Function of the human immunodeficiency virus types 1 and 2 Rev proteins is dependent on their ability to interact with a structured region present in env gene mRNA.

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0022-538X/90/094428-10$02.00/0

Function of

the Human

Immunodeficiency

Virus

Types

1

and

2 Rev

Proteins

Is

Dependent

on

Their

Ability

To Interact with

a

Structured

Region

Present

in

env

Gene

mRNA

PATRICK J. DILLON, PETER NELBOCK, ANN PERKINS,AND CRAIG A. ROSEN* Department ofMolecularOncology andVirology, Roche Institute ofMolecularBiology,

RocheResearch Center,

Nutley,

New

Jersey

07110-1199 Received 19April1990/Accepted 15 June 1990

The interaction of the humanimmunodeficiency virustype 1(HIV-1)Revproteinwithastructured region inenvmRNA(the Rev-responsive element [RRE])mediatestheexportof structuralmRNAs from thenucleus tothecytoplasm. We demonstrated that unlikeHIV-1 Rev, which functions with both theHIV-1and HIV-2 RREs, HIV-2 Rev functions only with theHIV-2 RRE. Rev-RREbinding studiessuggested thatthe lackof nonreciprocal complementation stems from the inability of HIV-2 Rev to interact with HIV-1 RRE RNA. Maintenance ofRNAsecondarystructure, ratherthan the primarynucleotidesequence, appearedto be the major determinant for interaction of both HIV-1 and HIV-2 Rev with the HIV-2 RRE.Moreover,thebinding domain of the HIV-2 RRErecognized by HIV-1 Revwasdissimilar to thebinding domain of the HIV-1RRE, intermsof both secondarystructureandprimary nucleotidesequence. Our resultssupportthehypothesisthat function of HIV RevproteinsandpossiblythefunctionallysimilarRexproteinsencodedbythe human T-cell leukemia viruses (HTLVs) HTLV-I andHTLV-II is controlled by the presence ofRNA secondarystructure generated within the RRE RNA.

Regulation ofhuman immunodeficiency virus(HIV)gene

expressionisdependentonexpressionofnumerouscell- and

virus-encoded trans-acting regulatory proteins. Expression oftwoofthesevirus-encodedproteins, Tat(1, 37)and Rev (12, 36),isrequiredforreplication (7, 15, 38). The evolution-arily and structurally related HIV type 2 (HIV-2) virus displays many similarities with HIV-1, including the pres-enceoftatandrevgenes (18).

The cis-acting elements responsive to Tat and Rev are

known. Both proteins function through RNA secondary structures present in either the 5' long terminalrepeat (14, 31)or envmessage, respectively (8, 11, 13, 19, 20, 27, 32).

Although the HIV-1 and HIV-2 Tat proteins function in a

reciprocal manner through the trans-acting responsive

re-gions ofeach other(10), Rev function appears to be more

restricted. For example, whereas HIV-1 Rev can function

withastructured regionin theenvRNA(the Rev-responsive element [RRE])ofHIV-2,thereciprocalcasedoesnothold true (26). Recent studies have shown thatHIV-1 Rev inter-actswith HIV-1 RRERNA(3, 6, 9, 27-29, 39)and that this interaction is primarily determined by the presence of a

hairpin loop structure within the RRE (9, 22, 28, 29). Once bound tothe RRE, Rev protein, eitherby itselforthrough

interaction with additional cellular factors, is thought to

mediate the export of viral structural mRNAs from the nucleustothe cytoplasm.

Tounderstandthemechanism for HIV-2Revfunctionand thebasisfor its inabilitytofunction with theHIV-1 RRE,we

examined the nature of HIV-2 Rev interactions with the different RRE elements. Our results show that, like HIV-1 Rev, theHIV-2 Rev protein interacts with the HIV-2 RRE and that maintenance of secondary structure, rather than primary nucleotide sequence, is the major determinant for

thisinteraction.Inaddition,the HIV-2RRE RNAstructures

* Corresponding author.

required for HIV-1 Rev interaction closely coincide with those required for HIV-2 Rev binding yet are completely dissimilar, in terms of both structure and nucleotide

se-quence, to the HIV-1 Revbinding domainidentified within the HIV-1 RRE.

MATERIALS AND METHODS

Plasmid constructions. (i) Construction ofeucaryotic Rev expression vectors. The construction of the simian virus 40-basedvectorforexpressionofRev, BLpSVRev,hasbeen described previously (5). For expression of HIV-2 Rev, a

synthetic cDNA wasfirst derived by using the polymerase chain reaction (PCR) method (34, 35). Exons 1 and 2 of HIV-2 Revwereamplified froman

HIV-2ROD

bacteriophage

plaque. The 5' primer used for amplification of exon 1 contained a BamHI site, and the 3' primer contained an

extension ofcodingsequenceforamino acids 25to 35,with

an engineered KpnI siteatamino acid 34. The 5' primerfor

exon 2contained anengineered KpnI site atamino acid 34, and the 3' primer contained a HindIII site. PCR-amplified exons were cut, ligated, and cloned into BL-KS (Strata-gene).Theresultantplasmid, BLHIV-2Rev,wassequenced

and contained a single nucleotide difference from the

pub-lished sequence. This difference changed the aspartic acid present atamino acid66toasparagine. TheHIV-2 Revgene was cloned into a simian virus 40 expression plasmid,

BLpSV, which contains the simian virus 40 promoter and polyadenylation signals. Briefly, the HindlIl site of BLHIV-2Revwas convertedtoBglII,andafragmentcontaining the

HIV-2RevcDNA wascloned atthe XbaI-BglII sitesin the polylinker of BLpSV. The resultant plasmid, pSVHIV-2Rev, was usedfortransfections ineucaryotic cells.

