Identification and characterization of intragenic sequences which repress human immunodeficiency virus structural gene expression.

Identification

and

Characterization

of

Intragenic Sequences

Which

Repress Human

Immunodeficiency

Virus

Structural

Gene

Expression

ALAN W. COCHRANE,' KATHRYN S. JONES,2 SARY BEIDAS,'t PATRICK J. DILLON,'

ANNA MARIE SKALKA,2AND CRAIG A.

ROSEN'*

Department ofMolecular Oncology & Virology, RocheInstituteofMolecular Biology, Roche Research Center,

340Kingsland Street, Nutley, NewJersey07110-1199,1 andInstituteforCancerResearch, Fox Chase Cancer Center, Philadelphia, Pennsylvania 191112

Received 15 April1991/Accepted 11July 1991

Examination of the lifecycle of the human immunodeficiency virus (HIV)hasshown that multiple levels of

regulation exist, includingsomewhich require the virus-encoded Rev protein.In the absenceof Rev,mRNAs

encoding thestructuralproteins remainuntranslated,aphenomenon whichappears, inpart, tobecausedby

nuclear entrapment of these RNA species. To examine the basis for repression of structural gene mRNA

expression,aheterologousassaysystemwasutilizedtodetermine whether regionspresentwithingagandpol

containelements capableofsuppressinggeneexpression whenpresentin cis. Bothgenes werefoundtocontain cis-acting repressor sequences (CRS) that block gene expression when present within the 3' untranslated portionofaheterologous genetranscript. Theelementwithinpolwasfoundtohave the strongestrepressive effect.While Rev alonewasunabletoreversetherepression observed with thepolsequence,addition oftheenv

Rev-responsive element(RRE) incisandRev in transdidcause reversal ofinhibition. Deletion mutagenesis

defineda260-bp element withinthe3' portionofpolthatcontainsapotentCRSwhich functions whenpresent

in thesenseorientation. Thecorresponding regioninHIV-2polwasfoundtocontainafunctionally similar CRS

element. Toexaminethemechanism ofrepression,theeffects oftheCRSelementsonboth the abundance and subcellular distribution ofthemRNAswereexamined. Neitherwasdramatically alteredwhenexaminedin the contextofaheterologousreporter (chloramphenicol acetyltransferase) mRNA. Theseresultssuggestthatthe CRS element defined within pol operates at a splicing-independent, posttranscriptional level to prevent

expressionofstructuralgenes.We suggest thatmRNA bearingtheseCRS elementscannot reactproductively with thetranslational machinery ofthecell.Theabilityof Rev,anuclearprotein,toreversethiseffectindicates that Rev must alterthe nuclearmetabolism of these mRNAssuch thatthe mRNAreachingthecytoplasmis capableofbeing translated.

Investigationof the humanimmunodeficiencyvirus(HIV) lifecycle has revealedacomplexpatternofgeneexpression.

In addition to elements common to other retroviruses, the

HIV genome encodes numerous regulatory proteins that

affect different facets of HIV replication and pathogenesis. At present, at least six nonstructural regulatory proteins which participate in the regulation ofgene expression or

replication havebeenidentified (29, 44). Ofthese

nonstruc-turalproteins,two,TatandRev, have beendemonstratedto

beessentialforreplication (7, 11, 20, 39, 41).Themechanism of action of Tat remains controversial, with both posttran-scriptionalandtranscriptionalmechanismsbeingconsidered (la, 17, 23, 27, 31, 33, 38). Studiesof the mechanism of Rev indicate that it operates at the posttranscriptional level to

permit synthesis oftheviralstructuralproteins (10, 39).How this isachieved isnottotallyclear. As with allretroviruses, expression of the full coding capacity of the viral genome requiresdifferential splicingofthefull-lengthgenomicRNA initially generated upontranscription. However, splicingof the genomic RNA, by necessity, must be an inefficient

process, sinceexpressionof thegagandpolgenesrequires

the transport and translation of the full-length genomic RNA. While with the simpleretroviruses, genomic RNA is

*Correspondingauthor.

t Present address: Mercer University School ofMedicine,

Ma-con,GA 31201.

transported from the nucleus tothe cytosol constitutively, with HIV, the genomic RNA and single spliced forms encoding env appear to be sequestered within the nucleus, whilethemultiplyspliced formsareefficientlytransportedto

thecytoplasmintheabsence ofRev (9, 15, 16, 25). Expres-sion of Rev results in the partial transport and subsequent translation of the sequestered mRNAs. Although it is well established that this release from sequestration requires binding of the Rev protein to its cis-acting target sequence

within env (26, 28), termed the Rev-responsive element

(RRE), the basis for the selective retention of the RNA encoding structural proteins is currently unclear. Work by Changand Sharp (2), usinga heterologous beta-globin sys-tem, has demonstrated that a mRNA can be rendered Rev responsive byboththe addition of RRE and the mutation of either the splicedonororacceptor site ofthe intron. These

results have ledtothehypothesisthatsequestration ofHIV

structural mRNA in thenucleusisduetotheinefficiency of the HIVsplicingreaction. However, the failuretoobservea

similar pattern ofregulation with thesimpleretroviruses,in

which a similar inefficiency in env mRNA splicing occurs

(12, 19, 40), suggests that either the basis for the splicing inefficiency of HIV must differ or that additional elements arepresent within the HIVgenomewhich contribute to this

novel form ofregulation.

