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VIROLOGY 184,187-196(1991) SyntheticPeptidesfromthePrincipalNeutralizingDomainofHumanhncideciency VirusType1(HIV-1)EnhanceHIV-1InfectionthroughaCD4-DependentMechanism ANITADEROSSI,* , ' MARCELLAPASTI,*FABRIZIOMAMMANO,*MARINAPANOZZO,*t MONICADETTIN,tCARLODIBELLO,t AND LUIGICHIECO-BIANCHI* `InstituteofOncology,lnteruniversiryCenterforCancerResearch ; tI .S.TBiotechnologySectionofPadova, and#instituteofIndustrialChemistry,UniversityofPadova,Padova, Italy ReceivedMarch18,1991 ;acceptedMay15,1991 Theprincipalneutralizingdomain (PND) ofHumanImmunodeficiencyVirustype 1(HIV-1)is mappedto a24-amino acidsequencelocatedinthehypervariableV3regionoftheviralenvelopeprotein .The PND ofNfV-1isSWafrom infectedindividualscorrespondsmostlytothatofthe HIV-1MN strain .Wefoundthatapeptide ft m:thePNo of HIV-1MN virusgreatlyenhancedviralinfection,whileapeptide-derived PNDofHTLV-1119; vwsshsvedatleast 10-foldlessefficientactivity;nosucheffectwasexhibitedbytheotherpeptidestestsd,including ofd from the PNDofHIV-1RF strain .Theobservedenhancingeffectoccurredintheearlystepsofviral infisaftinarsillwilenot strain-restrictedasboth MN- and1118-derivedpeptidesincreasedheterologausvirusexpression,indudng00ofthe RFstrain .The MN- and,toalesserextent,111118-derivedpeptidesalsoincreasedC1)4expressiononthe De*, anddifferentiallyinhibitedCD4down-regulationinducedbythephorbol saw TPAand/orbythemttitmfe GM1 ; thepeptidesshowingnoviralinfectionenhancementhadnosucheffects .Thesefindings ithatthe viralenhancementobservedtookplacethroughaCD4-dependentmechanismandsuggestthatthePNDisInvolvedin HIV-1 infectionandspread. ®1991AcademicPress .Inc . INTRODUCTION GeneticvariabilityisacharacteristicfeatureofHu- manImmunodeficiencyVirustype1(HIV-1),thecaus- ativeagentoftheAcquiredImmunoDeficiencySyn- drome(AIDS)(Alizonatat,1986 ;Hahn etat, 1986 ; Saag etal ., 1988) .Aminoacidsequencemutationsin theviralenvelopeprotein(env)mayplayacriticalrolein virusinfectivityandantigenicity,asenvproteinmedi- atesvirusbindingtoitscellularreceptorCD4(DaIg- leish etat, 1984 ;Klatzmannet at, 1984 ;McDougalat al, 1986 ;Lasky etal ., 1987)andinducesbothhumoral andcellularhostimmuneresponses(Bolognesi1989 ; Mills atal ., 1989) .Theprincipalneutralizingdomain (PND)ofHIV-1hasbeenmappedtoa24-aminoacid- sequencewithinadisulfidebridgedloopinthethird hypervariableregion,V3,oftheexternalenvproteingp 1 20(Goudsmitet al., 1988 ;Palkeretal.,1988 ;Rusche etal., 1988 ;Javaherian etal ., 1989) .ThePNDsofthe conventionalHTLV-1118,MN,andRFstrainsdifferge- neticallyandantigenically,butrecentfindingsdemon- stratethathumannaturalinfectionoccursmostlywith viruseswhosePNDiscloselyrelatedtothePNDofthe HIV-1MNstrain(Devash etal ., 1990 ;LaRosa etal., 1990) .PND-derivedpeptideselicittype-specificanti- ' Towhomcorrespondenceandreprintrequestsshouldbead- dressedatInstituteofOncology,UniversityofPadova,ViaGattame- late64, 35128 Padova . 187 bodiesthat invitro neutralizecell-freeinfectionand alsopreventfusionbetweenvirus-infectedcellsand uninfectedCD4-bearingcells(Palker etaL, 1988 ;Java- herianet al ., 1989,Kenealyetal .,1989);theythus representgoodcandidatesforthedevelopmentofa vaccineagainstAIDS(Goudsmit etal ., 1988 ;Rusche etat, 1988 ;Javaherian etal., 1989 ;LaRosa,1990) . However,despitethemanyinvestigationsintotheanti- genicpropertiesofthePND,thefunctionitmightplay inviralinfection,andtheimportancethatdifferencesin itsaminoacidsequencemighthaveinthisprocessare stillunclear . Inaseriesofexperimentstoevaluatethecapacityof syntheticpeptidesdesignedfromdifferentenvregions tocompetewithviralepitopesinvolvedintheinfection process,weratherunexpectedlyobservedthatpep- tidesderivedfromtheV3-PNDoftheMNand1118 strainsenhancedviralinfection ;nosucheffectwas seenwiththeotherpeptidesunderstudy .Theob- servedenhancementoccurredintheearlystepsof viralinfectionandapparentlyoperatedthroughamech- anisminvolvingtheCD4cellsurfacemolecule . MATERIALSANDMETHODS Peptidesynthesis PeptidesweredesignedfromthePND(307-330 aminoacidsequence)(Javaherian at al .., 1989)ofdiffer- 0042-6822/91$3 .00 Copyright W 1991byAcademicPress,Inc . Allrightsofreproductioninany1oin,reserved .