(ii)Constructionof Escherichia coli Revexpressionvectors.

The E. coli Rev expression plasmid, pRC-25 synart, has beendescribedpreviously (3). Expressionof HIV-2 Rev in E. coli wasachieved through the use of PCRtomodify the HIV-2Revsequencetocontaincodonsmostfrequently used

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FUNCTION OF HIV REV PROTEINS 4429 in E. coli (P. Dillon and C. Rosen, BioTechniques, in press).

Briefly, four overlapping oligonucleotides, each 105 base pairs in length, spanning the entire Rev coding sequence were synthesized. These oligonucleotides contained codons which were derived from an E. coli high-usage codon table. Thefour oligonucleotides were amplified in a standard PCR mixture for 8 cycles. A second PCR mixture containing short flanking primers gave rise to a 320-base-pair PCR product which was cloned as a BamHI-HindIII fragment into the procaryotic expression plasmid pDS56 (4). This plasmid contains sequences upstream from the BamHI site which code for a tag sequence, Met-Arg-Gly-Ser-(His)6-Gly-Ser, which precedes in frame the amino terminus of HIV-2 Rev. The resultant plasmid, designated pDSHIV-2H6Rev, was usedfor expression of HIV-2 Rev in E. coli. In addition, an EcoRI-XbaI fragment containing the HIV-2 Rev sequence and the extra amino-terminal amino acids from pDSHIV-2H6Rev was cloned into BLpSV and the resultant plasmid, pSVHIV-2H6Rev, was assessed for function in eucaryotic cells.

Subcloning and mutagenesis of the HIV-2 and SIV RREs. The RREs for HIV-2 and simian immunodeficiency virus

(SIV) wereobtained by PCR. The HIV-2 RRE (nucleotides 7608to7973)wasamplified froman

HIV-2ROD

phage plaque and cloned as a ClaI-XbaI fragment into the Bluescript vector(Stratagene). The SIV RRE (nucleotides 8086 to 8454) wasamplified from plasmid pBK28-SIV and was also cloned

into Bluescript as a ClaI-XbaI fragment. Stem-loop

muta-tions(PD-1 through PD-21) were generated by site-directed mutagenesis (25) of the Bluescript vector containing the HIV-2 RRE, BLHIV-2RRE. 5' and 3' deletion mutations weregeneratedby PCR. A 5' PCR primer which contained a 5' T7promoter-ClaI site RRE coding sequence was

synthe-sized. The 3' primer contained an XbaI site to facilitate

cloning directly from the PCR products. Amplicons were

used forboth T7 transcription in vitro and subcloning into

the RevindicatorplasmidpIlIAR(seebelow).

Construction of Rev-responsive vectors. The plasmid

pIIIAR has been described previously (32). Briefly, this

plasmid contains the HIV-1 long terminal repeat and the gene for chloramphenicol acetyltransferase (CAT) fused to the env sequence from HIV-1. Theenvgene has aBamHI

frameshift which prevents expression of Rev. In addition,

the env sequence has been modified to contain a ClaI site

(nucleotide 7204) upstream and an XbaI site (nucleotide 7684) site downstream ofthe Rev response element. The wild-type andmutantRRE sequencesdescribed abovewere cloned into the unique ClaI-XbaI sites of the

pIlIAR

plas-mid.

Expression and purification of Rev protein from E. coli.

Expressionand purification ofHIV-1 Rev

protein

has been

previously described (3). Expression and

purification

of HIV-2 H6Rev was accomplished by

using

methods de-scribedpreviously (4, 16).

Briefly,

expression

ofHIV-2Rev

proteinwasinduced bytheadditionof1mM

isopropyl-3-D-thiogalactopyranoside (IPTG) to a

logarithmically

growing

culture. The induced E. coli were

harvested,

lysed

with

guanidine hydrochloride, and

passed

over a nickel chelate

affinity matrix; and HIV-2 H6Rev

protein

was eluted from the column. The eluant

containing

HIV-2 H6Rev was

dia-lyzed into

phosphate-buffered

saline

containing

0.5 M NaCl and then filter

concentrated

(Amicon

Corp.).

The

purified

HIV-2 H6Rev protein was

analyzed by

sodium

dodecyl

sulfate-polyacrylamide gel

electrophoresis.

In vitro transcription ofthe

Rev-responsive

elements. BL-RREplasmidswerelinearizedattheirXbaIsite and usedas

templatesfor T7 RNA polymerasein vitro. PCR T7 ampli-cons wereuseddirectly forT7transcription in vitro.The T7

transcription reactions were performed by using the

sug-gested conditions of the manufacturer (Promega Biotec, Madison, Wis.). The T7 transcripts were labeled by

incor-poration of[32P]UTP during transcription. DNA templates were digestedwithDNase, and unincorporated nucleotides were removed by using Sephadex G-50 spun columns. The

integrity ofthe labeledtranscriptswasconfirmedbyanalysis

on6% denaturing polyacrylamidegels.

