In vivo binding of wild-type and mutant human immunodeficiency virus type 1 Rev proteins: implications for function.

0022-538X/92/095569-07$02.00/0

Copyright© 1992,AmericanSocietyfor

Microbiology

In

Vivo Binding

of

Wild-Type

and

Mutant

Human

Immunodeficiency

Virus

Type

1

Rev

Proteins:

Implications for Function

SALVATOREJ.ARRIGO,1* SHAUNHEAPHY,2 ANDJULIA K. HAINES'

Department of Microbiology and Immunology, MedicalUniversity ofSouth Carolina, Charleston,

South Carolina

29425-2230,1

andDepartment of Microbiology, University ofLeicester

SchoolofMedicine, Leicester, LEI9HNEngland2 Received 14 March 1992/Accepted 12 June 1992

The Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1) is required for protein expression from theHIV-1 RNAswhich containabinding site forthe Rev protein,termed the Rev-responsive

element(RRE). This transactivatoractsbothatthe level of splicing/transportof nuclearRNAs andatthe level

oftranslation ofcytoplasmic RNAs. We useda monoclonal antibody specific fortheHIV-1 Rev protein to immunoprecipitate cellularextractsfromHIV-1-infectedand-transfectedcells. High levels of specific binding of

wild-type

Rev totheRRE-containing RNAswerefound in

cytoplasmic,

butnotnuclear,extractsfromthese

cells. A Rev mutantwhichlacked both nuclear andcytoplasmic Rev function but retained RNA bindingin vivo wasgenerated.Thisbindingwasdetectablewithboth nuclear and cytoplasmicextracts.Theseresults verify the existence ofdirectbinding of RevtoHIV-1 RNAs in vivoand

conclusively

provethat binding of Rev is not

sufficient fornuclearorcytoplasmic Rev function.The results alsosupportadirect role for Rev in thenuclear exportandtranslation ofHIV-1 RNAs.

Human immunodeficiency virus type (HIV-1) encodes a

regulatory transactivator, termed Rev, which is essential for viral replication (14, 31). The inability of the virus to replicate without Rev is in part due to a lack of viral

structural protein expression (14, 31). The Rev protein

appears to act at two levels in allowing the production of

structuralproteins.Itfunctionsatthe nuclearleveltoinhibit splicingorfacilitate the nuclearexport of HIV-1 RNAs (2, 13,16, 17, 24, 25),and itfunctionsatthecytoplasmic levelto permit the translation of HIV-1 RNAs (1, 4, 7). We have previously demonstrated that cytoplasmic accumulation of thesingly spliced Rev-responsiveelement(RRE)-containing HIV-1 RNAs encoding Vif, Vpr, Env, and Vpuwas

unaf-fectedby the presence orabsence ofRev; however, these RNAs were not translated owing to a defect in polysome

formation(1).Acis-actingelement found intheenvgeneof HIV-1,the RRE, has been shownto mediate both of these effects of Revonviral RNAs(2, 13, 16, 17, 24, 25).

The Rev protein localizes to the nucleus of transfected cells, andpurifiedRev proteinbinds with highaffinityto in vitro-synthesized RRE-containing RNA (6, 10, 15, 18, 27, 28, 30, 33). This binding is dependent on an extensive secondarystructurewithin the RRE. Site-directed mutagen-esis of the RRE has localized importantstructures involved in this binding and has shown that these structures are

importantin vivofor Rev function(6, 9-12, 19, 20, 22, 27, 29, 32). Site-directedmutagenesisof the Revprotein has

delin-eated domainsof Revimportant for RNAbinding,

multim-erization,and function(4, 21, 23, 26,28).These studies have

shown thatmutagenesisof thefunctional domain of Revcan

resultin mutants whichretain the in vitrobinding properties ofwild-typeRev butlack Rev function. These mutants have

*Corresponding author.

been shownto exerttransdominant repression ofwild-type Revprotein.

