Integration is essential for efficient gene expression of human immunodeficiency virus type 1.

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0022-538X/93/031169-06$02.00/0

Microbiology

Integration Is

Essential for Efficient Gene Expression of

Human

Immunodeficiency Virus Type

1

HIROYUKI SAKAI,' MEIKOKAWAMURA,' JUN-ICHI SAKURAGI,12SAYURISAKURAGI,' RIRISHIBATA,1AKINORI ISHIMOTO,' NOBUMI ONO,2 SHIGEHARU UEDA,2

ANDAKIOADACHI1*

Institute forVirusResearch, Kyoto University, Kyoto 606,1andResearch Institute forMicrobial Diseases, Osaka University, Osaka 565,2 Japan

Received 7August 1992/Accepted 25 November 1992

A mutant of human immunodeficiency virustype 1 which carries a frameshift insertion in the integrase/ endonucleaseregion ofpolgenewasconstructed invitro. Upontransfection intocells, althoughthismutant exhibitedanormalphenotypewith respecttoexpressionofgag,pol,andenvgenesandtogenerationofprogeny

virions,noreplication-competent virus inCD4-positivecellsemerged.Anassayforthesingle-step replication ofa defective viralgenomedependent on trans complementation byrev proteinwasestablished and used to monitorthe early phase of viralinfection process. Viral cloneswith a mutation in the vif, vpr, or vpu gene

displayedno abnormality intheearly phase. In contrast, theintegrase mutantdid notdirecta markergene

expression afterinfection.Togetherwithanobservation that themutant lackedtheabilitytointegrate,these results indicated that theintegration wasrequiredforefficient viralgeneexpressionandproductiveinfection of humanimmunodeficiency virus type 1.

Thereplication cycleofretrovirusesconsists oftwomajor phases (4, 29). The first half of thecycleis directed toward theintegration ofacompleteDNAcopyof viralgenesinto hostcell DNA.Shortlyafterentryintosusceptible cells, the viral single-strandedRNAgenomeisconverted intoalinear

double-stranded DNA. The viral DNA intermediate then migratestothe cellnucleus and iscovalently integrated into

a host chromosome. The second phase begins with the expression of the genes encoded in the provirus and ends with theproductionofinfectiousprogenyviralparticles.The two events in the early phase of the replication cycle,

reverse transcription and integration, distinguish

retrovi-ruses fromother classes of virus. Thesetwo processesare

mediated by the two enzymes (reverse transcriptase and integrase) encodedbyavirussingleopen readingframe,pol

(5, 7).

IntegrationofaDNAcopyof theviralgenomeinto host

cellular DNA isan essentialstepinthe lifecycleofmost,if not all, retroviruses (4, 12, 29). Integration appears,

more-over,tobeimportantfor thetranscriptionof viral DNA into new copies of the viral genome and mRNAs that encode viral proteins (4, 12, 29). Integration-defective mutants of retroviruses are able to produce a DNA copy of the viral

genome,but thereplicationof themutantsinthetargetcells

is almostnegligible (6,14, 19, 20, 24).Therearesomereports

onthefunctional role of theintegraseof human

immunode-ficiencyvirus type 1 (HIV-1) inthe replication cycle. Viral clonescarryingamutation in theintegrase regionofpolgene wereunable to integrate (25)andwerereplicationdefective

(2, 3, 12, 25). Surprisingly, in one article (25), the uninte-grated HIV-1 DNA was reported to serve as an efficient template for HIV-1 antigen production. Because the

inte-grase of HIV-1 is very likely to function in the early

replication phase asdo those of the otherretroviruses, this result might represent a unique property of the HIV-1 integrase.

* Correspondingauthor.

In thiscommunication, an integration-deficient mutantof HIV-1 was constructed and analyzed in detail in several transfection and infection experiments. We demonstrate here thatthe HIV-1 integrase functions in the early replica-tion phase and that integration is important for viral gene expression.

