RREZ 1.8(1.4) 3.1(1.7) 6.3(2.7)

RREZ-MS 4.8(2.4) 2.8(1.4) 143(19.7) RREZ-MSAA 3.7(1.8) 3.7(1.8) 12.3(6.8) RREZ-MS(U/A) 7.8(3.2) 1.8(0.9) 49(7.8) RREZ-MS(A/C) 10.7(4.3) 4.7(1.3) 87(14.6)

a In addition tothe deletion of theentire MSsequence(RREZ), threepoint

mutationswereintroduced in theMS stem-loopsequenceinthe context of the

RREZ-MS target: (i) deletion of the single bulged A in the MS stem

(RREZ-MSA&A), (ii) substitution ofAforUin the -AUUA-loop sequence

[RREZ-MS(U/A)], and (iii) substitution of Cfor A in the -AUUA- loop sequence[RREZ-MS(A/C)].NormalizedGAG-expression valuesareshown.

(Fig. 4B).Thefaster-migrating immunoreactive bandsinFig.

4B probably represent a protein

breakdown product(s).

Whenthe

purified MBP

fusion

proteins

wereincubated with

[a32P]UMP-labeled

RRE or RREZ-MS

RNA,

ribonucleo-protein (RNP) complexeswereformed only with theMBP/ Rev/MS-C protein and not with the fusion containing the

antisense Rev/MS-C(Fig. 4C). Under the sameconditions, MBP/Rev(LE/PE)/MS-C fusion protein bound both RRE andRREZ-MSRNAs,whilepurified RevboundonlyRRE RNA (data not shown). When purified

MBP/Rev/MS-C

protein was incubated with RREZ, RREZ-MS, or RREZ-MSAARNAs,

RNP

complexeswere formed onlywith the

RREZ-MS RNA and not with the RREZ or the

RREZ-MSAA chimera withadeletedbulgedAresidue(Fig. 4D).

Definition ofthe minimalRRE/MSchimera that is respon-sive toRev/MS-Cfusionprotein in vivo. To examine whether the RRE sequences in the RREZ-MS target contribute to

activationby Rev/MS-Cfusionprotein(by recruitingcellular

factors, etc.), the stem-loops C, D, and E

and

the central

loopof the RRE were excised from RREZ-MS to generate

(MS)+A

and

(MS)+a. In (MS)+A, RREstem Asequences

correspondingtopositions31 to50 andpositions 204 to 222 were

placed

5' and 3', respectively, of the MS sequence.

RREsequences between positions 39 and 50 and between 1.VIROL.

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>ux

A~~~~~~~~~~>(

~ ~ ~ ~ ~ ~

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V) Uln -_

-A :;> E >1 m

cc cc X4

116 _m

97 _

66 _ .

45 _"

um

U

li c; a

L~2

;I tm

B 2 i

cc --j

MBP 66 _

PARAMYO

-MBP 45

31

22

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4 t

;

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RREZ-MS RNA

D

0510 025 10

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4-h*

+-"I

i.

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02 510

TABLE 2. Minimalheterologous RNA recognized by Rev/MS-C in vivo

p241a production (- SD)relative to RRE and RRE or RRE-MS Rev in the presenceof":

RNAchimerae

Notransactivator Rev Rev/MS-C

wt RRE 2.0 (0.4) 100 111 (17.8)

ERR(reverse RRE) 1(0.7) 3(1.3) 2(1.4)

RREZ-MS 2.3 (0.9) 2.8 (1.4) 143 (19.7)

(MS)+A 2.8 (1.5) 1.8(0.6) 32.8 (6.8)

(MS)+a 0.8(0.5) 0.8(0.6) 45.8(9.8)

(MS) 4.8(1.9) 3.7(1.3) 17.1(6.2)

(SM) 5.1(1.3) NDc 4.1(2.2)

MSAA) 6.1(4.4) 3.7(1.3) 3.1(1.2)