The binding of Rev to the RRE in vitro appears to

correlatewell with the function ofRev in vivo; therefore, the association of Rev withthe HIV-1RRE-containing RNAs in vivoisproposedtobeaprerequisite for Revfunction. Since Revlocalizes to and functions at the level of splicing and transport in the nucleus, Rev should initially interact with these RNAs in the nucleus. The binding of Rev to the RRE-containing RNAswould theneither inhibit the splicing

or facilitate the nuclearexportofthese RNAs. However, it is assumedthat binding of Rev isnotsufficient forfunction since transdominant mutants of Rev, which retain the in vitro binding and multimerization properties of wild-type Rev but donotretain invivofunction,have beengenerated (4, 21, 23, 27, 28). Since Rev has an effect on the transla-tional capacityof these RNAs in thecytoplasm, it seemed likely that Rev would continue its association with these RNAs afternuclear export.However,the associationof Rev with HIV-1 RNAs in vivo has notbeen established.

Using monoclonalantibodies raised against the Rev

pro-tein, we examined the in vivo association of Rev with the

HIV-1RRE-containingRNAs. We demonstratethat in HIV-1-infected and -transfected cells, Rev is found associated withhighlevels ofRRE-containingRNAs in thecytoplasm. This association isdependentonthepresenceofRev and the

RRE. The bulk of the wild-type Rev-RRE binding was

detected in the cytoplasmic, but not nuclear, fractions of

infected andtransfected cells. Resultswith anonfunctional Rev mutant, which retains RNA binding, indicate that in vivo binding of Rev is not sufficient for function. The

detection of nuclear bindingwith this mutant indicates that

thebindingof functionalRev toanRNA enhances itsexport from the nucleus. The continued association of Rev with

cytoplasmicRNAssuggeststhat Rev allowsthe association

of HIV-1RNAswith the translational machinery.

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5570 ARRIGO ET AL.

MATERIALS AND

METHODS

_RESULTS

Proviral constructs. The construction of

CSF/EBV-

and

ASal/EBV- has beenpreviouslydescribed(1).

TDBg/EBV-was generated by using polymerase chain reaction (PCR)

mutagenesis. The oligonucleotide primersTDBg andM112

were used first to amplify a PCR product containing the

desiredmutation.TheoligonucleotideprimersBamSandCR2

were used to amplify an overlapping PCR product. The

gel-purifiedPCRproductswerethenamplifiedwithBamSand M112oligonucleotideprimers.ThePCRproductwasdigested

with BamHIandXhoIand subclonedintopBluescript (Strat-agene).Thisfragmentwas thenisolated frompBluescriptand

cloned at these unique sites in the proviral construct. This

reconstructed a full-length provirus containing the desired

mutation.Thesequencesoftheseoligonucleotideprimersare

asfollows:TDBg,

5'-GCTACCACCGCTTGAGAGATCTAC

TCTFGAC-3'; M112,

5'-GCITACITTGTGATTGCTCCATG-3'; BamS,

5'-CACTCCTCAGGAGGGGATCC-3';

andCR2,

5'-CTCTCAAGCGGTGGTAGCTG-3'.

Tissue culture, infections, and electroporation. Peripheral

blood lymphocytes were prepared by using lymphocyte separation medium (Organon Teknika). These were

main-tained for3daysinRPMI 1640 mediumsupplementedwith

20% fetal calfserum (GIBCO or Whittacre) and 0.5 mg of

phytohemagglutinin(Sigma) perml.Nonadherentcellswere

infectedwith HIV-1culture supematantsinthepresenceof

20 ,ugofPolybrenepermlfor 1 to 2h.Cells werepelletedby low-speed centrifugation and resuspended in RPMI 1640

mediumsupplementedwith20% fetalcalfserumand5 U of

humanrecombinantinterleukin-2(GIBCO) perml.

The 729 B-cell line was maintained in Iscove's medium

supplementedwith 10%fetalcalfserum. Cells were electro-porated as previously described (2). The wild-type,

Rev-mutant, and RRE-mutant DNAs refertoconstructs

previ-ously described as pYKJRCSF/EBV-, ASal/EBV-, and

ABX/EBV-,respectively (1).

Cellular fractionation and immunoprecipitation. Nuclear

and cytoplasmic fractionswere prepared as previously

de-scribed(2).Nuclearfractionswerepreparedfor

immunopre-cipitation by being treated in a mini-beadbeater (Biospec

Products)with0.1-mm glassbeadsfor 2min. The

superna-tantswerecentrifugedtoremovedebris.Then 100 to200 U

ofRNasin(PromegaBiotec)wasaddedtoeachextract,and

the extractswere aliquotedintothe appropriatenumber of

tubes. All tubes contained 40 to 50

,ul

ofa 50% slurry of

protein A-Sepharose (Pharmacia) in Nonidet P-40 lysis

bufferand 5 p,gofrabbitanti-mouseimmunoglobulinG(18).