MATERIALSANDMETHODS

Cell culture and DNAtransfection. Ahumancolon carci-noma cell line, SW480 (1), and a monkey kidney cell line, COS-1 (9), were maintained in Dulbecco's modified Eagle

medium supplemented with 10% heat-inactivated fetal calf

serum.CD4+humanT-cellleukemic celllines, A3.01 (8)and H9 (21), were maintained in RPMI 1640 medium

supple-mented withheat-inactivated fetal calfserum.For transfec-tion,uncleavedplasmidDNAwasintroduced intoadherent (SW480 andCOS-1) andnonadherent (A3.01 andH9) cells bythecalcium-phosphate coprecipitation (11, 30) and mod-ifiedDEAE-dextran (27)methods, respectively.

Infection. The infectivityof progenyvirions produced in the transfected SW480 cells was assayed in A3.01 cells. Culturesupernatantswerefiltered(poresize,0.45 ,um),and

appropriatevolumes were added to 106 cells aspreviously described(8).

RT assays. Virion-associated reverse transcriptase (RT) activity was measured as described previously (31). For quantitation, spots on DE81 paper were cut out, and RT activitywas determinedbyscintillation counting.

CAT assays. The chloramphenicol acetyltransferase (CAT)assayhas beenpreviouslydescribed(10).CAT levels were

assaypd

in equivalent amounts of cell lysates from transfected SW480cells and infected A3.01 cells.

DNA constructs. An infectious proviral clone of HIV-1, pNL-432, and its mutants, designated pNL-Afl

(pol/inte-grasemutant), pNL-Nd(vif mutant), pNL-Af2 (vpr mutant),

andpNL-Ss (vpu mutant), have been previously described (1, 2, 17). pNL-nCATandthemutant clones ofpNL-nCAT

were constructed from pNL-432 (1) and itsmutant clones,

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which have been previously described (2, 17). To make a series of pNL-nCAT constructs, the CATcoding sequence of pSV2CAT (784-bp HindIII-Sau3AI fragment) (23) was inserted into the XhoI (nucleotide [nt] 8887) and

KpnI

(nt 9005) sites in the nef coding region of parental clones, and therev coding sequencewas inactivated byinsertion of an 8-bp XbaI linker (Toyobo Inc., Osaka, Japan) into the BamHI site (nt 8465) (see Fig. 2). pNL-nCAT-Nh was generated from pNL-nCAT by digestion with the NheI restriction enzyme, by blunt ending with T4 DNA poly-merase,andbyresealingwith T4 DNAligase. Construction of pNL-gCAT and prevl has been describedpreviously(22, 23).

EM.Transfected SW480 cells wereprocessed for electron microscopy (EM) by the standard method (2). Briefly, the cells were prefixed with Karnovsky's fixative (1.0% paraformaldehyde-3.0% glutaraldehydein 0.2 Mcacodylate buffer [pH 7.4]) at 4°C for 1 h, postfixed with 1% osmium tetroxide at 4°C for 1 h, and then dehydrated in a graded ethanol series and embedded in epoxy resin. Ultrathin sectionswereprepared,stainedwithuranylacetateandlead citrate solution, and observed under a Hitachi HU12A electronmicroscope.

Extractionof extrachromosomalandintegrated virus DNA. Extrachromosomal virus DNA in the infected cells was extracted at 10 h postinfection bythe method of Hirt (15). High-molecular-weightcellulargenomicDNAwasextracted from cells 2 days after infection by a standard proteinase K-phenol spooling method. RNAwas removed fromDNA solution by digestionwith RNase H and RNase T1 (Sigma Chemical Co., St.Louis, Mo.).