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VIROLOGY 184, 187-196 (1991)

Synthetic Peptides from the Principal Neutralizing Domain of Human hncideciencyVirus Type 1 (HIV-1) Enhance HIV-1 Infection through a CD4-Dependent Mechanism

ANITA DE ROSSI,* , ' MARCELLA PASTI,* FABRIZIO MAMMANO,* MARINA PANOZZO,* tMONICA DETTIN,t CARLO DI BELLO,t AND LUIGI CHIECO-BIANCHI*

`Institute of Oncology, lnteruniversiry Center for Cancer Research ; tI.S.T Biotechnology Section of Padova,and #institute of Industrial Chemistry, University of Padova, Padova, Italy

Received March 18, 1991 ; accepted May 15, 1991

The principal neutralizing domain (PND) of Human Immunodeficiency Virus type 1 (HIV-1) is mapped to a 24-aminoacid sequence located in the hypervariable V3 region of the viral envelope protein . The PND of NfV-1 isSW a frominfected individuals corresponds mostly to that of the HIV-1 MN strain . We found that a peptide ft m:the PNoof HIV-1 MN virus greatly enhanced viral infection, while a peptide-derived PND of HTLV-1119; vwsshsved at least10-fold less efficient activity; no such effect was exhibited by the other peptides testsd, including ofd

fromthe PND of HIV-1 RF strain . The observed enhancing effect occurred in the early steps of viral infisaftinarsill wile notstrain-restricted as both MN- and 1118-derived peptides increased heterologaus virus expression, indudng 00 of theRF strain . The MN- and, to a lesser extent, 111118-derived peptides also increased C1)4 expression on the De*,and differentially inhibited CD4 down-regulation induced by the phorbol sawTPA and/or by themttitmfeGM 1 ; the peptides showing no viral infection enhancement had no such effects . These findings

i that theviral enhancement observed took place through a CD4-dependent mechanism and suggest that the PND is Involved inHIV-1 infection and spread. ®1991 Academic Press . Inc .

INTRODUCTION

Genetic variability is a characteristic feature of Hu-man Immunodeficiency Virus type 1 (HIV-1), the caus-ative agent of the Acquired ImmunoDeficiency Syn-drome (AIDS) (Alizon at at, 1986 ; Hahn et at, 1986 ;Saag et al ., 1988). Amino acid sequence mutations inthe viral envelope protein (env) may play a critical role invirus infectivity and antigenicity, as env protein medi-ates virus binding to its cellular receptor CD4 (DaIg-leish et at, 1984; Klatzmann et at, 1984; McDougal atal, 1986 ; Lasky et al., 1987) and induces both humoraland cellular host immune responses (Bolognesi 1989 ;Mills at al., 1989). The principal neutralizing domain(PND) of HIV-1 has been mapped to a 24-amino acid-sequence within a disulfide bridged loop in the thirdhypervariable region, V3, of the external env protein gp

1 20 (Goudsmit et al., 1988; Palkeretal., 1988; Ruscheet al., 1988 ; Javaherian et al ., 1989) . The PNDs of theconventional HTLV-1118, MN, and RF strains differ ge-netically and antigenically, but recent findings demon-strate that human natural infection occurs mostly withviruses whose PND is closely related to the PND of theHIV-1 MN strain (Devash et al ., 1990; La Rosa et al.,1990). PND-derived peptides elicit type-specific anti-

' To whom correspondence and reprint requests should be ad-dressed at Institute of Oncology, University of Padova, Via Gattame-late 64, 35128 Padova .