(MS)(MS) 9.1(5.4) 3.9(2.2) 42.7(8.7)

(MS)(SM) 8.4(4.7) ND 29.7(9.2)

aThreetypesof minimaltargets were analyzed: (i) MS sequence flanked by

the 5' and 3' strands ofstem Aof RRE[(MS)+Aand(MS)+ainFig.1], (ii)

complete exchange of theRREwiththe MS sequence in the sense (MS) or

antisense (SM) orientationorwithanMSmutantwhichdeleted thesingle

bulgedA residue(MSAA), and (iii)ahead-to-tail [(MS)(MS)]or head-to-head

[(MS)(SM)]dimer oftheMSsequence.

bgagexpressionfromthe respectivetargets in response to Rev or Rev/

MS-Corthat in theirabsence is tabulatedas described in the legend to Fig. 3. cND, notdone.

RREZRNA RREZ-

RREZ-MS RNA MS. A RNA

FIG. 4. Characterizationof Rev/MS fusion proteins expressed in E. coli. Rev/MS-C and Rev(LE/PE)/MS-Cproteins were expressed in E. coli as MBP fusion proteins and purified as described in Materials and Methods. (A) Coomassieblue staining of the respec-tive fusion proteins purified by affinity chromatography and resolved by SDS-polyacrylamide gel electrophoresis with12% gels. Results obtained with Rev/MS-C fusedto MBPin the sense and antisense (underlined by arrow) orientations and MBP/Rev(LE/PE)/MS-C fusionareshown. LE- PEreferstothe substitution ofleucine for proline in the -LE- sequence in the activation domain of Rev. Molecular mass markerswere run on the left lane, and sizes (in kilodaltons) are indicated. Purified MBP and paramyosin-MBP fusionproteinswereobtainedcommerciallyandelectrophoresed as indicated on the right. (B) Immunoblot of the respective fusion proteins. Inclusion bodies fromE. coli expressing the variousfusion proteinsweresolubilizedand electrophoresedon12% gelsin SDS. Theproteinswereelectroblotted and reactedwithmurine anti-Rev monoclonal antibodies. Immunoreactive bandswerevisualized bya commercial chemiluminescence protocol (Amersham Corp.). The faster-migrating band probablyrepresentsabreakdownproduct(s). (C) Gel mobility shift analysis ofRNAbinding by Rev/MS-C fusion proteins.Theaffinity-purified fusion proteins described for panelA wereincubated with[a-32P]UMP-labeled RRE RNA orRREZ-MS RNA, and the formation ofRNA-protein complexes (RNPs) was evaluated by polyacrylamide gel electrophoresis as described in Materials and Methods.Lanes: 1and4, control lanes withoutE.coli proteins; 2 and 5, results obtained with MBP/Rev/MS-C fusion protein; 3 and 6, MBP ORF fused to the antisense strand of Rev/MS-C. Thearrowsdenotethe RNPs. Theslow-migratingband in lane 3represents aconformational isomer of theRREandnotthe RNP. (D) Sequence-specific binding by MBP/Rev/MS-C fusion protein. Increasing concentrations (top) of affinity-purified MBP/ Rev/MS-C proteinwereincubatedwithconstantmolarequivalents of [a-32P]UMP-labeled RREZ, RREZ-MS, and RREZ-MSAA RNAs, and theRNPcomplexeswereanalyzed bypolyacrylamide gelelectrophoresis.

positions 204 and 213 flanked the MS sequence in (MS)+a (Fig. 1). Both(MS)+A and(MS)+aweretrans-activatedat levels somewhat lower than the RREZ-MS activation(Table 2). As expected, neither chimeraresponded to Rev alone. This led us topursue whetherMS sequence alone may be sufficienttotargetthefusionproteinin vivo.Arecombinant

that completely exchanged the RRE for the MS sequence

(MS) was poorly activated by Rev/MS-C, while the MS sequence inreverseorientation (SM) and the MS sequence with the deleted Abulge (MSAA) were not responsive to Rev orRev/MS-C(Table 2).However,when a head-to-tail dimer