In support ofthe latterhypothesis, studiesusing a heter-ologousassay systemtoexamineRev functionhave shown 5305

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5306 COCHRANE ET AL.

that cis-acting repressive sequences (CRS) encoded in the envgene of HIV contribute to the inefficientexpression of this mRNA(34). In this system,theenvgenewasplaced3' of thetermination codonofareportergene(chloramphenicol acetyltransferase [CAT]), resultinginadramatic decreasein CAT expression, which could be reverseduponadditionof Revin trans. Deletionanalysisof theenvsequencesuggests that several regions within env contain negative regulatory elements, because their deletion resulted in an increase in gene expression in the absence of Rev. Furthermore, dele-tionof thesplicedonororacceptorsites in this construct had

noeffectonexpressionand the constructremained respon-siveto Rev. These observations, coupled with the demon-stration that expression of other HIV structural proteins displayedasimilardependencyonthepresence of theRRE and Rev (8, 15), suggest that functionally identical CRS elements might be present within thegag andpolgenes of HIV.

Toidentify potentialCRSelements within the gag andpol genes ofHIV, segmentsof thesegeneswerefused3' of the CATterminationcodon and theireffectsonCATexpression following transfection were examined. Our results indicate thatboth gag andpolcontainCRSelementsthatareableto repressgeneexpressionwhenpresentin cis.Repressionwas reversedbyaddition of the RRE in cis andexpressionof Rev in trans. Deletionanalysisof thepolgenedemonstrated the presence ofaninhibitory sequencewithina260-bp regionof theintegrase domain. RNAanalysis suggeststhatinhibition ofgene expression mediatedby this CRS element is alsoa

posttranscriptional effect.

MATERIALSANDMETHODS

Preparationofexpressionvectors. ToidentifyHIVgagand

pol sequences capable of inhibiting gene expression, an

approach similar to that employed to study the CRS ele-ments of the env gene was used (34). In brief, the SacI-EcoRI(gag andpol, nucleotides 227 to4193), SacI-EcoRV (gag, nucleotides 227 to 2524), EcoRV-EcoRI (pol, nucleo-tides 2524 to 4193), or KpnI-EcoRI (pol 490-bp fragment,

nucleotides 3703 to 4193) fragment of strain Hxbc-2 was

placed 3' of the termination codon of the bacterial CAT gene. Nucleotidepositions are numberedby thenumbering systemof Ratneret al. (32). Vectors utilized contained the small t antigen intron and early polyadenylation signal of simianvirus40 (SV40) andexpression wasdrivenbyeither

the HIVtype 1 (HIV-1) longterminalrepeat(LTR)orSV40

early promoter. The plasmid pSVAR is a derivative of

pIlIAR(34) and the only difference betweenthe two is the

replacementof the HIVLTRwith the SV40 originandearly promoter.

The3'mutants ofthepol490-bpsequenceweregenerated by using the site-directed mutagenesis protocol ofKunkel (22). The 5'deletionmutantsweregeneratedby polymerase

chainreactionamplification (37)of theregionof interest and

cloned 3' of the terminationcodonof the CATgene.

The vectors pBC12BI/HIN and pBC12BI/HIN+RRE

were preparedby isolation oftheBamHI-NdeIfragment of pCG6 (a bacterial HIV integrase [IN] expression clone) which contains, in addition to the IN codingsequence, the lambda PL promoter. This fragment was ligated into the pBC12eukaryotic expression vector3' of the rat

preproin-sulin intron. ThelambdaPLpromoter was theneliminated,

and the 5' end of HIV IN was repositioned immediately

downstreamfrom the insulin ATG initiation codon by oli-gonucleotide-directed mutagenesis (22), usinga 36-base

oli-gonucleotidethat contained the 15 nucleotides

immediately

upstream of the ATGcodon,theATGitself,and the first18 nucleotides of HIV IN. The RRE utilized for both

pBC12BI/

HIN+RRE andpSVCP490RRE corresponds tonucleotides 7204 to 7684 of Hxbc-2 (32) and was placed 3' of the

inhibitory sequence and 5' of thepolyadenylation

signal.

Transfections andanalysis of expression. For initialstudies,

vectorsdrivenbytheHIV-1 LTRwereintroduced into CHO zip tatIII cells (34), which stably express tatprotein, using

theDEAE-dextran calciumphosphatetransfection

protocol

(34). Subsequent experiments utilized COS cells whichwere transfected by the DEAE-dextran protocol (5). Cells were

harvested 48 h posttransfection, and CAT activity was determined as previously outlined (13). For quantitative

assessment of the extent ofchloramphenicol modification, thin-layer chromatogram plates were scanned by using a

Betagen imaging device and theimages generated were used todetermine percent conversion(chloramphenicolto

acety-lated forms). For the analysis of CAT RNA subcellular distribution, cells were cotransfected with pgTat, another Rev-responsive vector(25).

To examineexpression of HIVIN, COS cells transfected with theINexpression vectors were labelled with 300 ,uCi of [35S]methionine for 3 to 6 h 48 h posttransfection. Cellswere subsequently lysed in RIPA buffer (0.1% sodium dodecyl

sulfate [SDS], 1% Triton X-100, 1% sodium deoxycholate,

0.15NaCl, 0.01 M TrisHCI [pH 7.4], 1 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride) and immunoprecipitation

performed by using rabbit polyclonal antisera directed against bacterially produced HIV-1 IN. Antibody-antigen complexes were precipitated with Staphylococcus aureus protein A, the precipitated proteins were analyzed on SDS-polyacrylamide gels, and bands were visualized by autora-diography.