Controlimmunoprecipitationscontained 2 ,ugofanti-Act(a

cell surface antigen) or 1 ,ug of anti-albumin antibodies.

Specific immunoprecipitations of Rev-bound RNAs

con-tained 10 to 100 plof NR4/3C4.22 anti-Rev monoclonal antibodyculture supernatant (18). The sampleswere

incu-batedwithrockingat40Covernight.Theimmunoprecipitate

was preparedby low-speed centrifugation andfour washes ofthepelletwithNonidetP-40lysisbuffer.Transfectedcells

wereradiolabeled aspreviouslydescribed(1).Proteinswere

immunoprecipitated as above by using pooled serum from

patientswithAIDS (1).

RNApreparation andRNA PCRanalysis. RNAwas

pre-pared from the immunoprecipitates and

immunosuperna-tants aspreviouslydescribed (2). Yeast tRNA(40 p,g)was

added as carrier to each ofthe immunoprecipitates. RNA

PCR analysis of RNAswas performed as previously

de-scribed(3).

Rev

is bound in vivo to

high

levels of HIV-1 RNA. Since Rev

has

an

effect

at the

cytoplasmic

levelon the translation of

RRE-containing

RNAs, we

expected

that Rev

binding

to

these RNAs should

bedetectable in the

cytoplasmic

fraction

of HIV-1-infected

cells. All the

RRE-containing

RNAs

should be

capable

of

binding

Rev. These RNAs include the

gag/pol, vif,

vpr, and

envlvpu2

RNAs. In

contrast,

the

tat/rev RNA is

an

HIV-1 RNA

that does not contain the RRE

and should not bind

Rev.

Therefore,

we

initially

investigated

Rev-RRE

binding by using cytoplasmic

extracts from

phy-tohemagglutinin-stimulated

peripheral

blood

lymphocytes

infected with HIV-1.

The

cytoplasmic

extracts from two

separate infections

of

peripheral

blood

lymphocytes

were

aliquoted

and

analyzed

in

duplicate.

Monoclonal antibodies

generated

against the carboxyl terminus

of Rev were used in

the

immunoprecipitation

analysis

of these extracts. In this assay,

the antibodies

are used to

immunoprecipitate

Rev

protein,

a

fraction of which

might be bound

to RNAs

through

the RRE. If the

carboxyl terminus

of the bound Rev

protein

were

accessible to

the

antibody

and

binding

of the

antibody

did not

perturb the binding

of Rev to the

RNA,

specific

immunoprecipitation

of Rev

and the bound RNA should be

detected. RNA

prepared from the immunoprecipitate

was

analyzed

by using

a

quantitative RNA

PCR

procedure

(1-3,

5)

to determine the

specific

immunoprecipitation

of

RRE-containing

RNAs

(Fig.

1A).

High

levels of the

RRE-contain-ing vif

and

vpr

HIV-1 RNAs

werefound in the

immunopre-cipitate

only in the

presence ofanti-Rev antibodies and not

with the control antibodies.

Therefore,

it

appeared that

the

anti-Rev monoclonal antibodies

were able to

recognize

Rev

bound to

RRE-containing

RNAs in the

cytoplasm

of infected

cells. The

binding of the antibodies

did not

perturb

the

association of Rev with the

RRE-containing

RNAs.

To determine the

specificity

of the

immunoprecipitation

and to determine the level of HIV-1

RNAs associated with

Rev,

we

used anti-Rev antibodies in another

experiment

to

immunoprecipitate

a

cytoplasmic

extract

of

an

HIV-1-in-fected

peripheral

blood

lymphocyte culture.

In this

experi-ment, RNA

was

prepared

from both

immunoprecipitate

and

immunosupernatant

fractions. The

amount

of total

RNA

prepared

from each fraction

was

analyzed by

agarose

gel

electrophoresis

(Fig.

1B). Although

high levels of

28S and

18S rRNAs could be detected in the

immunosupernatant,

no

rRNAs

were

detected in the

immunoprecipitate.