Rev-RRERNAbinding assays. The conditions for assess-ment ofRev-RRE RNA interactions have been previously

described(3, 29). Briefly, Revproteinand

32P-labeled

RRE

transcripts

were incubated at 30°C for 10 min in binding

buffer (5 mM HEPES

[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic

acid]

[pH 7.0], 25 mM KCl, 2 mM

MgCI2,

3.8%glycerol)which contained 10 ,LgoftRNAas a

nonspe-cific

competitor. Following binding,

7 U ofRNase T1 was added and reactions were incubated at 37°C for 10 min.

Binding

reactionswereterminated and thenanalyzed by gel

electrophoresis

under

nondenaturing

conditions in 0.5x Tris borate-EDTA buffer. Polyacrylamide

(4%)

gels were used

for

analysis

ofHIV-1 RRE-RNAcomplex formation.HIV-2 Rev-RRE complexes were analyzed on 5% gels, which allowedfor betterresolution ofthe RNA-protein complex. Gels were fixed in 10% acetic

acid, dried,

and exposed for

autoradiography

at

-70°C.

Cell culture, transfections, and CAT assays. CHO cells

constitutively expressing

HIV-1 Tat

protein

(CHOZiptatiii)

(31) were maintained in Iscove modified Dulbecco medium

containing

antibioticsand10%fetal bovineserum. Transfec-tions were carried out

by

using

a modified DEAE-dextran and calcium

phosphate procedure (33).

Twenty-four

hours

priortotransfection,cells wereplatedat a

density

of2.5 x

105in 35-mm

(diameter)

dishes andtransfected with250 ng

ofeach

plasmid

DNA. Cellswereharvested 48 h

posttrans-fection,

and CAT assays were

performed

as

previously

described

(17).

RESULTS

Expression and

purification

of HIV-2 Rev

protein.

For

expression

of HIV-2

Rev,

a

synthetic

cDNA was

con-structed

by

using

the PCR method with

proviral HIV-2RoD

DNA (see Materials and

Methods).

The

amplified

insert

which was tailed with the

appropriate

restriction siteswas

thenclonedintoa

eucaryotic

expression

vector.The

ability

of this artificial HIV-2 Rev cDNA to

produce

a functional HIV-2 Rev

protein

in

eucaryotic

cellswasassessed

by using

a

previously

described

Rev-dependent heterologous

gene

expression

assay

(32)

whichwasmodifiedtomeasureHIV-2 Rev function.

Briefly,

HIV-1 env sequences

harboring

the

regions

thatmediatenuclear retentionofviral mRNAaswell asthe RRE are

positioned

3' tothe termination codon and

incorporated

into the 3'untranslated

portion

ofthebacterial CAT reporter gene

transcript.

Theresult of this modification is that CAT gene

expression

becomes

subject

to

regulation

by

Rev.

Expression

of

BLpSV-Rev,

encoding

the HIV-1 Rev

protein, fully

restored CAT gene

expression

directed

by

plasmid

pIlIAR,

which contains the HIV-1

Rev-responsive

sequences

(Fig.

1).

Cotransfection with

plasmid

pSVHIV-2Rev,

capable

of

expressing

the HIV-2Rev

protein,

didnot restoregene

expression

directed

by

pIlAR,

consistent with

findings

showing

that HIV-2 Rev cannot function

through

the HIV-1 RRE

(26).

However,

plasmid pIIIH2,

a

modification of

pIIIAR

in which the HIV-1 RRE was re-VOL.64, 1990

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4430 DILLON ET AL.

aAT-LJ i.t.

lXL CAT

-L

'Venj-

----A

B

Riev

_- - 1 2 - 2 - 2

*

**

*

*4

*

--...._... _ _ pl H2 pil

plJ31-111

pill AR plilH2 plllSIY

2 H6 Rev

/1

4_, i-_11

1

FIG. 1. Functionalanalysisof theHIV-1,HIV-2,and SIV RRE

elements in aRev-dependent heterologous geneexpressionassay.

The plasmid pIlIAR contains theHIV-1 RRE andis shown

sche-maticallyatthetop.TheHIV-1RRE present inplasmidpIlIARwas

replaced with the HIV-2 and SIV RRE elements to generate the plasmids, pIIIH2 and pIIISIV, respectively. (A) Transfection of CHOZip,ta,Ii wasperformed in the absence (lanes -)orpresenceof

HIV-1Rev,pSV-Rev (lanes 1),orHIV-2Rev,pSVHIV-2Rev (lanes

2).(B) Comparison of transactivation with HIV-2 H6 Rev, pSVHIV-2H6Rev(lane H6Rev)(all other lanesarethesame asin panel A).

placed with the HIV-2 RRE, wasresponsive toboth HIV-1 and HIV-2 Rev (Fig. 1). Furthermore, both HIV-1 and HIV-2 Revproteins werecapable of restoringgene

expres-siondirectedbypIIISIV in which the SIV RRE replaced the HIV-1 RRE. In addition to showing that the HIV-2 Rev proteinis functional, this result demonstrates that both the HIV-2 and SIV RREs can work within the context of the HIV-1 repressive sequences which mediate the nuclear retention of structuralmRNA thatoccurs in the absence of Revexpression.