Subcellular fraction and RNA extraction. Harvested cells were washed twice with ice-cold phosphate-buffered saline and then fractionated into nuclear and cytosolic fractions (14). Cells were resuspended inlysis buffer (10 mM TrisHCl [pH 7.5], 5 mMMgCl2, 10 mM NaCl, 0.50% NonidetP-40),

vortexedfor S s, and then placed on ice for Smin. Thelysate was then centrifuged at 2,000 rpm in a Sorvall RT6000B centrifuge for 5min at 4°C. The supernatant, designated the cytoplasmic fraction, was collected and immediately added to3 volumes ofasolutioncontaining6 M guanidine thiocy-anate,37.5 mM sodium citrate(pH 7.0), 0.75%Sarkosyl, and 0.15M

P-mercaptoethanol.

Thepellet was suspended inlysis

buffer, and the nuclei were pelleted by a second

centrifuga-tion at 2,000 rpmfor5 minto remove residual cytoplasmic material. The supernatant was removed, and the pellet (designated the nuclear fraction) was resuspended in a solution containing 4 M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% Sarkosyl, and 0.1 M 1-mer-captoethanol. Both fractionswere processed as previously described(3). Following the final precipitation, RNApellets were washed with 75% ethanol, allowed to dry, and then redissolved in diethylpyrocarbonate-treated water. RNA was quantitated by determining the A260 of the solution.

Subcellular distribution of RNA. ForSianalysis of Tat and CATmRNA,theBl-pgTat S/K and pGM4CAT AEco clones,

respectively, wereused. TheBl-pgTat S/K plasmid contains theSalI-KpnI fragmentof HIV-1 (nucleotides 5331 to 5893 of

Hxbc-2)and encompassesthefirst Tat exon and a portion of the env gene. The pGM4CAT AEco plasmid contains the

HindIII-EcoRI fragment of the CAT gene derived from

pSVCAT (13). For labelling, Bl-pgTat S/Kand pGM4CAT AEco were linearized with Sall and HindIII, respectively.

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HIV-1 LTR CAT PoiyA

i-i

-*0

pillCATgag/pol HV- 1LTR. CAT 4 PoIyA +

+

HIV-1 LTR; CAT Pol2//// y

227 2524

':'AA _ ,

pillCAT pol iIV-1LTRCAT poI PoyA

2524 4193.

'AA

-pillAR !HIV-1LCAT PoIyA]

5331 9177

-AA 7204 -S46.

pillCATpolRRE'HIV-1 LTR CAT RRE'PoyA

2524

FIG. 1. Detection of inhibitory sequences in the gag and pol genes of HIV-1. To the left are the schematic diagrams of the expression vectors used to identify the presence ofCRS-likeelements within the gag andpolregions ofHIV-1. Numbers correspond to the nucleotide

positions ofthe 5' and 3' termini of the HIV-1 fragments present in the vectors according to the numbering system of Ratner et al. (32). To theright,the thin-layer chromatograms used to analyze CAT activities present in celllysatesfollowing transfection of the vectors into CHO

zip tatIll cells in the absence (-) or presence (+) of Rev. Quantitation of the thin-layer chromatograms shown revealed the percent conversions: pIlICAT without Rev, 32.3; pIlIlAR without Rev, 1.12; pIlIAR with Rev, 31.0; pIIICATgag/pol without Rev, 4.2;

pIIICATgag/pol with Rev, 5.8;pIIICATgagwithout Rev, 4.29;pIlICATgagwith Rev,7.17;pIIICATpolwithoutRev, 0.8;pIlICATpol with Rev, 1.44;pIIICATpolRRE without Rev, 0.8;pIIICATpoIRREwith Rev, 35.3.

Thelinear DNA was then labelled by incubation in a solution

containing 50 mM Tris HCl (pH 8.3), 40 mM KCl, 6 mM MgCl2, 1 mM dithiothreitol, 200 ,uCi ofat-32P-labelled

deoxy-nucleoside triphosphates (50 pLCi [each] of dATP, dTTP, dGTP, and dCTP), and 15 U of avian myeloblastosis virus reversetranscriptase at37°Cfor 30 min. The reactions were terminated byaddition of EDTA to 20 mM, and unincorpo-rated label was removed by passage of samples through

Sephadex G-50 spincolumns. The eluant was ethanol

pre-cipitated andresuspended in restriction enzyme buffer, and

plasmids Bl-pgTat S/K andpGM4CAT AEco were digested with PvuII and NarI, respectively. The desired fragments were isolated from 5% polyacrylamide gels, denatured by

heatingat 100°C for5 min, and used for S1 analysis. DNA

probes were added to 10

[Lg

of RNA, and the mixturewas

ethanol precipitated and dissolved in 20

[lI

of a solution containing 80% formamide, 40 mM

piperazine-N,N'-bis(2-ethanesulfonicacid) (PIPES) (pH 6.4),400mMNaCl,and 1 mMNa2EDTA. Sampleswereheatedat70°C for 10 min and thenplacedinaH20bath setatthepredetermined annealing

temperature(49°Cfor Tat mRNA and42°Cfor CATmRNA).