An

HIV-1

RNA which does

not

contain the

RRE,

tat/rev,

was

exam-ined

by

quantitative

RNA PCR

(Fig.

1B). This RNA

was

almost undetectable in the

immunoprecipitate

and was

present

at

extremely

high

levels in the

immunosupernatant.

We estimate that less than 1% of this

RNA

was

nonspecifi-cally

immunoprecipitated

in this

experiment.

In

contrast,

analysis

of

RRE-containing

RNAs

demonstrated

that

high

levels

(10

to

50%)

of

vpr,

env/vpu2, and full-length gag/pol

RNAs

were

specifically immunoprecipitated

in

this

experi-ment.

Thus, binding

of Rev

to

RNAs

appeared to be

specific

for RNAs which contained the RRE. These

results

demon-strated that Rev remains associated with

ahigh proportion of

the

RRE-containing

RNAs after

transport of these

RNAs

from the nucleus

to

the

cytoplasm.

Thus these

results

indicated that the

binding

of

Rev to

these

RNAs was a

prolonged

association

and not

merely

a transient

event,

restricted

to

the nucleus.

If the antibodies

were

binding

toRev

which

was bound to

the

RRE,

the

immunoprecipitation

of RNAs should

be

specific

for

RRE-containing

RNAs

andshould occur onlyin J. VIROL.

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IN VIVO Rev BINDING 5571

A INF 1 INF2 NF 1 INF2

-- vpr

-_N-- vif

B >

E-.i-- 28SrRNA

-..-W--18S

_tRNA

rRNA

i

-**2- ---tat/rev

v.1

"O ....-* p

u.REV (.ACT

C: C: C

C: c: a:< a:CC

a ft- - - -w --*-

tat/rev

- - - - *

4o.

* _

-m-a

gag/pol

*0S

-* ienvlvpu2

-.s-gagpol

CLUn

a

FIG. 1. Detection of Rev bound to HIV-1 RNAs in vivo. (A) A total of 3 x

i07

peripheral blood lymphocytes stimulated for 3 days with phytohemagglutinin were infected with HIV-lJRcsF (7 ,ug of p24). Twoidentical infections (INF 1 and INF 2) were performed. At day 2 postinfection, the cells were harvested and cytoplasmic extracts were prepared. Immunoprecipitations were performed in duplicate with either anti-Rev or control (anti-ACT) antibodies. RNA was prepared from the immunoprecipitates, and RNA PCR analysis was performed with oligonucleotide primers specific for HIV-1 vif and vpr RNAs as previously described (3). (B) Infections, harvests, and immunoprecipitations were performed essentially as inpanel A. RNA was prepared from both the immunosupernatant (IMMSUP) and the immunoprecipitate (IMMPPT). Of the recovered RNA,

15%o

was analyzed on a 1%agarose gel stained with ethidium bromide. RNA PCR analysis of HIV-1 RNAs was performed as in panel A. RNA standards (STDS) were made by sequential dilutions ofRNA from transfected or infected cells.

thepresence of both an intact RRE and Rev. To determine whether these criteria were met, wetransfecteda B-lympho-blastoid cell line with a wild-type proviral construct, an RRE mutant, and a Rev mutant. The RRE mutant has been previously characterized in terms of its ability to still pro-duce a functional Revprotein andtruncated RNA species, deletedfor theRRE(1,2).The Rev mutant is a truncation of Rev whichremoves the activation domain and the antibody recognition site. Ourprevious resultshaveshownthat in this lymphoid cell type, the level of cytoplasmic HIV-1 full-length

gag/pol

RNA was decreased with both of these mutants (1, 2). Inaddition, thelevelof tat/rev RNA, which doesnotcontainthe RRE, was concomitantly increasedwith themutants; however, thelevels of theRRE-containing vif, vpr, and env/vpu2 RNAs were notaffectedbytheabsence of Rev or the RRE (1). Therefore we expected that, in this experiment, theseprevious results should be reflected inthe amount of nonspecific immunoprecipitation, since nonspe-cific immunoprecipitation should be independent of the presence of Rev or the RRE and constant within a given experiment. Cytoplasmic extracts from these cells were prepared and immunoprecipitated with anti-Rev antibodies or control antibodies. RNAextracted from the immunopre-cipitates was subjected to RNA PCR analysis (Fig. 2). Analysis of tat/rev RNA, which doesnot contain the RRE,