We have previously described a strategy for expression and purification of large quantities of HIV-1 Tat and Rev protein fromE. coli(4, 16). Briefly, asynthetic Rev cDNA

was constructed by the PCR method (34, 35) to contain codonspreferentially utilized inE.coli.This cDNAwasthen cloned into an E. coli expression vector (pDS [2]), which placed anoligonucleotide encoding six histidine residues at

the 5' end of the cDNA insert (4). Following induction of HIV-2H6Revexpression by the addition of 1mMIPTGto

logarithmically growingcultures, Rev proteinwasextracted

by guanidine hydrochloride lysis of theE. coliisolatesand purified by a single-column chromatography step with a metal chelate affinity resin that has selectivity for proteins

M

1

2

3 -!4.3-

-4 HIV-1 R, v

HIV-2 Rev

FIG. 2. Purification of HIV-1 and HIV-2 RevproteinsfromE.

coli. HIV-1 Rev and HIV-2 H6 Rev proteins were purified as

described inMaterials and Methods. Proteins wereanalyzed ona

15% sodiumdodecyl sulfate-polyacrylamide gelandvisualized by

stainingwithCoomassie blue. Lanes:M,molecularweight markers; 1,HIV-2 H6 Revproteinstoredat4°Cfor 6weeks; 2,HIV-2 H6Rev

protein storedat-20°Cfor 2months; 3,purifiedHIV-1 Revprotein. kDa,Kilodaltons.

containing neighboring histidine residues (23). A relatively

purefractionofHIV-2 H6 Revproteinwasobtained(Fig. 2).

The function of the HIV-2 H6 Rev protein was first assessed in mammalian cellsasdescribed aboveby usingthe

eucaryotic expressionplasmid, pSVHIV-2H6Rev.The HIV-2 H6 Rev protein retained function (Fig. 1B), although its activity was somewhat lower than that obtained with the

unmodified Rev, which is consistent with previous results obtained with a modified HIV-1 H6 Rev protein (4). It is

unclear ifthe loweractivity of HIV-2 H6 Rev is duetothe amino-terminal modification of theproteinorlower levels of H6 Rev expressionin transfection.

Interactionof HIV-2Rev with HIV-2 RREandHIV-1RRE RNAs. Toexamine Rev-RRE RNAinteractions, theHIV-2

envgeneregion containing theputative HIV-2RRE (nucle-otides 7608 to 7973) fromHIV-2RODwasamplified by PCR

and cloned downstream ofthe T7 promoter present in the Bluescript transcription vector. 32P-labeled transcriptswere

generated in vitro with T7 RNA polymerase. Aqualitative gelshiftassay wasused inwhich RNAswereincubated with

purifiedRevprotein in the presenceofnonspecific compet-itortRNA, digestedwith RNase T1, and then analyzed on

nondenaturingpolyacrylamide gels (see Materials and Meth-ods).RRE RNAwhich formscomplexes with Revproteinis protected from nuclease digestion, whereas those RNAs which fail to bind Rev protein are digested to small frag-ments and oligonucleotides. The HIV-2 H6 Rev protein boundtoboth theHIV-2 RRE RNAandRNAcorresponding to thenucleotides present in the putative SIV RRE (nucle-otides 8086 to 8454) (Fig. 3). The HIV-2 H6 Rev protein-RRE RNAcomplexwasvisualizedas aband migratingnear

the top of the gel. In some experiments, a second,

much-faster-migrating bandwas present; however, this bandwas

Ii'

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FUNCTION OF HIV REV PROTEINS 4431

cG

UCG

C G

A A

A C AAG

J U U

C A A ;:

GGG

G;CA A

JCGA C

;LC A

AGuAG AG

A

A c

GGU;CAAAA

U

CAA

A

LoI

B

Ln XI

-L

wF C: t:Oo

N:- Nv

tor z km nw) n J I 4 u e ir

St+-+-+.+.+-+.+

.+.+.+ -+-+-+

-+

FIG. 3. Complex formation of HIV-1 and HIV-2 Rev proteins

with the HIV-1, HIV-2, and SIV RRE elements and deletion

analysisof the HIV-2RRE.Thecomputer-predictedstructureofthe HIV-2RREis shown schematically atthe top, with the nucleotide positions ofthe deletions. 32P-labeled RRE RNAwas generated in

vitro by using T7 polymerase. RRE RNA was incubated with

purified HIV-2 H6 Rev or HIV-1 Rev proteins, and binding was

assessedby the qualitative nuclease resistance gelretardationassay.