187

bodies that in vitro neutralize cell-free infection andalso prevent fusion between virus-infected cells anduninfected CD4-bearing cells (Palker et aL, 1988; Java-herian et al., 1989, Kenealy et al., 1989); they thusrepresent good candidates for the development of avaccine against AIDS (Goudsmit et al., 1988; Ruscheet at, 1988 ; Javaherian et al., 1989; La Rosa, 1990) .However, despite the many investigations into the anti-genic properties of the PND, the function it might playin viral infection, and the importance that differences inits amino acid sequence might have in this process arestill unclear .

In a series of experiments to evaluate the capacity ofsynthetic peptides designed from different env regionsto compete with viral epitopes involved in the infectionprocess, we rather unexpectedly observed that pep-tides derived from the V3-PND of the MN and 1118strains enhanced viral infection ; no such effect wasseen with the other peptides under study. The ob-served enhancement occurred in the early steps ofviral infection and apparently operated through a mech-anism involving the CD4 cell surface molecule .

MATERIALS AND METHODS

Peptide synthesis

Peptides were designed from the PND (307-330amino acid sequence) (Javaherian at al.., 1989) of differ-

0042-6822/91 $3 .00Copyright W 1991 by Academic Press, Inc .All rights of reproduction in any 1oin, reserved .

1 8 8

TABLE 1

SEQUENCES OF THE PRINCIPAL NEUTRALIZING DOMAIN PEPTIDES

Note. Sequence of DB1 peptide is from the BH 10 molecular cloneof the HTLV-IIIB isolate (Ratner et al. 1985), sequence of D82 is fromthe HAT-3 molecular clone of the RF isolate (Starcich et aL 1986),and sequence of D83 is from MN isolate (Curgo et al . 1988) . Dashesrepresent identity with DB 1 .

ent HIV-1 strains (Table 1) . The polypeptide chainswere assembled according to a standard solid-phasemethod (Stewart and Young, 1984) on an automatedpeptide synthetizer 431A (Applied Biosystems) . Eachpeptide was purified by ion-exchange chromatographyand then by reverse-phase high-performance liquidchromatography (HPLC). Peptide purity was estab-lished by analytical chromatography and exceeded90%. Amino acid composition was examined with anautomated analyzer (Carlo Erba A28), which gave theexpected results .Cells and virusesMOLT-3 and H938 cells were employed for HIV-1

infection experiments . Both lines have a T lymphocyteorigin and express the CD4 molecule ; H938 cells werederived from an H9 cell line in which a genetic con-struct containing the HIV-1 long terminal repeat (LTR)ligated to the gene of chloramphenicol acetyl transfer-ase (CAT) had been permanently transfected (Felberand Pavlakis, 1988) . Cells were maintained in RPMI1640 medium (Flow Laboratories, Irvine, UK) supple-mented with 10% fetal calf serum (Flow), 2% L-gluta-mine (Flow), and 50 ug/ml gentamycin, and culturedunder standard conditions at 37° in humidified air con-taining 5% C0 2 .

HTLV-IIIB (Ratner et al., 1985), RF (Starcich et al.,1986), and MN (Curgo et al., 1988) HIV-1 strains weregrown in H9 cells . Supernatants from infected H9 cellswere collected, centrifuged at 2000 g (Minifuge GL,Heraeus ; Hanau, FRG) for 15 min to remove cells, andthen filtered through 0 .22-um filters (Millipore, Bedford,MA) . Virus titers were measured by Reverse Transcrip-tase (RT) assay, as previously reported (De Rossi et al.,1990), and the supernatants were stored at -80° .Viral infection

Following resuspension to a concentration of 2 .5x 106 cells/ml, MOLT-3 cells were plated at 200 pVwell

DE ROSSI ET AL .

in 24-microtiter plates (Costar, Cambridge, MA) andincubated with 100 µl of medium containing scalarequimolar doses of peptides . After 1 hr, the cells wereinfected with 50 ul of an HIV-1 preparation containing 1X 106 cpm/ml of RT activity (0 .01 cpm of RT activity/cell) and further incubated with gentle shaking for 1 hr,after which 1 ml of complete medium was added, Thecells were observed daily at the light microscope forsyncytium formation . After 4 days, the supernatantswere collected for RT assay . The H938 cells were incu-bated and treated in a similar manner, except that cel-lular extracts were prepared 48 hr after infection byfreeze-thawing three times and then submitted to CATassay .