Followingincubation for 4 to 6h, 200,ulof S1 buffer (30mM

sodium acetate [pH 4.6], 250 mM NaCl, 1 mM ZnCl2) containing 300 to 600 U of S1 nuclease was added. For

analysis of Tat mRNA, the S1 reaction mixtures were

incubated at 37°C for 1 h. For

analysis

of CAT

mRNA,

samples were incubated at room temperature for 1 h. S1 reactions were terminated by addition of 50

[lI

of S1 stop buffer (4 M ammonium acetate, 20 mM EDTA, 40 ,ug of tRNA per ml), and the samples were ethanol

precipitated.

Pelleted DNA was resuspended in 70% formamide-5 mM Tris HCI (pH8.0)-0.5mMEDTA,heatedat100°Cfor 5min,

and then run on a 5% denaturing

polyacrylamide

gel. Fol-lowingelectrophoresis, gelsweredried and used for

autora-diography.

Determination of HIV IN RNA distribution was carried

out by Northern (RNA) blot analysis (43). Aliquots (10 ,ug each) of nuclear andcytoplasmic RNAfrom control cells or cells transfected with pBC12BI/HIN+RRE in the presence orabsenceof Revwerefractionatedon1.2%

formaldehyde-agarosegels, andfollowing electrophoresis, the RNAs were transferred to nitrocellulose. The blots were baked at 80°C for 90 min and prehybridized in a solution containing 50% formamide, 6x SSC (lx SSC is 0.15 NaCl plus 0.015 M

sodium citrate) 1% SDS, 0.1% Tween 20, and 100 ,ug of

tRNA perml. Prehybridizationwascarriedoutat55°C for2 h, and then hybridization wasperformed in the same buffer at55°C overnight. The probe used for detection ofIN RNA was an antisense RNAprobe corresponding to nucleotides

3703to4193of Hxbc-2 (32). The blotswerewashed twice in 1x SSC-0.1% SDS at room temperature and twice in 1x

SSC-0.1%SDSat65°C.Toremoveresidualhybridizationto 28S rRNA, blots were washed three times in 2x SSC at roomtemperature, oncewith 2x SSCplus 1 ,ugofRNase A permlfor30min atroomtemperature, and thenonce with 0.1x SSC-0.1% SDS at 50°C. Signals were detected by autoradiography.

RESULTS

Identification of CRS-like elements in thegagpolregionof HIV-1. Having

successfully

used a heterologous assay to

examine the CRS present within the env region of HIV-1 (34), a similar strategy was utilized to

identify

functionally

identical sequences within othercodingregionsof the HIV-1 genome. gag,pol, or gagpol

regions

ofHIV-1 were fused downstream of the termination codon of the CAT gene and the effect of the fusion on CAT

expression

was assessed

followingtransfection

(Fig.

1).

Analysis

of the CAT expres-sion levels (Fig. 1) revealedthataddition of the gag and gag polregions resulted inan

approximately

sevenfold decrease

in the levelof CAT

expression.

However, additionof thepol

pill CAT

227

pIlCAT gag

S.

*

.

1- 0

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5308 COCHRANE ET AL.

A

TAA

pSVCAT SV40 CAT rPoIYA 1

-___

_

"AA

pSVCP490 SV40 I CAT iPo1yA

3703 93

rAA

pSVCPA967 SV40 CAT Pol

3703 3729

IAA

PSVCP,&968 USV401 CAT YA

3793 3817 TAA

pSVCPA971 [j0o CAT PoPolyA

3703 3918

TAA

pSVCPA972 isv40o =AT Poly

3703 4030

B

1 .3 4 5

1 2 3 4 5 6 7

C

_

.*

* *

t 2 3 4

TAA

pSVCP270

LSV4o

CAT

3795 4055

FIG. 2. Deletionmapping of thepolCRSelement. TomaptheCRSelementwithin thepolfragmentutilized inFig. 1,subregionsofthis fragmentweretestedforinhibitoryactivity. (A) Schematic diagramofconstructsusedtodefinetheCRSelement withinpol.Numbersshown indicatethe 5' and 3' boundaries of the fragmentstested andarenumberedbythenumbering systemof Ratner et al.(32). (B)CATactivities generatedfollowing transfection of 3' deletionconstructsinto COS cells. Lanes:1,pSVCAT, 91.9%; 2,pSVAR,7.1%; 3,pSVCP490,21.7%; 4, pSVCPA967, 91.5%; 5, pSVCPA968, 75.8%; 6, pSVCPA971, 56.3%; 7, pSVCPA972, 15.4%. (C) CAT activities generated following transfection of 3' and 5' deletionconstructs into COS cells. Lanes: 1, pSVCAT, 83.4%; 2,pSVCP490, 11.8%; 3, pSVCPA972, 8.0%; 4, pSVCP270, 13.9%. Percentages indicated refertopercentconversionofchloramphenicoltooneof theacetylatedforms.

region alone effected an approximately 30-fold decrease in the level of expression. The failure to observe a similar