Rev protein-RRE RNA interaction resulted in anucleaseprotected

complexwhichmigratedslower than RNaseTl-digestedproductsin

the nondenaturing polyacrylamide gel. (A)Complexformation with

HIV-2Rev protein and RRE RNA analyzedon a5% polyacrylamide

gel. (B) Complex formation withHIV-1 Rev proteinas analyzedon

a4%polyacrylamide gel. RRE RNAwasincubated intheabsence

(lanes -)orpresence(lanes +)of Revproteins.

most likely a partial RNase T1 digest product, since it was

alsoseentoalesser extentin the absence of HIV-2 H6 Rev protein. To rule out the possibility that contaminating bac-terial proteins from the HIV-2 H6 Rev protein preparation boundtothe RRE RNA,acrudelysatefromnontransformed E.coliwasusedin the gelshiftassay, andno bindingtoRRE

RNA was observed (data not shown), indicating that the RREbinding shown in Fig. 3 was specific to the HIV-2 H6 Revprotein. Very weak, ifany, binding was observed with

the HIV-1 RRE RNA,although the HIV-1 Rev protein was

abletointeract with all three of the RRE elements (Fig. 3). This observation is consistent with the inability of HIV-2

Revto regulate HIV-1 gene expression in vivo.

Delineation of HIV-2 RRE sequences necessary for HIV-1

and -2 Rev interaction. Ina previous study with the HIV-1 RRE RNA, it was shown that formation of a large stem structure isrequiredfor HIV-1 Rev-RRE interactions(3). In

subsequent studies, the actual Rev binding sitewas mapped to oneof thesmallerhairpin loopstructures(22,28, 29). It is

likely that formation of the larger stem is necessary for generationof the smallerhairpinloop structures in the RRE,

includingthe one required for HIV-1 Rev interaction. Simi-lar to the HIV-1 RRE, the HIV-2 RRE contains a Simi-large stem structure,asindicatedby the computer-predicted secondary structure of the HIV-2 RRE RNA (26). The importance of thelarge stem found in the HIV-2 RRE was assessed by the PCR method to generate deletions from the 5' and 3' ends of the HIV-2 RRE. A 5' deletion (5'A7679) upstream from the

large stem allowed binding of both HIV-1 and HIV-2 Rev proteins (Fig. 3). Furthermore, a 247-nucleotide HIV-2 RRE containing nucleotides 7679 to 7926 (5'A7679-7926), which maintained an intact stem, was sufficient for Rev-RRE interaction (data not shown). However, both Rev proteins

failed tobind HIV-2 RRE transcripts containing deletions of

either the upstream (5'A7728) or the downstream (5'A7679-7875) sequences which comprise the stem structure. In addition, deletion of both parts of the stem (5'A7728-7875)

failed to support Rev binding. These results indicate the importance of the HIV-2 RRE large stem in Rev binding, which is consistent withobservations reported for the HIV-1 RRE(3). Introduction of minor alterations (Fig. 4) consisting of five or six nucleotide changes had no dramatic effect on either HIV-1 or HIV-2 Rev interaction, with the exception of mutant PD-10, which failed to associate with either Rev protein. However, computer analysis of this mutation pre-dicts formation of an RRE with a markedly different RNA

secondary structure (data not shown).

Whereas major deletions in the stem abolished Rev

inter-action, small changes in base pairing within the stem struc-ture had little effect on Rev-RRE interactions. These data

indicatethat stem formation may be required for the overall

generation of the smaller secondary structures within the HIV-2 RRE shown in Fig. 5, of which one or more may be importantfor Rev interaction. This possibility was examined further by creating the mutations shown schematically in

Fig. 6and 7. The structures shown inFig. 6, 7, and 8 indicate the regions of the wild-type HIV-2 RRE affected by the mutations and are not intended to represent the actual RNA secondary structures formed by each mutated RRE.

Dele-tion ofthe first two hairpin structures (as shown in PD-2) caused a loss of interaction of the HIV-2 RRE with either Rev protein. Deletion of thefirsthairpin alone (PD-3) did not affect RRE interaction with the Rev proteins. In addition, a changeof sequence in the loop of the first hairpin (PD-8)had noeffect on Rev-RRE interaction. However, deletion of the second hairpin (PD-4) resulted in complete loss of binding with HIV-2 Rev and a very weak interaction of the RRE with the HIV-1 Rev protein. Deletion of the third hairpin struc-ture (PD-9) gave substantially weaker interactions with both Rev proteins. A mutation (PD-15) which disrupted a stem structure between the second and third hairpins showed weak interaction with HIV-2 Rev and an extremely weak interaction with the HIV-1 Rev protein. Deletions of the fourth and fifth hairpins alone (PD-7 andPD-6, respectively) or together (PD-5) had no dramatic effect on interaction of the RRE with the HIV-2 Rev protein. However, deletion of both hairpins (PD-5) abolished HIV-1 Rev binding and deletionof the fourth hairpin alone(PD-7) resulted inamuch weaker interaction of the RRE RNA with the HIV-1 Rev protein. Inaddition, disruption ofa stemin the fourthhairpin

5,

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A L

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u

A

A C

,GOG n U iUA_G, C

LUC A

A

:J aG

GG U 1 CLI

C G

A

A A

A C

A A G

a: D a:

X XcL a z X

a 7

XL :L

fl-.,. .- .i

0

I

FIG. 4. Analysis of the major stem involvement in Rev-RRE RNA complex formation. In vitro mutagenesis ofthe HIV-2 RRE

wasperformedas described in Materials and Methods. The

nucle-otidechangesareindicated in boldface in thediagramatthetop.(A) Complex formation of HIV-2 Rev protein with HIV-2 RRE

muta-tions.(B) Complex formation with HIV-1 Rev protein.

(PD-14) had no effect on either HIV-1 or -2 Rev protein binding.