CAT assay

The protein contents of the cellular extracts wereevaluated by a rapid immunoenzymatic assay (Bio-RadProtein Assay, Bio-Rad, Richmond, CA), according tothe supplier's instructions . Ten micrograms of proteinwere assayed for CAT (Gorman et al., 1982) in 160 ul ofreaction buffer containing 0 .125 uCi of 14C-labeledchloramphenicol (Amersham, 50 uCi/ml, sp act 50mCi/mmol), 2 mM of acetyl CoA (Sigma, St . Louis,MO), and 0.15 M Tris-HCI, pH 7 .8. The samples wereincubated at 37° for 2 hr, extracted with 1 ml of ethylacetate, spotted onto silica gel plates (Millipore), devel-oped in chloroform/methanol (95 :5, v/v), and autoradio-graphed. The percentage conversion of chlorampheni-col (Cm) into acetylated forms (AcCm) was determinedby liquid scintillation counting of spots cut from thesilica plates and calculated as : [cpm AcCm/cpm(AcCm + Cm)] x 100. According to this procedure,values were linearly related to CAT activity up to Cmconversion into mono and diacetylated forms; Cm con-version to the triacetylated form was out of range and isindicated in the figures as >99% Cm conversion .

Cytofluorimetric analysis

Anti-CD4 OKT4 and OKT4a monoclonal antibodies(MAb) (Ortho Diagnostic Systems, Raritan, NJ)wereflu-orescein-conjugated and used for direct staining . Anti-CD4 Leu3a (Becton-Dickinson, Mountain View, CA)and anti-HIV gpl 60 (Cellular Products Inc ., Buffalo, NY)were employed for indirect immunofluorescence with afluorescein-conjugated goat (Fab')2 anti-mouse Ig(NEN DuPont, Dreieich, FRG) . Every step was carriedout at 4° . After washing, the cells were fixed in 1 ml ofcold 1 % formaldehyde/PBS to prevent virus contamina-tion, and phenotype was then analyzed byflow cytome-try (EPICS-C flow cytometer Coulter Electronics, Hia-leah, FL) ; at least 5000 cells were collected in each

Peptide HIV-1 isolate Sequence

DB1 HTLV-IIIB NNTRKSIRIQRGPGRAFVTIGK IGDB2 RF ------- TK---- VIYAT-QI--DB3 MN Y-K--R-H- Y-TKNI--DB5DB6

--V-R-LS- R-R--I--

fluorescence histogram . Viable cells were gated on thebasis of forward and 90° light scatter parameters .

TPA and GM1 treatment

TPA (Sigma Chemical Company, St . Louis, MO)stock solution was prepared by dissolving 1 mg of TPAin 1 ml of ethanol ; working solutions were prepared inRPMI m edium . GM 1 was a gift of FI DIA Research Labo-ratories (Abano Terme, Italy). All experiments per-formed with GM1 were carried out in RPMI mediumwithout FCS (Chieco-Bianchi et al., 1989) .

RESULTS

PND-derived peptides enhance HIV-1 infection

The synthetic peptides designated DB1, DB2, andDB3 were prepared according to the amino acid se-quences of the V3-PND of the HIV-1 strains, HTLV-IIIB .RF, and MN, respectively . A 10 amino acid (10-mer)shortened derivative of DB 1 peptide, as well as a modi-fied 23-mer peptide, were also synthesized (Table 1) .MOLT-3 cells were incubated for 1 hr with scalar equi-molar doses of different peptides and then infectedwith strain HTLV-IIIB. Cultures treated with DB3 pep-tide exhibited large syncytia within 48 hr of infection ;this effect was dose-dependent and was observed atconcentrations as low as 2 .5 MM. Untreated cellsshowed only small syncytia up to 4 days postinfection(Fig . 1). Cultures treated with DB1 peptide showed anincrease in syncytium formation only at peptide dosesof 10 and 20 AM, while no such effect was observed inDB2-treated cultures nor in cultures treated with thetwo modified peptides, DB5 and DB6 . The DB3 and, toa lesser extent, the DB1 enhancing effects on HTLV-1118 infection were further reflected in the RT values ofculture supernatants collected 4 days after infection .RT assay of control-infected cells showed 9,846 cpm/ml, but in cultures exposed to a final DB3 concentra-tions of 201AM this figure rose 19-fold to 186,000 cpm/ml (Fig . 2). This upsurge in RT activity was peptidedose-dependent, as 0 .62 µM DB3 produced a twofoldincrease . Higher RT values were also found in culturescontaining DB1 at final concentration of 20, 10, and 5AM, but not in cultures treated with DB2, DB5, andDB6, even at maximum doses . These findings sug-gested that the enhanced cytopathic effect observedwas most likely the result of a more efficient cell infec-tion, as well as a more intense viral replication andenvelope protein expression on the surface of infectedcells . In fact, 48 hr after the HIV infection, whereas only18% of control cells expressed the gp 160 viral enve-lope protein, the cells preexposed to 20 AM of DB3 andDB1 peptides were 76% and 42% gp 160 positive, re-spectively .