[(MS)(MS)] linked by a 6-nucleotide palindromic Asp-718

site (-GGTACC-) was inserted in place of the RRE, the resulting RNA had a modest response compared with that of RREZ-MS(Table 2). Ahead-to-head dimer[(MS)(SM)]was less efficientfor the Rev/MS-C-dependent response (Table 2). These resultsimplythat the MS operator sequence alone is sufficient to tether the fusion protein if the operator structureisexposed for protein binding.

Invivo responsesofthe bivalent RNAs correlate with their predicted binding affinities for Rev and MS-C. Rev/MS-C trans-activatedboth RRE and RREZ-MSRNA,presumably

by its abilitytobindthe RREby the N-terminal end of Rev and the MS operatorby thecoatprotein moiety. An RNA moleculecontainingboth ofthesetargets maybeinactivated at one orboth ofthe sites ofprotein binding and used to uncouple the bindingand theactivationeventsforthe fusion

protein.Twobivalentchimerasweredesigned:(i) B/3G/MS/ B2, whichreplaced theB1stem-loopin the

Rev-responsive

B/3G/B1/B2RREsubdomainwith the operator sequence but leftthecriticalthree-Gsequenceintact, and (ii)

(MS)+B/B1/

B2, with tandemly placed MS and B/B1/B2 sequence ele-ments(Fig. 1). Neither of theseplasmids expressedgagin

the absence of Rev or Rev/MS-C. B/3G/MS/B2 was acti-vated by Rev/MS-C at 40% of the levels observed with

RREZ-MS

(Table 3).

Inthe presenceofRev,gag

expression

fromB/3G/MS/B2wasabout24% of thatfroman

equivalent

RRE plasmid (Table 3). Arecombinant that had

tandemly

placedMS and B/B1/B2 targets

[(MS)+B/B1/B2]

was acti-vatedby bothRevandRev/MS-Ctolevels

exceeding

those

obtained with the RRE and Rev or

RRE/MS-C

and

Rev/

MS-C,respectively(Table

3).

The above bivalent chimerasweremutatedtorestrict their

targeting for the Rev or MS-C component of the fusion

protein. When the three G's in the

Rev-responsive

subdo-mainwerereplaced bythreeA's,Revwas no

longer

ableto

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TABLE 3. In vivo Rev responses ofwtandmutant

bivalent chimeras

p240agproduction (± SD) relativetoRRE and

RRE or RRE-MS RNA Rev in the presenceofb: chimerasa

Notransactivator Rev Rev/MS-C

wtRRE 2.0(0.4) 100 111(17.8)

RREZ-MS 2.3(0.9) 2.8(1.4) 143(19.7)

B/3G/MS/B2 7.8(3.8) 24(6.5) 59.7(11.3) B/3A/MS/B2 7.8(3.8) 5.1(2.5) 43.7(9.3) B/3G/MSAA/B2 5.2(2.2) 22(8.2) 37.7(8.3) (MS)+B/3G/B1/B2 4.3(1.2) 137(21.8) 173(18.4) (MS)+B/3A/B1/B2 5.5(3.2) 7.2(1.8) 94.3(12.4) (MSAA)+B/3G/B1/B2 3.8 (1.8) 96.7(9.8) 87.3(6.7) (MSAA)+B/3A/B1/B2 6.3 (2.8) 2.7(0.8) 9.3(4.9)

aTwoplasmids containingtargets forbothRevandthecoatproteinwere

used.In B/3G/MS/B2,the MSsequence replacedtheBi stem-loop in the

Rev-responsiveRREsubdomainB/B1/B2butleft the three G's ofthe RRE

intact.In(MS)+B/B1/B2, the MS sequencewaslinkedtothe downstream

Rev-responsiveB/B1/B2subdomain of theRREbythree A's.Thechimeras

weremutagenizedtoreplace the three G's intheRev-responsivesubdomain

with three A's,todelete thesinglebulgedAintheMSoperator, or todo both.

bgagexpressionvalues in thepresenceof RevorRev/MS-Corthose in

their absenceareshown.

activate theresultingchimera (Table 3).Thechangeof three G'stothreeA'sin thecontextof the full RRE haspreviously

beendemonstratedtolose the Rev response withoutovertly affectingthefolding dynamicsof the RREstructure(23, 24).