Toexamine furtherthe importance of secondarystructure

asopposedtotheprimary nucleotidesequencefor Rev-RRE

interaction, single and compensatory mutations were made

inthe third and fifthhairpin structures(Fig.7).Surprisingly, disruption of thestem structure in the third (PD-17) orfifth

(PD-16) hairpin causedasubstantial loss of interactionwith

HIV-1 but not HIV-2 Rev proteins. Restoration of HIV-1 Rev-RRE interaction was observed when compensatory

mutations were made which regenerated the base pairing necessary for stem formation in the hairpins (PD-17C and PD-16C).

Rev responseof the RRE mutations in vivo correlateswith in vitro Rev-RRE binding. The ability of the HIV-2 RRE mutants tofunction in vivo was examined by replacing the intact HIV-2 RRE present in plasmid pIIIH2 with the individualHIV-2 RRE mutations andmeasuring their ability

FIG. 5. Schematicrepresentationofhairpinloopdeletion

muta-tions in the HIV-2 RRE. Thecomputer-predicted structuresof the wild-type HIV-2 RRE RNAs affectedbyhairpin loopmutationsare

indicated by differential shading. The first through fifth hairpins referredtoin the textareclockwise fromPD-3(i.e.,thefirsthairpin

contains PD-3and fifthhairpin containsPD-6).

to respond to either HIV-1 orHIV-2 Rev incotransfection assays. The resultsobtained fromCAT assayswere scored aspositiveornegativefor Rev response andarepresentedin Table1. Ingeneral,RREmutantswhich didnotbindRev in thebindingassaysdidnotdisplayRevresponsesin vivo and thosemutationswhichinteractedweaklyfunctioned weakly

invivo.Examplesof thisweremutationsPD-15, PD-16,and PD-17, which bound weakly to HIV-1 Rev in vitro and showed weak responses in vivo. Interestingly, these same RREs interacted with HIV-2 Rev protein but failed to

function in vivo with HIV-2 Rev. Interestingly, mutations PD-3 and PD-5 showed weak or no response in vivo, although PD-3 bound well to both HIV-1 and HIV-2 Rev

proteins and PD-5 bound well to the HIV-2 Rev protein.

These observations are addressed further below. Signifi-cantly, the compensatory

mutations,

PD-16C and PD-17C,

which restored secondary structure comparable to that of

the predicted wild-type HIV-2 RRE, responded to both HIV-1 and HIV-2 Rev proteins to the same extent as the

wild-typeRREitself.Therefore, as waspreviouslyobserved

with the HIV-1 RRE,maintenance of secondary structure is a major determinant for both Rev-RRE interaction and function.

DISCUSSION

The HIV Rev proteins, as well as the Rex proteins encoded by the human T-cell leukemia viruses (HTLVs), mediate the export of viral structural mRNAs from the nucleus tothe cytoplasm (11, 19-21, 24, 27). The ability of the HIV-1 Rev protein to interact directly with the RRE RNA(3, 6, 9, 27, 29, 39) isconsistent with the direct role of Revin the nuclear export of these mRNAs. Here we show that theHIV-2 Revproteininteracts with HIV-2 RRE RNA. Unlike the HIV-1 Rev protein, which can interact with the

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PD-2 PD-3

aai

C; r ;E t

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A . C;AS_CfiCC£l

S. a c

~~~~~~~~~~~~~~~~~~~~~~~A

r

A * t~~~~~~~~~~~~~~~~~~

-A S AffI,

A~~~~~~~~~~~~~~6 a a;

CC~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I

m

w~~~~~~~~~~~~~~~~~w

C CrcC

~~~~~~~~~~~~~~~~~~~~~~~~AS BIL

A

a3

a a D

mI

_,I_ w@,1 r ---CS.

, r

;

S Ca

ASC | eWt Ca

6,1 CCA

CF

PD-,

a6a

A

I:C

-cc

* r;LO

c co

a~~~~~~~-FIG. 6. Mutational analysis of the first three hairpins of HIV-2 RRE and Rev-RRE complex formation. Mutations are indicated

schematicallyonthe basis ofcomputer-predicted wild-type HIV-2 RRE RNA secondarystructure. These diagrams (Fig. 6-8) indicate the

structures affectedby the mutations andarenotintended torepresent actual RNA secondarystructuresformed by these mutations. Also

shown iscomplex formation of HIV-2 RREmutantswith HIV-2 H6 Revprotein (A) and HIV-1 Revprotein (B).

RRE of HIV-1 and HIV-2, significant binding of the HIV-2 Rev proteinwasonlyobserved with theHIV-2 RRE andto

a lesser extent with the SIV RRE. This observation is consistent withbiologicaldatashowingthat,whereasHIV-1 Rev can complement HIV-2 gene expression, HIV-2 Rev

onlyfunctions with its ownRRE and that of SIV.