30-folddecrease with thepIIICATgag/polconstructsuggests that positional effects may influence the expression of the

inhibitory elements and that the CRSelements within both

gag and pol probably act independently of one another. Alternatively, the presence ofa major splice donor within

the gag fragment used in these experiments may have permitted excision of the repressive sequences by using a

downstream cryptic splice acceptor, thus permitting CAT expression. While addition of thepol region was found to

elicitthesamedecreaseasthatpreviouslyobserved withenv

(pIIIAR), in the case ofpIIICATpol, expression of Rev in

trans did not rescue gene expression. This observation suggested that either the mechanism ofrepression effected

bypolwas different from that ofenv or that the construct

lackedtheelement requiredtomediatetheeffect ofRev.To

test the latter hypothesis, the RRE within env was added ontothepIIICATpolconstruct(generatingpIIICATpolRRE) and theeffectof Revexpressionwasexamined. Additionof

Revin trans, andthe RREin cisresultedinrescue of CAT expression to levels observed with pIIICAT (Fig. 1). No

rescueofexpression wasevident uponaddition of theRRE

alone.Together these data suggestthat the CRS element(s) identified withinpol are at least functionally equivalent to those previously characterized in env.

Deletion mapping of CRS elements within pol. Deletion analysis was subsequently used to identify a minimal CRS

element in pol. To facilitate subsequent RNA analysis, experimentswereperformed inCOS cells, necessitating the

use of SV40 promoter-based vectors. A 490-bp region, corresponding to nucleotides 3703 to 4193, was found to significantly repress CAT expression when placed in cis (Fig. 2). Whilethe490-bpsequenceisinhibitory, itsextentof inhibition isnotasgreatasthatobserved usingthefull1.7-kb

pol region. This observation suggests that additional

se-quences within the 1.7-kb pol region may operate in

con-junction with the CRS element in the 490-bp fragment to effect the full inhibitory capacity of the region (indeed, a second inhibitory sequence has beententatively mapped to the deleted region in subsequent experiments;Cochraneand Rosen [4a]). Subsequent work focused on the 490-bp

frag-ment. By introducing further deletions,we found that (Fig.

2)greatestinhibitionwasobserved withaconstruct contain-ing nucleotides 3703 to 4030 (pSVCPA972), with further 3' deletions resulting in a significant increase in CAT expres-sion. Having established a 3' boundary for the repressive element,asimilarstrategywasemployedtocharacterizethe 5' boundary. The conclusion of these studieswasthe dem-onstration that a 260-bp sequence, comprising nucleotides 3795to4055, wascapable ofinhibiting CAT expressiontoa

similar extent as that observed with the 490-bp fragment

(pSVCP270; Fig. 2).

While the repression observed with pSVCP490 was not

relievedby expression of Rev intransalone, addition of both the RRE in cis and Rev intranselicitedamarked increasein

CAT expression (Fig. 3). These observations indicate that the490-bp regiondoes containabona fideCRS-like element.

The repressive effect of this sequence was found to be orientation dependent; inversion of the 490-bp sequence

resulted in an increase in CAT expression. The orientation

dependence of the sequence and its location within the construct strongly suggest that its effect ismediated at the posttranscriptional level.

The presence ofa CRS element within one of the

struc-turalgenesofHIV-1suggestedthatasimilarelementmaybe present within homologous regions of related viruses. To

test this hypothesis, regions of HIV-2 (Rod) and simian immunodeficiency virus (SIV) with extensivesequence

sim-ilaritytothe490-bp sequencedefined abovewere cloned 3'

ofthe termination codon of CAT and their effects on CAT

expression following transfection were examined. The

se-quence from HIV-2 inhibited CAT expression (pSVCAT

Rod490)toasimilarextentasthatobservedwiththe490-bp

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A

pSVCP490 RRE

pSVCP094

pSVCPRod 490

pSVCPSIV490

TAA 7204 7684 SV40 CAT | pol tRRE PoyA

3703 4193

TAA

SV40 CAT pol PolyA

4193 3703 TAA

SV40 CAT 1 pol JPolyA|

3969 4489

TAA

SV40 CAT pol |PolyA

4416 4936

B

1 9 A r R

C

IL 123 4 5 7* 8910

1-

-o

o o o_ ..0... 0

I ' a Lf D 0 1 2 3 4 5 6 7 8 9 10

FIG. 3. Effect ofRevexpressionandsequence polarity on the inhibition mediated by thepol490-bp sequence of HIV-1 and examination

of its homologs inHIV-2 andSIV.(A)Schematic diagram of constructs used to assess the capacity of Rev to reverse the inhibitory effect of

thepol 490-bp sequence and the dependency of the inhibitory effect on thepolarityof the sequence. The RRE used in theindicated constructs

corresponds to theregion ofenv encompassed by nucleotides 7204 to 7684 (32). For constructs pSVCPRod490 and pSVCPSIV490, the

indicated nucleotidesdelineate the regions of HIV-2 (Rod) or SIV found to have extensive homology to the HIV-1pol490-bp sequence

(numbering ofRod andSIVregions according to GenBank sequence). (B) Effect of Rev expression in trans on the inhibition mediated by the

pol490-bpsequence. Lanes: 1, untransfected cells, 0%; 2, pSVCAT, 85.1%; 3, pSVAR without Rev, 11.5%; 4, pSVAR with Rev, 92.3%; 5,

pSVCP490 withoutRev,1.61%;6,pSVCP490 with Rev, 4.57%. (C) Effect of pol 490-bp sequence inversion and addition of RRE. Lanes: 1,

pSVCAT withoutRev,96.5%;2,pSVCATwithRev, 95.0%; 3, pSVAR without Rev, 2.8%; 4, pSVAR with Rev, 23.0%; 5,pSVCP490RRE without Rev, 10.5%; 6, pSVCP49ORRE with Rev, 54.2%; 7, pSVCP490 without Rev, 15.7%; 8, pSVCP094 without Rev, 35.8%; 9, pSVCPRod490 without Rev, 4.6%; 10, pSVCPSIV490 without Rev, 45.1%. Percentages shown indicate the percent conversion of

chloramphenicolto one of the acetylated forms.