The three-G-to-three-Atransition mutantresulted onlyin a slight to moderate diminution in the activation responseto Rev/MS-C(Table3), since thesemutants retainthebinding

potential for MS-C. In contrast,amutationwhichdeletes the

bulgedAresidue maylosecoatproteinbindingbutcanstill beboundbyRev. Accordingly, deletion of thebulgedAin MS induced only a modest diminution of the in vivo re-sponse toboth Rev and Rev/MS-C (Table 3). Mutatingthe

(MS)+B/B1/B2RNAatboth the three G's and the Abulgein the MS operator causeda severeimpairmentof activationby Rev/MS-C andacomplete loss of the Rev response (Table 3).

A comparison of the in vivo responses of [(MS)(MS)],

(MS)+B/3A/MS/B2,

and RREZ-MS to Rev and combina-tions of the Rev and Rev/MS fusionproteinsis shown inFig.

3. All three chimeras lack Rev recognition sites and are exclusively activatedby theRev/MS-C fusionprotein (Fig.

3, row 5) but not by Rev, MS-C, or MS-N, alone or in combination(Fig.3,rows1to4).All threeunivalenttargets also failed to respond to the Rev/MS-D fusion protein containingaprematurely terminated MScoatprotein(Fig.3, row6). The differential responses of the three chimeras to

Rev/MS-C probably reflect the intrinsic stability of the MS RNA secondary structure in the various molecules rather than the requirements for additional sequence to facilitate

protein binding. Therefore, ifthe RNA can bind the fusion protein, Rev moiety will activate that RNA. A large deletion at the Nterminus of Rev in the fusion protein (A3-5ORev/ MS-C)abolished trans activation(Fig. 3, row 7), most likely because of the loss of nuclear targeting (Fig. 2C). Insertion ofthe HIV-1 Tat nuclear homing signal in the coat protein

moiety

ofthe above deletion mutant generated

A3-50Rev/

MS-N,whichwasdistributed in both the nuclear and

cyto-plasmic compartments (Fig. 2C). However, with the

A3-5ORev/MS-N

protein, onlyafraction of the trans-activation

potentialofRev/MS-Cwasrealized(Fig.3, row 8), implying that foroptimaltransactivation of evenaheterologous RNA

TABLE 4. Repressionof MSoperatoractivationbyMS-N coatprotein'

p2401 productionb

Type oftransfection

- MS-N + MS-N

RRE+ Rev 100 89

RRE + Rev/MS-C 117 84

RREZ-MS + Rev/MS-C 135 23

[(MS)+B/3G/B1/B2] + Rev/MS-C 144 87

[(MSAA)+B/3G/B1/B2] + Rev/MS-C 87 73

[(MS)+B/3A/B1/B2] + Rev/MS-C 82 17

aAnMS-Nexpressionplasmidwascotransfected intoHeLacells withRev

orRev/MS-C transactivatorplasmidswith therespectivegagtargets.Ineach

experiment,theMS-Nplasmidwastransfectedat asixfoldmolarexcess over

Rev orRev/MS-CDNA.

bNormalized gagexpressionvaluesareshown. Dataareaveragevalues

from two orthreeexperiments.

target,thestructural

integrity

of the N terminus of Rev has tobepreserved.