- - - w * -- < vif

- - -*

-- I---

--*-env/vpu2

a: Z cnco

i: < 06

I

FIG. 2. Detection of specificRevbinding requires Rev, the RRE, and anti-Revmonoclonal antibodies. Atotal of2 x 107cellsof a B-lymphoblastoid cell line (line 729)wereelectroporated with 100 ,gof the indicated DNA. At 2daysposttransfection, cytoplasmic extractswereprepared andimmunoprecipitationswereperformed essentiallyasinFig.1.Anti-human albumin antibodieswereusedas anegative control.RNA waspreparedfromtheimmunoprecipitates andanalyzedbyRNAPCRasinFig. 1.

revealed little or no difference between immunoprecipita-tions with anti-Rev or control antibodies of any of the constructswhichwere tested. Thisanalysis requiredalong exposure timetoenhance the detection of the low levels of nonspecific immunoprecipitation. As expected, the level of this RNA waselevatedcompared with thewild-type levelin all samples from the mutanttransfections. Analysisof full-lengthgag/pol RNA revealed that specific immunoprecipita-tion ofthis

RRE-containing

RNAwasdetectableonly in the transfections with the wild-type proviral construct. As

ex-pected,

the level of

full-length

gag/pol RNAwasdecreased in the mutantsamples compared with

wild-type levels.

The vifandvprRNAsbothshowedspecific immunoprecipitation only when an intact RRE and Rev were present in the transfected construct. The level of these RNAswas similar inthewild-type control

sample,

themutantcontrol samples, andthemutantanti-Rev

samples, indicating

thatthe nonspe-cific

immunoprecipitation

was

independent

of

the

presence of Rev or the RRE and the antibody used. These data

indicate

that,

in

vivo,

Rev is

specifically

bound to RNAs which containan intact RRE. The RREmutant

produces

a functionalRev

protein,

butnoincrease in

nonspecific

immu-noprecipitation

wasseenwith thismutantinthe presence of anti-Rev antibodies, eliminating the possibility of nonspe-cific interaction of Rev with RNAs.

Consequently,

the

specific

immunoprecipitation

of

RRE-containing

HIV-1 RNAs from infected-cell extracts represents a biological association of Rev with HIV-1 RNAs in vivo which is

absolutely dependent

onthe presence of Rev and the RRE. Rev is bound to RNAs in the

cytoplasm

but not in the nucleus. Since Rev exhibits an

effect

at the nuclear level

(splicing

and

transport)

and has been shownto

localize

tothe

nucleus

(2, 8, 13, 15, 16, 17, 24, 25,

30), it is generally

accepted

that Rev must

initially interact

with

the

RRE-containing

RNAs in thenucleus.

Therefore,

we

attempted

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[image:3.612.201.415.116.238.2] [image:3.612.204.411.494.618.2]

5572 ARRIGO ET AL.

IMMPPT IMMSUP

C:

e

C

E E EE

>. D )> > n >

<_.:Ec: <: _>.Er <: a:r_£2 Er

Wild-Type

Rev

74 75 76 77 78 79 80 81 82 83 84

...GlnLeuPrProLeuGluArgLeuThrLeuAsp... ...CAGCUACCACCGCUUGAGAGACUUACUCUUGAC...

i. v .e

a#

w

4

_. envvpu2

-* vif

nuci cyto nucl cyto

FIG. 3. Rev-RREbindingiscytoplasmicand not nuclear.

HIV-1-infected cells (asinFig. 1)wereharvested,and nuclear(nucl)and cytoplasmic(cyto)extractswereprepared.The fractionswerethen

immunoprecipitated, and RNAwaspreparedfrom the immunopre-cipitates (IMMPPT) and immunosupernatants (IMMSUP). This

RNAwassubjectedtoRNAPCRanalysisasinFig. 1.

detect Rev binding to RNAs in the nuclear extracts of infected cells. HIV-1 infected cells were separated into nuclear and cytoplasmic fractions and immunoprecipitated with anti-Revorcontrol antibodies. RNAwaspreparedfrom the immunoprecipitate and immunosupematant fractions andsubjectedtoRNA PCRanalysis (Fig. 3). High levelsof theRRE-containing env/vpu2andvifRNAswerespecifically immunoprecipitated from the cytoplasmic fraction of these cells.However, littleor noRevbindingwasdetectedinthe nuclear fraction. Thiswasreproduciblyobserved withcells infectedwith wild-type virus or transfected with the wild-typeproviralconstruct.These results indicated thatthebulk of the Rev-RREbinding,incells infectedortransfected with wild-type HIV-1, is in thecytoplasm.