PCR analysis of viralDNA.DNAsampleswerepretreated withDpnI restriction endonuclease (New England Biolabs, Inc., Mass. USA) to remove plasmid DNAs potentially contaminated. Polymerase chain reaction (PCR) amplifica-tionwasperformedwithtwoprimers,V3DF(nt6990to7011 ofpNL-432) and SK69 (nt 7927 to 2907 ofpNL-432) (18). Samples(2,ugofDNA)weresubjectedto24cyclesof PCR ina50-,u reaction mixture. Compositionofreaction mixture was as the supplier suggested for Tth DNA polymerase (ToyoboInc.). Eachcycleconsisted of 1 min of denaturation (93°C), 2 min of annealing (57°C), and 3 min ofextension (72°C). Amplifiedproductswere runthrough a3%NuSieve GTG agarose gel (FMC BioProducts, Rockland, Md.) and analyzed by Southernblottinghybridization.The

32P-labeled

SalI (nt 5785)-XhoI (nt 8887) fragment produced by the random primerDNAlabeling kit (Takara Shuzo Co., Ltd., Kyoto,Japan)wasused asaprobe.

Nucleotide sequence accession number. Nucleotide se-quence data of pNL-432 are from GenBank data base accession number M19921.

RESULTS

TransfectionanalysisofHIV-1integrase mutant.Aproviral DNAclone of HIV-1, designated pNL-Afl, which carriesa frameshift insertion within the integrase region of pol gene (2)was transfected into CD4-negative SW480 cells (1) and examinedforitsexpression.Wepreviously reported that the mutant expressed viral structural proteins and produced progeny virions normally in transfected SW480 cells, as judged byWestern immunoblotting analysis and RTassay, respectively(2). As shownin Fig. 1A, this clone could also direct the synthesis of progeny virions morphologically similartowild-type (wt) particlesupon transfection, andno

significant

difference between the amounts of progenies

AfA1 (IN)

4432

(WT

432(WT)

B

input 1 3 5 7 9 11 14 16

mock

Af 1

(IN)

*

[image:2.612.309.545.80.382.2]

432(WT)

,*

0

FIG. 1. Characterization of an integrase mutant of HIV-1. SW480 cells were transfected with pNL-432 (HIV-1 wt) (1) or pNL-Afl (HIV-1 integrasemutant) (2), and48 hlater,sampleswere prepared forEM(A) and for infection (B). (A)EMpicture of virions produced by transfected SW480 cells. Afl(IN),virus from pNL-Afl;432(WT),virus frompNL-432.Bar = 100nm.(B) Infection of CD4-positivecells withvirus.Forinfection,A3.01 cells(106)were infected with 105 RT units of cell-free virus obtained from trans-fected SW480cells (input). RTproductionin the culture fluidsof infected cellswas monitored atintervals. Virus designationis the

sameaspresentedinpanelA.Values indicatedaysafter infection.

produced by wt and mutant clones was observed. The NL-Afl virus obtained from transfection, however, was unable to establish a productive infection in

CD4-positive

A3.01 cells. RT activity was not detected in the cells inoculatedwith the NL-Afl onanydayspostinfection (Fig. 1B). These resultswere consistent with those presented in previousreports,includingours(2, 3, 12,25), andsuggested that the viralintegraseprotein playsanimportant functional role in theearly phase of viral replication cycle, atleast in the SW480-A3.01 system. To examine theearly

replication

phasefurther, anewassay systemtoquantitateviral infec-tivity (from virus entry to transcription) similar to those previously described (13, 28) was established. This system utilizedreplication-defectiverev-minusproviralclones con-taining the CAT gene in the nef region (Fig. 2) and was

dependent on trans complementation for

production

of in-fectiousvectorvirus(Fig. 3).Whentherev wassupplied in trans, infectiousviruses,which carrythe CAT gene andare capable of undergoing one round of replication, would be generated (Fig. 3). Infection of cells with the virus and the CAT activity obtained would provide a measure of the efficiency withwhich the early infection phase of the virus

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Stop codon

Irevf1F-n

Fv7lf

-t-at

-l nef|

pol

11 jIdl

Rjejnv

Aflii NdeIAflii SSpI Nhei

Frameshift

_ - rev - ]

vif

|1

-tat - |nef

j \ pol Ivprl

ElI

env

3D

O 50 100

% CONVERSION

I

I~~~~~~~~~~~~~~~~~~~~

rev

I+

pNL-432 (WildType)

0 0.5 1

[image:3.612.69.308.77.435.2]

RELATIVE RT PRODUCTION

FIG. 2. Replication-defective proviral clones carrying a CAT

geneused forasingle-roundinfectionassay.(A)Genomestructures

of pNL-nCAT and pNL-gCAT. pNL-nCAT constructed in this

studycontainaCATgenein thenefgeneregionandanXbaI linker

insertion in the 2ndcodingexonof therevgene.Asaresultof the linkerinsertion, therevproteinwould stopatthe60th aminoacid,

and theenvprotein wouldhavea4-amino-acid insertionatthe site.