Coatproteininhibits thetransactivation of theMSoperator by Rev/MS-C. To examine whether the MS operatorwas boundby the coat

protein

in

vivo,

we cotransfectedHeLa cells with the MS-Nexpression plasmidunder conditions of trans activation

by

Revor

Rev/MS-C.

MS-N

protein

con-tained the HIV-1 Tat nuclear localization

signals

and was distributedin both the nuclear and the

cytoplasmic

compart-ments. MS-N would be expectedto complete

directly

with the fusionproteinfor the targets

containing

the MS operator butnotfor the

Rev-responsive

target. Asshownin Table

4,

asixfold molar excess of MS-Nover

Rev/MS-C

induced a sevenfold decrease in the trans activationof RREZ-MS

by

the Rev/MS-C fusionprotein; activation of the RRE either

by Rev or by the fusion

protein

was not

significantly

af-fected. Activation of the bivalent RNA and the mutant chimera restricted to Rev

binding

was alsonot

significantly

influenced by MS-N coexpression

(Table 4).

In contrast,

MS-N caused a fivefold reduction in Rev/MS-C-dependent

gagexpression from achimera containingthe three-A mu-tation in the Rev-responsivemotif

(Table

4).

Activationby the fusion protein requires the Rev activation domain. Genetic studies have identified an Leu-rich motif near the C terminus of Rev as the activation domain. Mutations inthis domain were not only inactive fortrans activation but alsodominantlyinterfered with the

function-ingofwtRev (31, 34).Toexaminewhethertransactivation

of the heterologous RNAbythe fusion protein was

depen-dent on the

integrity

of the activation domainof Rev, we designedtwosubstitutionmutantswith

changes

in the Leu-rich motifof Rev inthecontextofthefusionprotein. Both

Rev(78-79LE/EE)/MS-C

and Rev(78-79LE/PE)/MS-C pro-teinswereinactive for gagexpression from RREplasmids;

theyweremarginallyactive(10to20% of the levelsobtained with Rev/MS-C) on the RREZ/MS-C plasmid (data not shown). However, when increasing amounts of either mu-tant werecotransfected with thewtRev/MS-C, therewas a dose-dependent repression oftrans activation (Fig. 5). Un-der similarconditions, cotransfection ofamolar excess of

A3-5ORev/MS-C was without an inhibitory effect (data not

shown).Themutant(-LE-to-PP-)wasmoreinhibitorythan the change (-LE- to -EE-). trans activation of the RREor

(MS)+B/3A/B1/B2

containinggagplasmids bythewtfusion

protein

was repressed more efficiently (90% inhibition at a fivefoldmolarexcess) bythesameLeumotifmutants(data not shown). These results, which parallel the previously J. VIROL.

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Cn

z

4L

CD

0 3 6 9 12 15

MOLAR EXCESS of COMPETITOR OVER REV/MS-C

FIG. 5. Effects of dominant interfering mutations near the C

terminus of Rev on trans activation by Rev/MS-C. Increasing

amountsof RSV LTR-linkedRev/MS-C proteinswith substitutions

at the 78 and 79 residues of the RevORFwere cotransfected with fixed amounts ofRev/MS-C and RREZ-MSgagplasmid. Normal-ized values ofp24,a' production arepresentedas afunction of the

competitor concentration. Conditions are as described for other

experiments.

reported behavior of activation domain mutants of Rev on

the RRE (31, 34), imply common biochemical pathways of activation for both Rev and Rev/MS-Cfusion proteins.

DISCUSSION

We have demonstrated that direct interactionof Rev with the RRE RNA is not an absolute requirement for trans

activation. Rev will activate a heterologous RNA if it is directed to it by another RNA binding protein. When the Rev-binding domain of the RRE was replaced bythe MS2 phageoperatorsequence(RREZ-MS), Rev/MScoatprotein fusion activated therespectivegagmRNA at levels slightly greater than those for the corresponding activation of the RRE by Rev. Mutations in the MS operator known to abolish or reduce in vitro binding by the coat protein demonstratedacorrespondingloss of in vivoresponseto the fusionprotein. Thispoint was directlyconfirmed by the in vitrobinding preferences of thepurified fusionproteinwith the various RNAs. The purified fusion protein bound the RRE and RREZ-MS RNAs but not the RREZ or RREZ-MSAARNAs.