HIV-1 element. In contrast, the homologous region of SIV

wassubstantially less efficient in repressinggeneexpression

(Fig. 3), with only a maximum twofold repression in CAT

expression observed.

Demonstration of the inhibitory effect ofthepolsequences

using an INexpression system. Theuseof the CAT

expres-sion systemto testforthepresenceof CRS elements, while sensitive, suffers from the caveat that results obtained in sucha heterologous system may notreflectwhat occurs in virus infection. To address this possibility, a second assay

system was employed to verify the repressive effect ofthe pol region. The 490-bpsequencedefined aboveencompasses alargeportionof the HIV-1 IN domain.Therefore, onemay

anticipate that IN expression would display a Rev

depen-dencysimilartothatobserved forpSVCP49ORRE. By using thepBC12vectorsystem(5), the regionofpol corresponding

totheINgenewascloned 3'oftheratpreproinsulinIIintron andINexpressionwasexamined after transfection into COS

cells. No IN expression was detectable after transfection with either pBC12BI/HIN or pBC12BI/HIN+RRE in the

absence of Rev (Fig. 4). However, a significant level of IN

expressionwasdetected from thepBC12BI/HIN+RRE con-structwhen Rev was expressed in trans. The second band observed in cells transfected with plasmids pBC12BI/ HIN+RREand pSVRev would appeartobe a degradation

product, given its absence in any of the other lanes. This

observation supports the previous findings using the CAT

assay system andconfirms that this domain ofpolcontains

repressive elements whoseaction can be reversedby addi-tion ofthe RRE and Revin cis and trans, respectively.

Examination of the mechanism of pol 490-bp sequence inhibition. In light ofprevious studies which indicate that inhibition of viral structural gene expression is due to the

sequestrationof thecorrespondingmRNAsinthe nucleus of thecell(9,15,16,25),itwasofinteresttodeterminewhether a similar mechanism applied to the inhibitory effect of the

CRSelements definedabove. Accordingly, cellstransfected

with plasmidscontainingthepol490-bp CRS elementwere

fractionated into nuclear andcytosolic componentsand the distribution of expressed mRNA was determined. Analysis

of CAT mRNA generated from plasmids pSVCP490 and

pSVCP49ORRE

(Fig. 5)revealed that incontrast to

previous

studies (9, 15, 16, 25), the inhibition of CAT

expression

effectedby thepol490-bp sequence could notbe

explained

by sequestering of mRNA containing the sequence in the

nucleus ofthe cell, despite the marked reduction in CAT

expression. AsignificantamountofCAT-containingmRNA was detected in the cytoplasm. In addition, the inhibition observed couldnotbeattributedsolelytoalterations in CAT RNA steady-state levels, because the abundance of RNA

generatedfrom thepSVCP490vector wascloseto that from

pSVCAT. Furthermore, rescueof CAT

expression,

effected

by addition of the RREwith Rev in trans, wasnotfound to

be associated with a dramatic alteration in CAT RNA

subcellular

distribution,

although

some increase in CAT

mRNAabundancewasnoted. To address the

possibility

that

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5310 COCHRANE ET AL.

A. pBC12BI/HIN

5

SV40 RSV LTR

on

sd 3'sa II

HIV IN

B.

pBC12BI/HIN+RRE

5'sd 3'sa

SV40

RSV

LTR

on

polyAs BstEll/(CIal) (Srmal)/(BstEll)

HIV IN

...:.

rS:'alwEma:

HIV RRE (Ndel)/(Smal)

FIG. 4. Dependency of HIV-1 INexpression onRREand Rev.

(A and B)SchematicofexpressionvectorsusedtoexamineHIV IN

expressioninCOScells.Abbreviationsandsymbolsareasfollows:

SV40 ori, SV40 origin of replication; RSV LTR, LTR of Rous sarcomavirus;5'sdand3'sa,5' splice donor and 3' spliceacceptor

sites, respectively,of the ratpreproinsulin intron; HIVIN, coding

region of the HIV-1 IN; RRE, RRE (nucleotides 7204to7684) of HIV-1; polyA+, polyadenylation site. (C) Analysis of HIV IN expressionfollowing transfection into COScells. At 48h

posttrans-fection,cells weremetabolically labelled with [35S]methionine and

theresultinglysatewasusedforimmunoprecipitation using antisera raised against bacterially generated HIV IN protein. Lanes: 1, pBC12BI/HIN+RRE cotransfected with pSVRev; 2, pBC12BI/

HIN+RREtransfectedwithoutpSVRev; 3,pBC12BI/HIN;4, cells

transfected withpSVRev alone.