A mutant which lacks Rev function. Owingto thenuclear localization of the Revprotein, itwasinitially expectedthat

Revbindingto RRE-containingRNAs should be detectable in the nucleus. Sincehighlevels ofcytoplasmicbindingand littleor nonuclearbindingwereobserved withthewild-type Revprotein, it seemed likelythatthis subcellular localiza-tionmightbe correlated with theinvolvement of Rev in the nuclear export of HIV-1 RNAs. If this were the case, a

nonfunctional Rev which retained RNA-binding capacity should bereadilydetectedboundto RRE-containing RNAs in the nucleus. Such a mutant would also provide direct evidence thatnuclearRevbinding exists andthatthebinding ofRevto anRNAinvivoisnotsufficient for Revfunction. Dissection ofthe Rev protein haselucidated RNA binding, proteinmultimerization, nuclearlocalization, and activation domains. Although the binding, multimerization, and local-izationdomains overlap, theactivation domainappearstobe separate. Mutants have been generated with mutations in thisdomain and showntoretainnuclearlocalizationandin vitrobindingandmultimerizationbuttobenonfunctional (4, 21, 23, 26, 28).Apoint mutant, TDBg/EBV-, in whichthe conserved leucine at amino acid81 of Rev is changedto a

serine,wasgenerated(Fig. 4).This mutation didnotalterthe aminoacidsequenceof the overlappingEnv reading frame. This leucine had been previously mutated to an alanine, abolishingRev function in fibroblast cells (26). The TDBg/ EBV- mutant preservedthe RNA-binding, protein multim-erization, nuclear localization, and antibody-binding do-mains ofthewild-type Rev protein.

TDBg Mutant

Rev

74 75 76 77 78 79 80 81 82 83 84

[image:4.612.98.274.78.228.2]

...GlnLeuProP

roLeuGluArgSerThrLeuAsp...

...CAGCUACCACCGCUUGAGAGAUCUACUCUUGAC... FIG. 4. RNAandproteinsequencesofwild-type andmutant Rev constructs. Theaminoacidsequenceandcorrespondingnucleotide sequenceofRevbetweenresidues 74 and 84 isshown. Changesin nucleotide and amino acid sequencesbetween the wild type and mutant areunderlined.

To determine whether the

TDBg/EBV-

mutant was de-fective for Rev

function,

we transfected it into

lymphoid

cells in

parallel

with

previously characterized

wild-type

and Rev mutant constructs

(1). The cells

were

radiolabeled,

and HIV-1

proteins

were

immunoprecipitated and analyzed by

polyacrylamide gel

electrophoresis. The results

are

shown

in

Fig. 5A.

The

wild-type

construct,

CSF/EBV-, produced

high

levels of

p24 Gag,

p55 Gag

precursor,

and

gpl20/160

Env. The

ASal/EBV-

Rev mutant did not

produce

detect-able

levels

of

these

proteins

but,

rather, produced

elevated levels of Nef

compared with the wild

type.

These results

are consistent with our

previously published results with

these constructs

(1). The

TDBg/EBV-

mutant

produced

a

protein

profile

indistinguishable from that of

ASal/EBV-, indicating

that the

TDBg/EBV-

mutantwas

exhibiting

a

lack of

struc-tural

protein expression

and an

increase in Nef

production

characteristic

of a

Rev-

mutant.

The

TDBg/EBV-

mutant was

also

analyzed for

cytoplas-mic

RNA

production.

Mutant and

wild-type

constructswere

transfected into

lymphoid

cells, and

cytoplasmic

RNA was

prepared

48 h

posttransfection.

This RNA

was

subjected

to

quantitative RNA PCR

analysis

to

determine the level of

specific HIV-1 RNAs (Fig.