The mutant clones of pNL-nCAT, designated pNL-nCAT-Afl, pNL-nCAT-Nd, pNL-nCAT-Af2, pNL-nCAT-Ss, and pNL-nCAT-Nh, have frameshift mutations at the restriction enzyme sites

indicated. Generation and characterization of pNL-gCAT were

described previously (22). (B) rev dependency of marker gene

expression.pNL-nCATandpNL-gCAT (5 ,ug)weretransfected into

SW480 cells with(+)orwithout (-)revexpression plasmidprevl (10p.g).Twodays after transfection, CAT activityin the celllysates (upper graph)andRTproductioninto culture supernatantsrelative

tothatby pNL-432 (5jig)(lower graph)weredetermined. RTcounts produced bypNL-432were2,885cpm.

proceeds. As shown in Fig. 2B, while the pNL-nCAT construct (wt) forthis assay expressed CATactivity inde-pendently of the presence or absence of the rev protein withintransfected SW480 cells, itsprogenyproduction into aculture supernatantwas revdependent. Aseries of pNL-nCAT mutant constructs (Fig. 2A), which carry the same alteration in eitherthepol (integrase)orthe othergenes(vif,

vpr, vpu, andenv [gpl2O])aspreviously reported (2, 17) in

addition to the rev gene mutation, were transfected with

prevl (HIV-1 rev expression plasmid [22, 23]) into SW480 cells todetermine the effect of the mutationon the

expres-pNL-nCAT(rev mutant) prev 1

transfecton

SW480cells

virion production

trevmutant)

infection

CD4'cells

CATassay

(noprogenyproduction)

FIG. 3. A single-step infection assay. Virions recovered from SW480 cells cotransfected with replication-defective pNL-nCAT constructs(Fig. 2)andrevexpressionvectorprevl(22, 23)areable to undergo one round of replication but not multiple cycles of infection in CD4-positivecells. Earlyphaseof viral replication can be monitoredby the highlysensitive CAT assay.

sion of the marker CAT gene and the progenyproduction. Asis clear inFig. 4,noneof the mutations affected CAT and virusproduction, which is in agreement with our previous data(2, 17).To determine whether these results representa cell-specific phenomenon,several celllines, including CD4-positive A3.01 and H9, were transfected with wt or the integrase-minus mutantclone andmonitored for RT produc-tion. As shown inFig. 5,nosignificant difference, again, in the levelof progenyproductionin anyof thecell lines tested was observed. None of the progeny viruses produced in transfectedCOS-1, A3.01, and H9 cells were infectious (data notshown).

Ofparticular note is that the integrase mutant exhibited the wtphenotype in all transfection experiments described above. Because transfection ofaviral DNA clone into cells would bypass the early phase of viral replication (adsorp-tion, penetra(adsorp-tion, uncoating, and reverse transcription), these resultsarecertainly consistent with the requirement of the integrase proteinin theearlyinfection stage.

Analysisofearlyinfection phase ofthe mutant clones.The effect of mutations in the viral genes on the early stage of virusreplicationwasstudiedby the system described inFig. 3. Cell-free virussamples, collected onday 2 posttransfec-tion into SW480 cells, were inoculated into CD4-positive A3.01cells, and2 days later, celllysateswere prepared for CAT assay.Becausethegpl20envplaysacriticalrolefor the first step in the infection cycle, the env mutant carrying a frameshift insertion in the upstream region ofCD4-binding domain (Fig. 2A) was used as a negative control for this experiment. As shown in Fig. 6, approximately 100-fold increases in A3.01 cellCATactivitywereobserved between the positive (wt) and negative (env) control samples. In agreementwith thefindingthat the vpuproteinisrequired in the late infectionphase