DISCUSSION

1 2 3 4

thepresenceof RNA in the cytosol wasduetoleakage from

the nucleus during fractionation, cells were cotransfected

withthepgTatvector(25). The pgTatvectorconsists ofthe

twoTatexons separated by an intron corresponding to the

env gene of HIV-1. Previous work with this vector has established that in the absence ofRev, the spliced form of the RNAis transportedtothe cytoplasm while theunspliced form remains entrapped in the nucleus. Analysis of the distribution ofthe unspliced form ofpgTat RNA withinthe fractions used to examine the distribution of pSVCP490-generated RNA (Fig. 5) revealed that unspliced RNA was

detectable only in the nuclear fractions. Thus, results with pSVCP490cannotbe attributed toleakage of material from thenucleus into the cytoplasm.

In contrast to our findings with the CAT-based vectors,

analysis of the subcellular distribution of pBC12BI/HIN+ RRE-generated RNA (Fig. 5) gave results similar to those previously reported (9, 15,16, 25). Inthe absence of Rev, IN mRNA was detectable only in the nuclear fraction of the

cell.Addition ofRevresultsin theappearanceof IN mRNA

in thecytosol.

In previous studies using the env sequences of HIV-1, CRS elements which functionto repressgene expressionin

the absence of Revwereidentified(34). Since therepression could be alleviated by expression ofRev, it was suggested

that these sequence elements are involved in regulation of HIV structuralgeneexpression. Other studies (8, 15), dem-onstratingasimilardependency uponRev forexpression of the gag gene products, suggested that CRS elements are

likely present within other structural genes of HIV. In this study, wehaveverified this hypothesis usingaheterologous

assaysystem. Weobserved that addition of either thegag or

pol sequences in cis resulted in reduction of CAT gene

expression. Sequences present within pol were found to

elicit the greatest inhibitory affect. To delineate the CRS elements, subregionsof thepolgene wereexaminedforthe

presenceofinhibitorysequences. Initialexperiments

identi-fieda 490-bpsegment thatcontained a CRSelement which wasfurthermapped to a260-bp sequence correspondingto nucleotides 3795 to 4055 of strain Hxbc-2. The deletion mapping demonstrates that the splice acceptor within this region is not required for the repressive capacity of the

sequence, since equivalent inhibition of CAT expression is

observed with mutant pSVCPt\972, which lacksthe known splice acceptor. Thiswould suggest that splicing signals do not contribute to the inhibitory effects of this region. Our datadonotruleoutthepossibilitythat theCRS elementsare

capable ofinhibiting splicing events which occur in other

regions of the vector. However, experiments designed to

examine thispossibilityhavefailedtodetect suchanactivity

(data not shown). Furthermore, deletion of the intron from

poIA+

BsiEll

(Ndel)/(Smal)

C.

kD

HIV

IN-- 30

-21.5

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I

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pSVCAT

I

-'--CP490

+neo +rev +neo

N

C

N

C

N

C

e No

S_

cmO

C o

0.O

CO

- - + -

re

C

pSVCAT pSVCP49ORRE

r--

+

I _ 1 [

F--REV: N C N C N C N C

_ *

p

a

pSVCAT pSVCP49ORRE +

thepBC12BI/HIN+RREconstructhadnoeffecton

expres-sion orRev responsiveness (datanot shown). Thefunction of theCRS elementis orientationdependent,because inver-sion ofthe sequence alleviated a portion of the repressive

effect. Since the HIV-2 virus isvery similartoHIV-1 in its

d

b

pSVCAT

pSVcp49D

+neo

+Rev

+neo

I

I

111

N

C

N

C

N

C

M

HIN

ARRE

Rev +

.. I

Nuclei

-

probe

- unspliced

-

spliced

M HIN

RRE

FI771

Cytosol

FIG. 5. Analysis of subcellulardistribution of RNAs containing

pol CRS element. (a) (Topgel) Aliquots of RNA from nuclear (N)

andcytoplasmic (C) fractions of cells transfected with pgTat

to-gether with pSVCAT or pSVCP490 and pSVNeo (+neo) or Bl-SVRev(+rev). TheseRNAs were hybridized with a CAT-specific

probeand treated with S1,and samples were run on adenaturing polyacrylamide gel. (Bottom gel) CATassays ofcells utilized for RNAanalysis. (b) Aliquots ofRNAtreated as described above were

hybridized with aprobefor spliced andunspliced Tat RNA. The

three bands visible following S1 treatment represent reannealed

probe, the unspliced form ofTatmRNA,and thesplicedform of Tat mRNA. AfterS1treatment, sampleswereanalyzed ondenaturing polyacrylamide gels.(c)(Topgel) Aliquotsof RNA from nuclear (N) andcytoplasmic(C) fractions of untransfectedcells or cells

trans-fected with pSVCAT or pSVCP49ORRE in the presence (+) or

absence(-) ofRev were used forhybridization withCAT probe. (Bottomgel) Corresponding CATassaysfrom cells used forRNA

analysis. (d) Analysis of subcellular distribution ofHIV IN mRNA.