PND-DERIVED PEPTIDES ENHANCE HIV-1 INFECTION

189

HIV-1 gene expression depends mostly on the regu-latory sequences in the LTR region ; LTR may be acti-vated by viral factors such as the transactivator protein(tat), which interacts with the responsive elements(TAR) in the LTR, and by cellular factors that recognizetarget sequences in HIV- 1 -LTR (Nabel and Baltimore,1987) and are induced by several T cell mitogens(Greene et al., 1989) . The peptide effect on HIV-1-LTRwas evaluated using H938 cells . As these cells arepermanently transfected with HIV-1 LTR ligated to theCAT gene, activation of the LTR results in activation ofCAT expression (Felber and Pavlakis, 1988) . Parallelcell cultures were incubated for 1 hr with scalar pep-tide dilutions, and one culture for each dilution wasthen infected with HTLV-11113 strain . After 48 hr, bothinfected and noninfected cells were processed for CATassay. As shown in Fig . 3, CAT expression, evaluatedas the percentage of Cm conversion into AcCm forms,was heightened in DB3-treated infected cultures, andsince CAT is more sensitive than RT assay, 0 .31 AM ofthis peptide sufficed to bring about almost completeCm conversion into AcCm ; at doses as low as 0 .15AM, a twofold increase in CAT activity was still ob-served . DB1 peptide was less effective, and total Cmconversion was observed only at concentration of 20and 10 AM; 2 .5 AM was needed to increase CAT ex-pression twofold . DB2 (Fig . 3A), as well as D85 andDB6 (data not shown), had no effect on CAT expres-sion, and values were very close to control levels . NoCAT expression was detected in uninfected culturestreated with even maximum peptide doses (Fig . 3B) .These results suggested that LTR activation was mostlikely due to viral tat protein overexpression by initialenhancement of HIV-1 infection, rather than a peptidemitogenic effect on infected cells .

Enhancement activity occurs in the early steps ofHIV-1 infection

The accumulated data implied that the enhancingeffect exerted by the peptides occurred in the earlysteps of viral infection. To address this point, wetreated H938 cells before, during, and after HIV-1 in-fection with different doses of DB3 . Cells simulta-neously exposed for 1 hr to both virus and peptide (Fig .4A) and cells exposed to peptide 1 hr before infection(see Fig . 3) exhibited a similar enhancement effect . En-hancement also occurred when cells were treated withpeptide for 1 hr and then washed before infection (Fig .4B). However, no enhancement was observed whencells were infected, then washed twice to remove vir-tually all virus particles still present in the medium, andfinally exposed to peptide (Fig . 4C). That enhancementtook place only when peptide was present in the cul-tures before or during, but not after, HIV-1 infection

1 9 0

Fra . 1 . Enhancement of HIV-1 (HTLV-IIIB)-induced syncytiurn formation by DB3 peptide . Optical magnification x200 . MOLT-3 cells wereinfected with a dose of virus corresponding to 0 .01 corn of RT activitylcell after 1 hr exposure to 20µM DB3 (A and B, 2 and 4 days postinfection,respectively), and without DB3 treatment (C, 4 days postinfection) . Controls were performed by incubating MOLT-3 cells with medium alone (D)and with 20 AM DB3 for 4 days (E) .

means that the peptide action occurred in the earlysteps of viral infection .

Enhancement activity is not virus-restrictedThe antigenic properties of the PND region are

known to be virus-specific ; antibodies elicited by MN-derived peptide do not neutralize HTLV-1116 virus and

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treated for 1 hr with peptides at scalar concentrations and then in-fected with HIV-1 (HTLV-IIIB) as described in the legend to Fig . 1 . RTwas performed on culture supernatants 4 days post-HIV-1 infection .