(maturation/release)

(16), the

muta-A pNL-nCAT

gag

pNL-gCAT

gag

B

rev

pNL-nCAT

pNL-gCAT

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A

pNL-nCAT (wild type)

pNL-nCAT-Nh (env)

pNL-nCAT-Af1 (in t)

pNL-nCAT-Nd (vif)

pNL-nCAT-Af2 (vpr)

pNL-nCAT-Ss (vpu)

-~~~~---

~~~~~~~~~~~~~~~~~~~~~~~~~I

O 0.5 1.0

RELATIVE CAT ACTIVITY

I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

I

~~~~~~~~~~~~~~~~~~~~~I

mock

pNLnCAT

pNLnCAT-Afl

mock

pNLnCAT

CD4-negative SW480

CD4-negativeCOS-1

pNLnCAT-Afl

B

pNL-nCAT-Nh (env)

pNL-nCAT-Af1 (in t)

pNL-nCAT-Nd (vif)

pNL-nCAT-Af2 (vpr)

pNL-nCAT-Ss (vpu)

0 0.5 1.0

mock

pNLnCAT

I1

CD4-positive A3.01

pNLnCAT-Afl

mock

pNLnCAT

CD4-positive H9

pNLnCAT-Afl

[image:4.612.55.299.71.354.2]

FIG. 4. Expression of marker gene by transfectionofaseries of pNL-nCAT constructs. Theprovirus-CATclones(Fig.2)and prevl (revexpressionvector) were cotransfected into SW480cells,and 48 hlater, CAT activityin the cell lysates (A)and RTactivityin the culture supernatants (B) were determined. Relative activity is shown (that of wt pNL-nCAT = 1). CAT and RT activities

ex-pressed by the cellscotransfected with pNL-nCAT and prevl(black bars)were 24.8% (conversion ratio) and 3,124 cpm, respectively. Mutant designations and mutated genes (inparentheses) are indi-cated.

0 0.5 1.0

FIG. 5. Progenyproductionin variouscell lines transfected with theintegrasemutant.Four cell linesweretransfected withapair of pNL-nCAT(wt) plusprevl(rev supplier)orpNL-nCAT-Afl (inte-grase mutant) plusprevl, and 48 h later,RTactivityin the culture supernatantswasdetermined. Relative RT titer is shown. RTcounts

produced by various cell lines cotransfected withpNL-nCAT and prevl were 3,285 cpm (SW480), 1,898 cpm (COS-1), 1,825 cpm (A3.01), and 965 cpm(H9).

tion invpuhadnoeffectontheproductionofCAT. A similar

level of CAT activity was also observed in A3.01 cells infected with the viforvprmutantviruses. In contrast, no

significant increase in A3.01 CAT activity (relative to the activity of the negative control) was detected when the

integrasemutantwasusedas aninoculum. Theexperiments wererepeated three timeswithessentiallythesameresults.

Furthermore, the results were reproduced in two other T-cell lines(H9 andM8166)andamonocyticcellline, HL60

(data not shown).

To determine whether viral DNA is synthesized and integrated into achromosomal DNAof A3.01 cells infected with variousvirusclones,PCRanalysiswasperformed(Fig.

7). Hirt's DNA (15) and high-molecular-weight genomic DNAwereextracted from theinfected cellsatthe

appropri-atetime. Theenvsequence inthesampleswasamplified by thePCR procedure andprobed by a

3"P-labeled

viral DNA

fragment. While a937-bptargetof the HIV-1envsequence could be amplified in both Hirt and genomic fractions of A3.01 cells infected with wt virus, the env sequence was

detected inneitherfraction of thecells infectedwith theenv mutantvirus,asexpected (Fig. 7B). Consistent with thedata inFig. 6,mutantviruses of thevif,vpr,andvpugenes were able to synthesize viral DNA and integrate into host cell DNA, Although HIV-1 DNA was produced normally in

A3.01 cells infected with the integrase mutant, no proviral

DNAintegrated into cellular DNA was found. This result demonstrated theintegration-defective propertyof our inte-grasemutant.