Following fractionation of cells into nuclear (nuclei)orcytoplasmic (cytosol) fractions, aliquots ofRNA were analyzed by Northern blotting. Lanes: M, mock-transfected cells; HIN+RRE, pBC12BI/

HIN+RRE transfected cells inthepresence(+)orabsence(-)of

Rev.

regulationof geneexpression,weanticipatedthepresenceof

functionally equivalent CRS elements within the

corre-sponding regionof the HIV-2polgene. Ouridentification of ahomologous sequenceinHIV-2,withinhibitory properties

similartothoseof the element inHIV-1,suggeststhat these

a

a

I-A 5* a

REV:

ireAM

q

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on November 10, 2019 by guest

http://jvi.asm.org/

[image:7.612.328.551.89.461.2]

5312 COCHRANE ET AL.

elementsare likely important in regulation of virus structural gene expression. As observed with the entire

pol

sequence,

reversal of the inhibition with the 490-bp element could be achieved by addition of the RRE in cis together with

expressionof Rev in trans. This ability of Rev to reverse the

inhibition imparted by these sequences indicates that the

repressive element within pol is at least functionally related tothose previously characterized in env (34).

Upon analysis of the CRS-containing CAT mRNA, no gross alteration in abundance or subcellular distribution was

observedthat could account for the lack of gene expression. However, analysis of RNA generated from the IN expres-sion vectors was consistent with previous observations and

suggested that RNA containing a CRS element can be

entrappedin the nucleus in the absence of Rev. The basis for the difference between the two expression systems is un-clear. There is some support for the notion that sequence

context can affect the function of particular sequences. The iron-responsive element can affect mRNA translation or

stability, depending on the mRNA species analyzed (42). Theability to replicate previous findings in our system using the pgTat vector (25) as well as the IN expression vector

indicates that methodological problems such as gross nu-clear leakage of RNA did not occur during fractionation.

Therefore, our inability to detect similar changes in subcel-lular distribution with the heterologous CP490 transcripts is noteasily explained. One possibility is that because of some

peculiarity of sequence context in the

pSVCP490

transfec-tants, a significant amount of CAT mRNA is transported to the cytoplasm through a nonproductive route. In this case, Rev might still effect transport but we would not be able to

discriminate between RNA entering the cytosol through a

nonproductive route and the Rev-mediated exported RNA which is eventually translated. Regardless of the specific

mechanism, observations made with the heterologous sys-tem clearly showed that the requirement for Rev in gene

expressionis not solely in RNA transport from the nucleus but that Rev is capable of affecting the utilization of target mRNA by the translational machinery within the cytoplasm. A similar observation has been made by Ariggo et al. (1).

Analyzing the subcellular distribution of env mRNA, these

investigatorsobserved that RNA is present in the cytoplasm inthe absence and presence of Rev but is found associated with polysomes only after Rev expression. The ability of Rev toalter the translational properties of target mRNAs has alsobeenobserved in a similar heterologous system utilizing

CRS elements from the HIV-1 env gene (23a). The observa-tionthat Rev can affect the translational capacity of its target mRNA bears striking similarity to the effects of Tat on

TAR-containing mRNA, as demonstrated in the Xenopus

oocyte system (la). In this system, it was observed that

injected mRNA containing the TAR element is translated only when RNA is injected into the nucleus in the presence ofTat. The requirement for Tat and nuclear

injection

sug-gests that Tat interaction with the RNA results in the mRNA being translationally competent upon transport into the

cytoplasm. The close similarity of the effects of Tat in the Xenopus oocyte system and those of Rev on the reporter system used here are of particular interest, because both

proteins demonstrate strong accumulation in the nucleoli of

transfected cells (21, 24, 30, 36) and both are RNA-binding

proteins (4, 6, 18, 26, 35, 45), which have the potential of

modifyingthe interaction of other cellular factors with their

respective target mRNA.

Results from other Rev-dependent expression systems have suggested that nuclear entrapment of HIV structural

mRNA is the

by-product

of an inefficient splicing process.

Results obtained

by Chang

and

Sharp

(2),

demonstratingthat

mutation of either

the

beta-globin

splice

donor or acceptor

sequence

in a

heterologous

expression

vectorelicits nuclear

entrapment

of

mRNA,

which can then be efficiently

ex-ported

to the

cytoplasm

by placement

ofthe HIVRRE incis

and

expression

of

Rev

in

trans,

lendsfurther support to this

hypothesis.

The

ability

of the

CRS

elements, which

appar-ently

lack functional

splicing

signals,

to elicit the same

outcome

as that obtained with inefficient splicing does not

necessarily

conflict

with

this

interpretation.

We speculate

that cellular factors which

interact with the HIV splicing

machinery may

also interact with

cryptic

elements present

within the CRS

sequences.

In

support

of this hypothesis, we

have found

that

specific

cellular factors interact with the

HIV-1

and HIV-2 CRS elements and that monoclonal

anti-bodies directed

against

specific

splicing

components can

interfere with

complex

formation

(unpublished

data).

Fur-ther studies on

the nature of these interactionsand their role

in nuclear

entrapment

of HIV

RNA will likely add to our

understanding

of those

processes

that controlmRNA metab-olism.

ACKNOWLEDGMENTS

We thank Tina Rose for

preparation

of this manuscript and

J.

Kulkosky

for

providing

antibodies to HIV IN. Alan Cochraneis the

recipient

of an

AmFAR/F.L.

and

E.L.

Cummings ScholarAward.

This work was

supported

in

part

by a National Drug Discovery

Award(C.A.R.),

by

National Institutes of Health grant CA49042,

CA-06927, andRR-05539, a

grant

from the Pew Charitable Trust,

and also

by

an

appropriation

from the Commonwealth of Pennsyl-vania (A.M.S.).

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