DE ROSSI ET AL .

vice-versa (Javaherian et al ., 1989 ; Scott et al., 1990) .Our observation that DB3 (i .e ., MN-PND-derived pep-tide) more greatly enhanced HTLV-IIIB infection thanDB1 (i.e., IIIB-PND-derived peptide) suggested thatthe effect was not virus-restricted . This aspect wasexamined in experiments using MN and RF HIV-1strains. The D83 peptide effect on MN and RF infectionresembled findings with HTLV-IIIB infection (Fig . 5) . RTvalues in MOLT-3 cells infected with MN and RF viruswere increased 20- and 12-fold, respectively, by DB3at a final concentration of 20 AM, and this effect wasstill evident with 0 .62 and 5 uM in MN and RF infec-tions, respectively (Fig . 5B). These findings were sup-ported by the results of CAT assay in H938-infectedcells, where the increase in transactivation activity wasalso peptide dose-dependent (Fig . 5A) .As observed with HTLV-IIIB strain infection, DB1

was less effective, and significant increases in bothparameters were obtained only at maximum doses inboth MN and RF infections . Interestingly, DB2 exertedno effect on heterologous MN or homologous RF virusinfections .

Amplification of HIV-1 expression is related to viralinfectious dose

To examine whether the DB3 enhancing effect de-pended on viral infectious dose, serial dilutions ofstock virus, 10-fold concentrated with respect to H9/HTLV-IIIB supernatants, were evaluated for infection

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FIG . 3 . Enhancing effect on HIV-1 transactivation . H938 cells weretreated with peptides at scalar dilutions and then infected (A) withHIV-1 (HTLV-IIIR), as reported in the legend to Fig_ 1 or not infected(B) . CAT assay was performed 48 hr post-HIV-1 infection .

on H938 cells in the presence of 10 AM of final peptidedilution by CAT assay . We found that Cm conversion inuntreated cells decreased linearly according to theviral dilution, and at 10 -3 the signal was comparable tothe negative control (Fig . 6A). Peptide-treated cellsshowed almost 100% Cm conversion up to 10-2 vi-ral dilution, and at 10-4 Cm conversion was 13 .9%(Fig . 6B) .

Peptides enhancing viral infection modulate CD4expression

Our findings implied that viral enhancement oc-curred in the early steps of viral infection, possibly atvirus entry into the cells . Since the CD4 molecule is theviral receptor on T lymphocytes (Dalgleish et al ., 1984 ;Klatzmann et al., 1984), we exposed MOLT-3 cells for1 hr to scalar doses of different peptides and then ana-lyzed them by cytofluorimetry for CD4 expression us-ing OKT4 and OKT4a MAbs. An increase in CD4 meanfluorescence intensity (MR) was recorded in cellstreated with DB3 (Fig. 7); interestingly, this increasewas registered within 30 min of DB3 treatment and

PND-DERIVED PEPTIDES ENHANCE HIV-1 INFECTION

191

was also found in D83-treated cells 1 hr after peptideremoval by washing .

The initial step of viral infection likely results from the

interaction between conserved regions of gp 120(Lasky et al., 1987; 0lshevsky et al., 1990) and cellCD4 epitopes located in the V1 domain and recog-nized by OKT4a and Leu3a MAbs (Sattentau et al.,

1986) . To see whether PND-derived peptides interferein gpl20-CD4 binding . MOLT-3 cells were incubatedfor 1 hr with Leu3a (10 Ag/5 x 10 5 cells) before HIV-1infection, which was accomplished in the presenceand absence of 20 MM DB3 and DB 1 peptides . Syncy-tia and FIT activity were not detected in either peptide-treated or untreated cells up to 4 days after infection .Experiments in which cells were preincubated for 1 hrwith these peptides (20 AM) and then treated withLeu3a for 1 hr prior to infection gave similar negative

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FIG . 4. Enhancing effect of DB3 peptide on HIV-1 (HTLV-tltB) tran-sactivation . H938 cells were infected with a dose of virus corre-sponding to 0.02 cpm of RT activitylcell . Scalar concentrations ofDB3 were (A) added together with virus infection, (B) added for 1 hrand removed by washing before infection, and (C) added after HIV-1infection, before cell washing to remove virus particles in the me-dium. See details in the text .