Although manystudies have addressed the role of Rev in

the export of structural mRNAs from the nucleus to the cytoplasm, the mechanism for nuclear entrapment of these mRNAs, whichoccurs inthe absence of Rev expression, is

notunderstood.Similarly, althoughweknow thatsequences

present in both the env and gag-pol regions of HIV can

repress expression of heterologous genes when present in the 3'untranslatedregion,it isnotknownhow thisoccurs(8,

11, 13, 32). The studiespresented here do notaddress the mechanism for repression. However, we observed that replacement of the HIV-1 RRE with that of HIV-2 or SIV rendered the indicatorplasmid responsivetoboth HIV-1 and HIV-2 Rev proteins. Therefore, although the HIV-2 Rev proteincannotfunction with the HIV-1 RRE,inpartdue to its inability to bind, the interaction ofHIV-2 Rev with its

own RRE can overcome the repressive effects exerted by HIV-1 repressiveelements.

Inanefforttounderstand thenonreciprocal relationshipof

a

S

a a

U'

I

-.5.

. )i..--.

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4434 DILLON ET AL.

PO-5

A

- A'C G

-A-P

CAC-"A ' C G

A~~~~~~~iA

PD-6

A

PO-7

CbricGC A

;A

CAG

CCL,G

C.AdACs C

CAAACCGA CA ACA' C

LCCCCA

G

CC r AC

C CCS,A CA G

.CACAA A)ACCC. CCAA

CI5

G~~~~~~u

CG.AA,ACCCACAAC

C A

A C C'

A G AA

G C w

cCAAC-CC AG C --G A Cl C ; AAwG> GAD A A

A C~ A

A,C~~~~~~~~~~~~~~~~~~~~~~~~~~~~~A

w

c:

cc

S *

-+o- +

ID r

S I I

- + _+ - +

_____

|.'K..>">-

-.s

i

.,X,,j,,.R

|

w

w...

w+.,, .x<+.

w .'

:_ 2{

a:

Ba:

C1 Ln rl (0

I I IS a

-

.r

+ M + + +

c 6

CL CL

r r

-+ -+

FIG. 7. Mutationalanalysis of fourth and fifth hairpins of the HIV-2 RRE and Rev-RRE RNA complex formation. Mutations of HIV-2

RREareindicated schematically (the diagramsarenotintendedtorepresentactualRNAsecondarystructuresformed by these mutations). Alsoshown is complex formation of HIV-2 RREmutantswithHIV-2 H6 Revprotein(A)andHIV-1 Revprotein (B).

HIV-1 and HIV-2 Rev in binding the HIV-1 and HIV-2 RREs, mutational analysis and functionalassays were used

to identify and characterize the HIV-1 and HIV-2 Rev-binding sites within the HIV-2 RRE.Inarecentstudy, itwas

shown that binding of HIV-1 Rev to its own RRE is

determinedtoalargeextentbyformation ofasingle hairpin

loopstructurepresentwithin the RRE(22, 28, 29). Notably, altering the primary nucleotide sequence of the structure, whilemaintaining theproperRNAsecondarystructure,had littletonoeffectoneitherHIV-1Revinteractionorfunction

A

J. VIROL.

J- A_ MA

6A

-. L.

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U;

A

4

JG

ce

1- uUuu

G C A

u A C

GC GGCG GU

a-r

Gc cCG

GUC G CGG

GGA A`

U A A

PD-16&PD-16C C G

U A bU' G

G CAACAGCAA

~AG,.

G

A ACAG uGU ACG UUGUCGAC

r, A G G U

C,~ A U

A A rUC

luA C-- Gu-G I A

CC,_CGA~.,.,,,E E 6_UALJ.-GA_CAAU C_I GC---G GCGCAA_U _A

u

s sA U,a A

G--

CG

CA A

G c AG

CG

~

CA

A A A A

A C AA G

TABLE 1. Rev proteinbinding andfunctionalactivityofRREsa

Binding and function with:

RRE HIV-1 Rev HIV-2 Rev

Binding Function Binding Function

HIV-1 +++ +++ +/-

+/-HIV-2 +++ +++ +++ +++

SIV +++ +++ ++ ++

5'A7679 +++ +++ +++ +++

5'A7728 - - -

-5'A7679-7875 - - -

-5'A7728-7875 - NT - NT

PD-1 +++ +++ +++ +++

PD-10 - - +/-

-PD-11 + +/_ +

+/-PD-12 +++ +++ ++ +++

PD-2 - - - _

PD-3 +++ + +++

-PD-4 +/- +

-PD-9 +/- +/- +/- _

PD-15 +/- + +/-

-PD-8 +++ +++ +++ +++

PD-5 - - +++

PD-7 + ++ + +++

PD-6 ++ ++ +++ +++

PD-18 +++ +++ +++ +++

PD-14 ++ ++ ++ ++

PD-17 +/- + ++

-PD-17C +++ +++ +++ +++

PD-16 +/- + ++

-PD-16C +++ +++ +++ +++

PD-17& PD-17C

A'U

CG

UCG

CG

A A A A

A -A A G

C.)

CL N

r6.

Q.+

{D {D

6 6

0.0.

I

49I,P

9

w

cc cc

6_0.

rw

-

.t-aRev-RREbindingwasassayedbygelretardation analysis,andfunction

wasassessedby aRev-dependent heterologous gene expression assay (see text). Resultsareaveragesfrom several separate experiments: +++, full;

++, intermediate; +, weak; +/-, very weak;and -, no Revresponseor

interaction. NT,Nottested.

AG

through

the

HIV-1

RRE

(29).