Althoughthe RREZ-MS chimerawas fullyfunctional for the Rev response, RNA with a complete exchange of the

RRE with the MS sequence was activated by the fusion

protein to less than 20% that of RRE activation by Rev.

Activation of the MS stemwas slightly improved when it

was extendedtoincludethesequence correspondingto the

RREstemA. This improvementwasnot attributable to the

sequence content of the RRE, since similar enhancement

was also observed by lengthening the MS stem by an

unrelated sequence (data not shown). A head-to-tail or a

head-to-head MS dimer also showed a modest increase in

response over the MS monomer response to the fusion

protein.A deletionmutantof the RRE(RREZ)that removed

the

Rev-responsive

domain

(B/B1/B2)

wasactivated neither

by

Rev nor

by

the fusion

protein.

Therefore, the MS sequence alone is sufficient to tether the fusion protein

during

trans activation if this sequence forms a stable

secondary

structurein thecontextof the rather

large

HIV-1

gag-polmRNA,asinthecasesof RREZ-MS

and,

to alesser

degree,

theotherminimalMSchimeras.

The fusion

protein

was

equally

effective

against

both the RREandthe RREZ-MStargets.Cotransfection ofRREand

RREZ-MS

expression plasmids

resulted in an additive re-sponse when the fusion

protein

was not

limiting (data

not

shown).

Direct

proof

for the dual-RNA attachment

potential

of the fusion

protein

was obtained witha bivalent chimera that contained the MS operator and the RREsubdomain in tandem

[(MS)+B/3G/B1/B2].

As summarized in

Fig. 6,

the

bivalent targetwas activated

by

both Rev and

Rev/MS-C.

Substitution of the three G's

by

three A's in the

Rev-responsive subdomain selectively abolishedthe responseto Rev but not that to

Rev/MS-C.

When the coat

protein

sequencein

Rev/MS-C

was

truncated,

the

resulting

mutant,

Rev/MS-D,

failed to activatethe

MS2-only

targets,

empha-sizing

the

requirement

of the coat

protein

end for RNA

binding.

Invivo

binding

of the fusion

protein

to MS2RNA was

indirectly

demonstrated

by

competition experiments

with excess

quantities

of the coat

protein,

MS-N. MS-N

inhibited the activationof

operator-only

targets

by

thefusion

protein

but hadnoeffectonRREactivation.

Thus,

the RNA

binding

andactivation

properties

of Rev may be

structurally

and

functionally

dissociatedwithout the

fidelity

of the acti-vation process

being

lost.

The invivo reactivitiesof the bivalentchimerasreiterated that RNA attachment

by

either

binding

protein

is

highly

sequence

specific. Although

sequence-specific

interactions

of MS coat

protein

with the MS operator have been well

documented,

the situation

regarding RRE/Rev

interactions

hasbeen

equivocal.

Recent reportshave in factclaimed that

Rev

binding

of the RRE may be

specified

not

by

a

unique

sequence in the

B/B1/B2

RREsubdomainbut rather

by

the

unusual distortions in the sugar

phosphate

backbone

im-posed by

noncanonical

base-pairing

schemes

(3, 18,

28).

We havedemonstrated

previously

that Rev

binding

and the Rev response are

preserved

in mutants that

exchanged

the

Bi

stem-loop

for a

perfectly

Watson-Crick

base-paired

MS2

helix

loop

both with Rev

(24)

and with

Rev/MS-C

fusion

proteins.