SB). The levels of unspliced

gag/pol

RNA

produced by

both Rev mutants were

reduced

and the levels of

spliced

tat/revRNA

produced

by both Rev

mutantswere

concomitantly increased

compared

with

wild-type

levels.

The

levels

of

env/vpu2

RNA were

similar in

all constructs.

These results

were

in

agreementwith our

previ-ous

results

(1) and

demonstrated that the nuclear

exportand

splicing function of Rev

was

abolished

in

the

TDBg/EBV-mutant. Although the level of cytoplasmic

env/vpu2

RNA was

similar

to

that produced

by wild type, no Env protein was

detectable

with

this

mutant.These results demonstrated

that this

mutant was

deficient

in both the nuclear export/

splicing

andtranslation functions of Rev.

Rev binding is not sufficient for function. Although the

TDBg/EBV-

mutant produced a nonfunctional Rev, the mutantprotein

retained

the RNA-binding, protein multimer-ization, nuclear localmultimer-ization, and antibody-binding domains of the

wild-type

Revprotein. Functionally impaired mutants similar to the

TDBg/EBV-

mutant have been shown to retain the binding properties of wild-type Rev in in vitro

binding

assays; however, it has not been demonstrated whether

this binding

occursin vivo. To determine whether the mutant Rev protein was capable of binding in vivo to J.VIROL.

ON IP

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a)

CL

A

2a1

co > > tL -1

m

cc w w >- m0

0u

200i-- g

97.4

69

c c

E E

_ > : >

*-s---gaglpol

m u-0 vif

_- gpl20160

-a 0 0

-_-

env/vpu

2

_ p55

46 _ ^

_{so_}

_{- -.*-tat/rev}

30

--.

_4- Net

_-. p24

CL a

B -a

:

co > >D m

>- 0

*-gag/poi

so -4 tat/rev

o w_ _ onvlvpu2

FIG. 5. Effect of the Rev mutationon proteinandcytoplasmic RNA expression. (A) Lymphoid cells were transfected with the indicatedconstructs. At 48 hposttransfection, cellswere radiola-beled with35Stranslabel. Cytoplasmiclysateswere immunoprecip-itated with pooled serum from AIDS patients and analyzed by polyacrylamide gelelectrophoresis. (B) Lymphoidcellswere trans-fected with the indicated constructs. At 48 h posttransfection, cytoplasmicRNAwaspreparedandsubjectedtoRNA PCRanalysis for the indicated RNAs.

RRE-containing RNAs, wetransfected lymphoidcells with theTDBg/EBV- mutant. Nuclear andcytoplasmic extracts

were prepared and immunoprecipitated with anti-Rev or

control antibodies.RNAwaspreparedfrom the immunopre-cipitates and analyzed by RNA PCR for specific HIV-1 RNAs (Fig. 6). No specific immunoprecipitation oftat/rev

RNA was seen. In contrast,

gag/pol,

env/vpu2, and vif

RNAswere all specifically immunoprecipitated bythe

anti-Rev antibodies, indicating high levels of binding of the

mutant Revprotein to the RRE-containing RNAs. As

op-nuci cyto

FIG. 6. Mutant Rev is bound to HIV-1 RNAs in the nucleus. Lymphoid cells were transfected with TDBg/EBV-. At 48 h post-transfection, cellswere harvested and nuclear (nucl) and cytoplas-mic(cyto) extracts were prepared. The fractions were immunopre-cipitated, and RNA was prepared from the immunoprecipitates. The RNAwassubjected to RNA PCR analysis as in Fig. 1.

posed

to results with the wild-type protein, binding was

readily

detected in the nucleus as well as in the cytoplasm. This nuclearbinding was reproducibly seen in nuclear frac-tions from all experiments performed with this mutant.

These

results show that the binding of the mutant Rev to

RRE-containing

RNAsis both nuclear and cytoplasmic and

that this

binding is

not

sufficient

for either nuclear or

cytoplasmic

Rev

function.