The data obtained for the

A HIV-2 RRE are consistentwith those observed for HIV-1.

cG AA Forexample,while nosingle stem-loopstructure was found

uGcA

_U A_G to be solely responsible for HIV-2 Rev-RRE RNA

interac-CAA tions,ourdata suggest that a groupofstructures mayconfer

the ability to bind. Consistent with observations for the HIV-1 RRE,alteration ofoneside of a stem structure shown to be required for Rev interaction prevented Rev binding.

However, generation of compensatory mutations in the same structure restored Revbinding and responsiveness in the functional assay. Thus, as was observed with HIV-1, D

6)

C: RNA

secondary

structureappearstobea

major

determinant

for HIV-2 Rev-RRE interaction. Significantly, the region of --I the HIV-2 RRE RNA identified as being important for

interaction with HIV-1 Rev bears no resemblance with

regard to primary nucleotide sequence or secondary struc-ture tothe elementrequiredfor binding ofHIV-1 Rev in the g I, HIV-1 RRE. Moreover, asobserved with HIV-2 Rev bind-ing,

generation

of

alternate

secondary structures through

compensatory

mutation restored

HIV-1

Rev

binding.

Taken

together,

theseobservations offer

compelling

evidencethat RNA secondary structure is a major determinant for both

_I

HIV-1 and HIV-2 Rev binding and further suggest that FIG. 8. Analysisofcompensatory mutations ofHIV-2 RRE in

complex formation. Mutations ofHIV-2 RREare indicated sche-matically (the diagramsare notintended to represent actualRNA secondary structures formed by these mutations). Mutant PD-16 containsbasechangesindicatedontherightsideofthe fifthhairpin. The compensatorymutantPD-16C contains basechanges indicated

onboth sidesofthefifthhairpin.InPD-16C,theuridineresidue with

ablack background wasdeleted. MutantPD-17 contains the indi-catedbasechangesontheupper sideofthe thirdhairpin,whilethe compensatory mutant PD-17 contains changes on the upper and lower sides. Also shown is complex formation of HIV-2 RRE mutantswithHIV-2H6 Revprotein(A) and HIV-1Revprotein(B).

w cc rr

A

>

I

_

_k-

Mdoll

_

. 4P

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different secondary structures can be recognized by the HIV-1 Rev protein.

Since HIV-1 Rev can interact with the HIV-2 RRE, the remaining question concerns the inability of HIV-2 Rev to interact with the HIV-1 RRE.Althoughthe samesecondary

structuresarerequiredfor HIV-1 and HIV-2 Rev interaction

with the HIV-2 RRE, it appears that a smaller, more

well-defined structure is utilized for HIV-1 interaction with itsownRRE(22, 28, 29)which is insufficient forHIV-2 Rev protein binding.

Our mutational analysis of the secondary structures presentwithin the HIV-2 RRE indicates thatthe second and third hairpin structures are important for both HIV-1 and HIV-2 Revprotein interaction. Furthermore, itappearsthat the overall structure of the RRE RNA may be necessary

either forgeneration of the Rev protein-RRE RNA binding siteorforstabilityofacorrectcomplexformation necessary

for function. It could be postulated that binding of Rev protein tothe HIV-2 RREprovidesacomplex whichcanbe recognized byacellularfactor which facilitates transportof viral structuralmRNAs toacytoplasmicsite where transla-tionoccurs.Alternatively,Rev-RREinteractionmaycause a

conformationalchangein the mRNA which createsasite for interaction with cellular factor(s) that mediates nuclear

ex-port of structural mRNA. Either of thesepossibilities could explain the observation that mutations PD-3 and PD-5 are

capable ofbinding Rev but fail tofunction, presumably by formation of an inactive complex which is nonfunctional. Furthermore, the inabilityof HIV-2 Rev tointeract with the HIV-1 RRE may reflect the extent or type of secondary structuresthataregeneratedwithin the HIV-1 RRE. Indeed, only one stemstructure wasfound to berequiredforHIV-1

interaction with the HIV-1 RRE, while the data presented here demonstrate that interaction with the HIV-2 RRE is probably more complex and involves more than a single hairpin.

Our resultsmayalsoprovide insight relatingto theability of HTLV-I Rex protein tocomplement HIV-1gene

expres-sion versus the inability of HIV-1 Rev to complement HTLV-I(21, 30). Thepredictionis that HTLV-I Rex

recog-nizesanalternatesecondarystructurein the HIV-1 RRE and that a suitable binding site for HIV-1 Rev is absent in the HTLV-I RRE.

We propose that HIV Rev proteins and HTLV Rex proteins belong to a class of regulatory molecules that function through recognition of RNA secondary structure. Ourfindings would also be consistent withagroupofRNA secondarystructuresformingabindingdomain in which Rev

orRex would have severalcontact points.

ACKNOWLEDGMENTS

We thank Chein-Hwa Chen for providing purified HIV-1 Rev

protein. The following reagents were obtained through the AIDS

Research and Reference Reagent Program, Division of AIDS, NationalInstitute of Allergy and Infectious Diseases: reagent HIV-2ROD phage from contributor Ronald Desrosiers andreagent

pBK28-SIVfromcontributorJamesI. Mullins.

This work was supported in part with funds from a National

Cooperative Drug Discoverygrant.

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