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results (not shown) . Thus, PND-derived peptides prob-ably do not compete with Leu3a MAb for the CD4 bind-ing site and most likely are not involved in the initialvirus binding to its CD4 receptor. To further explore thepeptide effect on CD4 expression, experiments wereperformed using well-known down-regulators of CD4expression, the phorbol ester TPA (Acres et al., 1986 ;Maddon et al., 1988; Jaekyoon et at, 1990) and themonosialoganglioside GM 1 (Off nor et at, 1987; Naka-kuma etal., 1989; Chieco-Bianchi et al., 1989). Follow-

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FIG. 5 . Effect of peptides on MN and RF HIV-1 isolates . H938 and MOLT-3 cells treated with scalar concentrations of peptides were infectedby MN and RF at viral dose of 0 .01 corn RT activity/cell . CAT assay on H938 cells was performed 48 hr postviral infection (A) . RT assay wasperformed in MOLT-3 culture supernatants 4 days postinfection (B) .

ing the incubation of 5 X 10 5 MOLT-3 cells with 100ng/ml of TPA for 4 hr, we found that less than 10% ofcells expressed CD4 on their membrane surface. How-ever, if TPA exposure was preceded by 1 hr treatmentwith DB3 peptide (20 µM), or carried out simulta-neously, almost 50% of cells were still CD4 positive(Fig. 8 and Fig. 9). Even at highest dosage, DB1 did notblock TPA activity (Figs . 8 and 9) .One-hour incubation of 5 X 10 5 MOLT-3 cells with

50 pg/ml of GM 1 resulted in an almost complete block

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FIG. 8. Antagonist activity of peptides to TPA-and GM 1-induced CD4 down-expression . 5 X 10° MOLT-3 cells were incubated in 1 ml of finalvolume with peptides (20 MM) and TPA (100 ng/ml) or GM 1 (50 Mg/ml). Cytofluorimetric analysis was performed using the OKr4 Mob, after 4 and

1 hr in TPA- and GM1-treated cells, respectively . Dark profile, treated cells ; light profile, control cells .

DE ROSSI ET AL.

Des DSI DS7

We observed enhancement when cells were ex-posed to DB3 peptide before or during virus infectionbut not following virus entry into the cells . This indi-cated that the phenomenon occurred in the early stepsof viral infection, and most likely through a mechanisminvolving the CD4 receptor. In fact, while the nonen-hancing peptides exhibited no activity, DB3 and, to alesser extent, DB1 peptides induced an increase inCD4 epitopes on the cell membrane and, with different

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FIG. 9 . Dose-dependent effect of peptides DB3 on GM1 and TPAactivity . 5 x 10° MOLT-3 cells were incubated in 1 ml final volumewith scaler dilutions of peptides and 50 Mg/ml of GM 11-) or 100

ng/ml of TPA (----) . After 1 and 4 hr, respectively, cells were analyzedin flow cytometry for CD4 expression using OKT4 Mob . These re-sults are from a single representative experiment . Each peptide wastested in at least three separate experiments .

0.62

efficiencies, were able to inhibit Cf74 down-regulationexerted by TPA and/or GM 1 . CD4 modulation by PND-derived peptides probably occurred at the post-tran-scriptional level, as no increase in CD4 mRNA was de-tected in treated cells (not shown) . In line with the rapidrise in CD4 MR observed in D83-exposed cells, it islikely that the CD4 cell membrane pathway was modi-fied by the peptides. Consistent with this view are theresults of TPA and GM1 studies . In this regard, it isnoteworthy that TPA induces CD4 internalizationthrough a protein kinase C-dependent phosphorylationof amino acid residues in the ON cytoplasmatic do-main (Maddon et al., 1988; Jaekyoon et al., 1990),while GM 1-induced CD4 down-regulation seems to bephosphorylation independent (Offner et al., 1987; Na-kakuma et al., 1989). Therefore, the different capacityof DB3 and DB1 peptides to interfere with TPA andGM t effects might reflect their different levels of inter-action with CD4 cell surface molecules . In any case,these findings confirm once again that CD4 endocyto-sis is not required for HIV-1 infection (Stein etal., 1987 ;Maddon et al., 1988) .Whatever the mechanism underlying peptide-CD4

interaction, it is clear that the resulting increase and/or"stabilization" of CD4 moieties on the cell membranemay facilitate HIV-1 entry into target cells . Whether theproperties of PND-derived peptides reflect some bio-logical features of the viral strains from which they arederived remains yet to be demonstrated . The peptidesdid not compete with Leu3a MAb for the CD4 bindingsite, and antibodies against PND do not block gp 120binding to CD4 (Skinner at al., 1988). These data thusindicate that the PND is not involved in initial virus bind-ing to receptors ; nevertheless, as peptide-induced en-hancement occurred in the early steps of virus infec-tion, it is conceivable that the PND interferes with CD4recycling on the cell membrane and indirectly deter-mines a selective advantage for the virus by increasing

Cm

Ac Cm

A

AcCm

conversion 78.6 873 51 .0 249 18.4 78 3.0 2I7 2.5 25ss

Cm • • ; e.I

I .