DISCUSSION

The results presented in this study demonstrate that unintegrated viralDNAis anextremely inefficienttemplate for HIV-1expressioninthe natural infection system and that integration mediated by viral integrase protein is essential for HIV-1 productive infection. An integration-defective (Fig. 7) integrase mutant constructed in vitro (Fig. 2B) displayedthe wtphenotypein alltransfection assays(Fig. 4 and5),as wasthecasefor theoriginal integrasemutant(Fig. 1)(2). Theintegrasemutantvirus obtainedbytrans comple-mentation (Fig. 3), however, was unable to express the markerCAT gene after infection ofCD4-positivetarget cells in a single-step replication system (Fig. 6). Although the conclusion described above represents a sharp contrast to that ofaprevious report(25), it is consistentwith the data obtained with the other retroviruses (6, 14, 19, 20). If integration is not required for efficient expression of viral genes,asclaimed in aprevious article (25),onemust assume that the integrase has some functional role in the late infection phase because all integrase

mnutants

described so farareunabletoestablishaproductiveinfection. Our data in

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pNL-nCAT-Nh (env)

pNL-nCAT-Afl (in t)

pNL-nCAT-Nd (vif)

pNL-nCAT-Af2 (vpr)

pNL-nCAT-Ss (vpu)

0 0.5 1.0

RELATIVE CAT ACTIN

FIG. 6. Analysis ofearly phase of viral replicati4 round infectionassay. Virussamples obtainedfrom(

ofpNL-nCAT constructs and prevl (Fig. 3) were CD4-positive A3.01 cells, and 48 h later, CAT acti lysates was monitored. Relative activity is shown detected in thecellsinfected with the wt NLnCAT v (conversion ratio). Mutant designations and mut; parentheses)are indicated.

Fig. 1, 5, and 6 do not support this hypothesis ularbasis forrequirementofintegration toeffici viralgenes remains tobe investigated.

Anotherimportant finding in this report istU

vif,

vpr, and vpu proteins appear not to have role in theearlyinfection phase (Fig. 6). As fo vprproteins, this result was well expected. The mutantwasreportedtobe defective in the

ver3

viral replication cycle (16). The growth kineti mutantviruses in CD4-positive target cells were delayed relative to those of wt virus (17), anr difficult todetect the mutational effect. Incon twoproteins,the

vif

proteinwaspostulated to t theearly phaseofHIV-1infection in CD4-posit

A

Ftatv

tatJi

revE

SailI V3D-F

4-SK69

B

-- Z

HIRT GENON

FIG. 7. PCRanalysisofgenomic and HirtDNAs

A3.01 cells infected with various viruses. (A) L

primers V3D-F and SK69 within an env gene. L

restrictionsites (SallandXhoI) used to cut out a

32P-labeledprobe (usedforpanel B) is alsoindicatec

blot analysis ofthe amplified products by PCR. A2 infectedwithvirus asdescribed in the legendtoFi1

was extracted from infected cells for PCR ampli

appropriate time (see Materialsand Methods). The

were run through a 3% agarose gel and analyze( hybridization. Theresultsobtainedwith DNAsamp

chromosomalandgenomicfractionsareshown inpa

GENOMIC, respectively. Arrowheads indicate thes

amplified productspredictedfromthe location ofth

VITY

on by a single-cotransfections

The

vif

mutant virus obtained from transfected SW480 cells was normal with respect to infectivity as judged by a single-step infection assay (Fig. 6). This result showed that the

vif

protein isnot required in the early infection stage. A more detailed studyis necessary toprecisely determine the functional role of the HIV-1

vif

protein.

ACKNOWLEDGMENTS

M.K. is an awardee of a Research Resident Fellowship from the Japanese Foundation for AIDS Prevention. This work was sup-ported in part by a grant-in-aid for AIDS research from the Ministry ofEducation, Science andCulture ofJapan.

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