DISCUSSION

Using

monoclonal

antibodies

generated against the Rev

protein,

wehave analyzed the association of Rev with HIV-1 RNAs invivo. Wehave demonstrated that these antibodies canbeusedto

specifically immunoprecipitate

HIV-1 RNAs which contain the RRE,depending on the presence of Rev, the

RRE,

and anti-Rev antibodies. These RNAs include those

encoding Gag, Pol, Vif,

Vpr, Vpu, and Env. These results

indicate

that a

high

level of the

RRE-containing

RNAs

is associated

with Rev in

cytoplasmic

extractsfrom

infected and

transfected cells and that Rev

is

capable

ofa

prolonged association

with the

RRE-containing

RNAs. We

conclude

from these

observations

that Rev is

capable

of

maintaining

its

association

with the

RRE-containing

RNAs after their transport from the nuclear to the

cytoplasmic

fraction. Since

these

cytoplasmic

RNAs are

incapable

of translationin the absenceofRev

(1),

theseresults suggesta direct rolefor Revin this process.

The detection of both nuclear and

cytoplasmic

binding

with the

TDBg/EBV-

mutanthasseveral

important

implica-tions.

First,

the

binding

ofRevto

RRE-containing

RNAsin vivoisnot

sufficient

foreither the nuclear

export/splicing

or

translational role of Rev.

Second,

the presence of nuclear

binding

with the

TDBg

mutant and its absence with wild-type Revindicatesadirect role for Rev in thenuclear

export

of HIV-1 RNAs.

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[image:5.612.385.494.77.293.2] [image:5.612.101.259.79.529.2]

5574 ARRIGO ET AL.

FIG. 7. Model forbimodal Rev function. Revwouldbe translated fromanRNA which is notdependenton Rev for itsexpression.Rev

would thenmigratetothenucleus,where it would bind toRRE-containingRNAs andpromotetheirexportfrom the nucleus. Revwould remain bound to the RNA during this process. In the cytoplasm, Rev would allow the association of the RNA with the translational

machinery.

The detectionofwild-type Revbindingin the cytoplasm, but not in the nucleus (although a large amount of RRE-containingRNAs ispresumablyavailable assubstrateinthe nucleus),of infected andtransfected cellscannotcompletely exclude a role for Rev in the inhibition of splicing of RRE-containingRNAs.However,the resultsarenotstrictly in agreement with what might be expected from a simple

blockto splicing byRev. If the roleof Revwere simplyto inhibitsplicing,onemightexpecttofindahighlevel of Rev bindingwiththe wild type in the nuclear fraction. TheRev mutantwould be expectedto have alower level ofnuclear Revbinding asaresult ofanincrease in the removal of the

RREand thebound Revproteinby splicing.It isformallya

possibilitythat Rev inhibits thesplicingofHIV-1RNAs and that these RNAs are concomitantly exported from the

nu-cleus;however, thesimplest interpretation ofthedata leads tomodel inwhich thebindingof Revto anRRE-containing

RNA in the nucleus enhances the nuclear export of that RNA (Fig. 7). In the cytoplasm, Rev would allow the interactionofthat RNA with thetranslationalmachinery. By interacting with the RRE-containing RNAs in the nucleus and continuing its associationin thecytoplasm, Rev could provide both nuclear and cytoplasmic functions. In the absence of Rev and its associated enhancement of nuclear export, complete splicing ofprecursorRNA would be

aug-mented. Intheabsence of Rev, unspliced (atareduced level)

andsingly spliced HIV-1 RNAs would still accumulate inthe cytoplasmthroughan alternate (slower)transportpathway. WithoutRev, these RNAs would be incapable ofassociating withthetranslational machinery. Thus Rev wouldbe

capa-bleofabimodal effecton nuclearexportandtranslation of RRE-containing RNAs. More research is requiredtoanalyze

the precise logistics of Rev-RRE binding and the cellular

processesinvolved.

ACKNOWLEDGMENTS

We thank A. Lowe and S. Green, Laboratory for Molecular Biology, Cambridge, England,forproductionof theantibodyNR4/ 3C4.22. Wealso thank Irvin S. Y.Chen,in whoselaboratorysome

of thebasic concepts for this studyevolved, and M. Schmidt, P. Arnaud,andK.Arrigoforhelpfuldiscussions.

S.H. is a Medical Research Council Senior AIDS Research Fellow. This workwas supported inpart by grant 001524-11-RG fromtheAmericanFoundationforAIDSResearch(toS.J.A.).

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