I

1

I

I

I

I

I

Iconversion >99 >99 >99 >99 89 .8 460 33D 193 139 2.5

10 1607 10 1 1017 1d2 1e7103 1037 10 0

viral dilutionsFIG. 6 . Enhancement of HIV-1 infection was viral dose-dependent .

H938 cells were infected with scalar dilutions of virus stock (derivedfrom a 10-fold concentrated H9/HTLV-IIIB supernatant) in the ab-sence (A) and in the presence (B) of 10 µM D83 . Initial virus dilutioncorresponded to a viral dose of 1 cpm of FIT activity/cell .

of CD4 expression (Fig. 8). When GM 1 exposure fol-lowed 1 hr incubation with DB3 or DB1 peptides (20pM final concentration) or was carried out simulta-neously, the cells expressed CD4 levels similar tothose of GM -1-untreated controls . On the other hand,20 pM of DB2 peptide did not block GM 1-induced CD4down-regulation (Fig . 8). The DB3 and DB1 effectswere dose-dependent : 10 pM of either peptide inhib-ited GM1-induced CD4 down-regulation completely .At lower doses, however, DB3 peptide was more ac-tive than DB1 in blocking GM 1 activity ; following expo-sure to 1 .25 pM DB3 and DB 1, respectively, 45 and 5%of the cells expressed CD4 molecule on their cell sur-face (Fig. 9) .

DISCUSSION

PND-derived peptides have been successfully em-ployed in a number of studies to examine the antigenic

PND-DERIVED PEPTIDES ENHANCE HIV-1 INFECTION

193

properties of the PND region of HIV-1 envelope protein,and their possible use as immunogens for HIV vaccinehas been advanced (Goudsmit et al., 1988; Palker etat, 1988 ; Rusche et al., 1988; Javaherian 1989 ; Ken-ealy et al., 1989 ; Devash et at, 1990 ; La Rosa et at,1990) . We unexpectedly found that the DB3 syntheticpeptide designed from the PND of the HIV-1 MN strainexerted a strong enhancing effect on HIV-1 infection .DB1 peptide, designed from the PND of the HTLV-IIIBstrain, exhibited the same effect, but the intensity wasat least 10-fold less, while the other peptides tested inthis study, including DB2 derived from the PND of theHIV-1 RF strain, did not exert any such activity . Al-though neutralization antibodies elicited by the PND-derived peptides are type-variant-specific (Javaherianet al., 1989; Scott et al., 1990), the observed enhanceractivity was not strain-restricted and was also detectedduring infection with heterologous viruses, includingthe RF strain . Moreover, when a very low amount ofvirus was employed for infection, virus expression wasalso amplified . This finding may have a practical appli-cation, as the addition of DB3 peptide to CD4-positiveindicator cell cultures could improve the sensitivity ofvirus detection, as well as virus isolation from HIV-1-in-fected subjects .

25 -

20 10 1 .25 0.e2

0

peptide concentration (uM)FIG. 7 . Effect of peptides on CD4 expression. 5 x 105 MOLT-3

cells were incubated in 1 ml final volume with scalar dilutions ofpeptides for 1 hr and then analyzed in flow cytometry for CD4 ex-pression using the OKT4 (-), and the OKT4a (---) MAbs. Theseresults are from a single representative experiment . Each peptidewas tested in at least three separate experiments .

and/or "stabilizing" its receptors. The evidence thatnatural infections in humans occur mostly with MN-re-lated HIV isolates (Devash et al., 1990 ; La Rosa et al.,1990) seems to assign the PND an important role invirus infection and spreading .

ACKNOWLEDGMENTS

We thank R . C . Gallo for providing III B, MN, and RF strains and G .Pavlakis and B . Felber for providing H938 cells . We are grateful to D .Bolognesi and J . Guardiola forcritical and helpful advice . We thank P .Segato for help in preparing the manuscript . This work was sup-ported by grants from Istituto Superiors della Sanita', Progetto AIDS1989, 1990, and by CNR, Progetto Strategico "Sviluppo di farmaciper I'AIDS," and Associazione Italians per Is Ricerca Sul Cancro .

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