The loss of both the in vitro and in vivo Rev responses as aconsequence of the three G's

being

mutated upstream of the MS sequence in theseconstructsargues in favor of

sequence-specific binding

of Rev to the

B/B1

stem-loop

region,

both in its native context and as the

Rev/MS-C

fusion

protein.

Severalstudies have

implied

thatfor

optimal

activation

by

Rev,hostcellular factors

binding

toRRE

RNA, Rev,

orboth may be

required.

Several RRE RNA

binding

nuclear pro-teins were demonstrable

by

a UV

cross-linking

procedure

(36a), including

the

previously reported

56-kDa RBF

protein

(44).

The case for the

RRE-binding

Rev

helper

factors is

supported by

the

inability

ofcertain RREmutantsthat bind Rev to be trans-activated

by

it

(23)

and

by

the restricted

tropism

of the Rev response

(43).

Certain RREmutantsthat bound Rev

poorly

andwerenotRev

responsive

in vivo still

facilitatedgag

expression

inthe absence of

Rev,

implying

the presenceofacellular

surrogate(s)

for Rev

(24).

Our best non-RREconstructswere rescued

by

the

Rev/MS-C

fusion

protein

to

only

about 42% of the RRE

by Rev,

suggesting

that RRE sequence

recognition by

cellularfactors may also

improve

activation

by

the fusion

protein.

However,

our

1%

1%1.

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[image:9.612.64.303.75.283.2]

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[image:10.612.91.521.73.296.2]

MS/B/3G/B1 /B2

MS/B/3A/B1 /B2

MSAA/B/3G/B1 /B2

MSAA/B/3A/B1

/B2

FIG. 6. Schematicrepresentation ofRNAmotifsrecognized byRev and MScoatproteins.Achimeralinkingthe MSoperator sequence totheRev-responsiveB/B1/B2subdomain oftheRREwasmaximallyresponsivetobothRevandRev/MS-C.The cognate targetsforthe MS-C and Revproteinsaredenotedbythelightlyandheavilyshadedareas,respectively.Theasterisksrefertothechangeof threeG'sto threeA's intheRREsubdomainandtothe deletion that removed thebulgedAinthe MSoperator.The relativeRev responsesofthevarious mutants areshown.

substitution mutation of three G'stothree A's

(3G/3A)

inthe bivalent chimerawas notactivatedbyRevortheRev/MS-D

mutant that truncated the coat protein. Besides the MS2

RNA,the3G/3Amutantcontainedonlythe

non-Rev-respon-sive subdomain of the RRE. Furthermore, a

3G/3A

substi-tution mutant,B/3A/MS/B2, thatcontainedan evensmaller

Rev-responsive sequence (B and B2 stem-loops of the

B/B1/B2 subdomain) was also nonresponsive to Rev,

al-though not tothe fusion protein. While these observations underscore the fact that Rev binding to the target is the

primary event during activation, it is still possible that

RRE-binding cellular factors may regulate the overall effi-ciencyof the Rev response.

The amino-terminalpeptidedomain(s)of Rev is necessary for RNAbinding, nuclear-nucleolar localization, and

oligo-merizationproperties(8, 25, 27, 31).

A3-5ORev/MS-C

witha 48-amino-acid deletion at the N terminus of Rev accumu-lated preferentially in the cytoplasm and was therefore inactive with both the RRE and the MS chimeras. When the Tat nuclear localization sequence was inserted at the third codon of the MS-C ORF in the abovedeletion,theresulting

fusion protein,

A3-5ORev/MS-N,

was distributed equally

between thecytoplasm and the nucleus andpossibly in the nucleolus.

A3-5ORev/MS-N

failed to activate the RRE target andwasonly partiallyactive with the MS2 operator. Since A3-5ORev/MS-N lacks the RRE-binding site, its failure to activate the RRE is understandable. But its partial in vivo effect with the MS operator targets implies that the amino terminus of Rev may possess an additional function(s)

re-quiredfor maximal activation.BindingtoheterologousRNA targets is probably not one of these properties, since the

Rev/MS-D fusion, with anintact Rev ORF but a truncated coat proteinORF, activated only the RRE but not the MS targets. Since

A3-5ORev/MS-N

is not excluded from the

nucleus, a loss ofprotein oligomerization potential may be the critical defect in

A3-5ORev/MS-N.

However, theMScoat

protein itselfcandimerize and formhigher-orderstructures upon specific RNA binding (4, 48). It is likely that the A3-5ORev/MS-N fusion may have retained this property of theMS2coatprotein.Therefore, theA3-5ORev/MS-N dele-tionmutant mustlack yet another function encodedbythe N terminus of Rev. Even substitution mutants of Rev/MS-C

thatreplacedone or morearginineresidues in the N termi-nusof Revalso hadanegativephenotype withthe RRE and were only marginally active with the MS operator targets

(45a). Assuming that the various mutations in the Rev N terminus donotdrasticallyaffect fusionproteinfolding,we can speculateonthepossible roles of the Rev Nterminus. First,evenif RNAbindingoccursatthecoatproteinmoiety,

thefusionproteinmaystill havetooligomerize by usingthe N-terminal domain of Rev. Alternatively, activation may

require cooperative intramolecular interactionsbetween the

heterologousRNAbindingsite and theintactNterminus of

Revorintermolecular interactions between theNtermini of Revindifferent fusionproteinmolecules.Finally,an

authen-ticNterminus of Rev may berequiredforbindingof cellular factors.

In the life cycle of HIV, the Rev protein regulates the

temporal switch from the earlyregulatoryphase to the late

lytic phase (2, 13, 37,41). HIVreplicationmayberestricted atthe level of Rev from constraints of the threshold require-ments of Rev (33, 38, 39), by the requirement of species-specific cellular helper factors (43), or by both. Rev is

presumedtobea determinant of virallatencyand reactiva-tion during natural infection (38, 39) and is therefore an excellent candidate for targeted drug development. Like manyactivators, Revis a modularprotein, withthe N-ter-minal and theC-terminal halvessubservingthe RNA binding and the activation functions, respectively. Mutants with

changesin the leucine-rich activation domain bind the RRE, buttheyaredefectivefor activation anddominantly interfere with the Rev function (31), presumably by forming mixed J. VIROL.

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oligomers (26, 33). However, it is equally plausible that the activation domain interacts with specific cellular factors modulating the RNA spliceosome assembly or transnuclear RNA transport (11, 30, 31). The activation domain mutants also inhibited activation by the fusion protein, implying common biochemical pathways of activation for both Rev andRev/MS-C fusion proteins. While the activation domain is being explored as a potential target for drug development, the fusion protein system we have developed is useful for examining the various RNAs aspotential Rev decoys. Using adistant RNAbinding site in the fusion protein rather than the RRE-binding domain may uncover the in vivo RNA

bindingparameters and also identify cross talk between the heterologous RNA binding site and the Rev oligomerization domain at the N terminus. The need for an authentic

N-terminal domain of Rev for the activation of even the

heterologous targets prompts us to examine the contribu-tions of thedifferent residuesatthisdomain to the activation process. These studieswillallow us to synthesize "design-er" peptides that may interfere with the discrete steps leading to RNA binding, protein-protein interaction, and activation.

ACKNOWLEDGMENTS

We thank AliciaBuckler-White foroligonucleotide synthesis.We

aregratefultoPaulWingfield for the purifiedRevprotein expressed in E. coli.Wethank Barbara Felber ofNCI-FCRCand MarieLou Hammerskjold of SUNY, Buffalo, for anti-Rev antisera.Wethank Anthony S. Fauci, Bernard M. Moss, Damien Purcell, and Keith Peden ofNIAIDfor their criticalreviews andcomments.

This work was supported fully by a grant to S.V. from the intramural AIDS targeted anti-viral program administered by the OfficeoftheDirector, National Institutes of Health.

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