Vif Is Largely Absent from Human Immunodeficiency Virus Type 1 Mature Virions and Associates ...

14 Pages·2001·5.75 MB·English

JOURNALOFVIROLOGY,June2001,p.5504–5517 Vol.75,No.12
0022-538X/01/$04.0010 DOI:10.1128/JVI.75.12.5504–5517.2001
Copyright©2001,AmericanSocietyforMicrobiology.AllRightsReserved.
Vif Is Largely Absent from Human Immunodeficiency Virus
Type 1 Mature Virions and Associates Mainly with Viral
Particles Containing Unprocessed Gag
PAVELSOVA,*DAVIDJ.VOLSKY,LINGWANG,ANDWEICHAO
MolecularVirologyLaboratory,St.Luke’s/RooseveltHospitalCenter,ColumbiaUniversity,NewYork,NewYork
Received30October2000/Accepted26March2001
Vif is a human immunodeficiency virus type 1 (HIV-1) protein that is essential for the production of
infectious virus. Most of Vif synthesized during HIV infection localizes within cells, and the extent of Vif
D
packagingintovirionsanditsfunctionthereremaincontroversial.Hereweshowthatasmallbutdetectable o
amountofVifremainsassociatedwithpurifiedvirionsevenaftertheirtreatmentwiththeproteasesubtilisin. w
However, treatment of these virions with 1% Triton X-100 revealed that most of the virion-associated Vif n
segregated with detergent-resistant virus particles consisting of unprocessed Gag, indicating that detergent- lo
a
soluble, mature virions contain very little Vif. To investigate the control of Vif packaging in immature virus d
particles,wetesteditsassociationwithGag-containingvirus-likeparticles(VLPs)inaVifandGagcoexpres- e
d
sion system in human cells. Only a small proportion of Vif molecules synthesized in this system became
f
packaged into VLPs, and the VLP-associated Vif was protected from exogenous protease and detergent ro
treatment, indicating that it is stably incorporated into immature virion-like cores. About 10-fold more Vpr m
thanVifwaspackagedintoVLPsbutmostoftheVLP-associatedVprwasremovedbytreatmentwithdetergent. h
MutagenesisoftheC-terminalsequencesinGagpreviouslyshowntoberesponsibleforinteractionwithVifdid tt
p
notreducetheextentofVifpackagingintoGagVLPs.Surprisingly,shortdeletionsinthecapsiddomain(CA) :
/
ofGag(aminoacidresidues284to304and350to362)increasedVifpackagingover10-fold.The350to363 /jv
deletionintroducedintoCAinHIVprovirusalsoincreasedVifincorporationintopurifiedvirions.Ourresults i.
a
showthatVifcanbepackagedatlowlevelsintoaberrantvirusparticlesorimmaturevirionsandthatVifis s
not present significantly in mature virions. Overall, these results indicate that the Vif content in virions is m
tightlyregulatedandalsoargueagainstafunctionofvirion-associatedVif. .o
r
g
/
Humanimmunodeficiencyvirustype1(HIV-1)isacomplex known cellular function preventing replication of vif2 HIV-1 o
n
retrovirus that contains a number of genes not present in (38,39,58). J
oncogenic retroviruses. The protein product of one of these Vifisaphosphorylated23-kDaprotein,whichisabundantly a
n
genes,Vif(virioninfectivityfactor),isessentialforproductive expressed in infected cells. Vif has been shown to interact or u
a
HIV-1 infection in primary T cells and macrophages in vitro copurifywithmembranes(24,26),intermediatefilaments(32), r
y
andforpathogenesisinmodelsofAIDS(10,18,22,23,29,55, HIV-1 Gag (5, 31, 57), HIV-1 protease (PR) (2), and most
63,66,68).Incellculture,vif-defective(vif2)HIV-1isableto recentlywithviralRNA(14,72).VifcaninhibitPRactivityin 5,
2
replicate in some T-lymphoblastoid cell lines termed permis- severalmodelsofproteolysis,andVif-derivedpeptidesinhibit 0
sive (CEM-SS, SupT1, C8166, and Jurkat) whereas Vif is re- Gag cleavage in HIV-1-infected cells (2, 34, 49). Vif function 1
9
quired in other cell lines, such as H9 or MT-2, termed non- has been ascribed to most of these interactions, but each of b
permissive(18,22,37,64,66,69).Itisgenerallyacceptedthat theseactivitiesremainscontroversial. y

Vifactsinlatestepsofthevirallifecycletoensureproduction g
of infectious virions (3, 12, 53, 56, 61, 64, 69). vif2 HIV-1 is sevWerhaillereVpoifrtisshparveedosmugigneasntteldy tahnatinatrfarcaecltliuolnaropfrtohteeipnro(t5e4i)n, ue
s
impairedinendogenousreversetranscriptionandinitsability becomes incorporated into virions (32, 36). Liu et al. (36) t
toformproviralDNAinnewlyinfectedcells(4,25,59,64,69).
reported that virions contained approximately 60 to 100 mol-
Theunderlyingreasonforthesedeficienciesisunknown.Ithas
ecules of Vif that were incorporated into viral cores. Others
been proposed that Vif participates in the processing of viral
reportedarangeof20to100Vifmoleculespervirion(8,60).
structural proteins and in the assembly of virus particles. An
Observationsthattheamountofvirion-associatedVifdepends
influence of Vif on the processing of viral structural proteins
on the level of its expression within virus-producing cells (32,
(4, 28, 53, 56) as well as on the maturation and stability of
60) and that Vif can be packaged into murine leukemia virus
virion cores has been observed (4, 30, 43) although not con-
particles (8) led to the conclusion that Vif is not specifically
firmed by all investigators (6, 19). Another model suggested
incorporated into HIV virions. Recently, it was reported that
that Vif is needed in nonpermissive cells to overcome an un-
highly purified, infectious HIV-1 virions do not contain Vif
(15).
Resolving the question of Vif incorporation into virions is
*Correspondingauthor.Mailingaddress:MolecularVirologyLab-
important for understanding the site of Vif activity. Here we
oratory,St.Luke’s/RooseveltHospitalCenter,432W.58thSt.,Room
709, New York, NY 10019. Phone: [phone]. Fax: (212) 582- demonstratethatVifassociatesstably,albeitatlowlevels,with
5027.E-mail:[email protected]. highly purified virions produced from several cell lines with
5504
VOL.75,2001 HIV-1 Vif, VIRIONS, AND VIRUS-LIKE PARTICLES 5505
different permissivity to the replication of vif2 HIV-1. Inter- a tag was constructed by cloning the vif gene amplified by PCR using Pfu
estingly,thevirion-associatedVifwasfoundprimarilyinaber- polymerase and primers VIFNCO1F (59 CAGGGACCATGGAAAACAGAT
rant, Pr55Gag-containing virions resistant to detergent treat- GGC39)andTRAP13(59CGAGGAGATTCAGCTGATCACAGG39)inNcoI
andSalIsitesofpCITEHA,removingtheHAtag.Vifwasalsoexpressedin
ment, indicating that mature virions that are soluble in S-taggedformfrompCITE4a(1)/Vif.pCITEHA/Vprwasconstructedbyinsert-
detergent contain little Vif. To analyze the specificity of the ingthevprgeneamplifiedbyPCRfromforwardprimerVPRU(59CTGACA
associationofVifwithimmaturevirions,wecreatedaGagand GAGGATCCATGGAACAAGCCCCAG 39) and reverse primer VPRD (59
Vif coexpression model system to produce virus-like particles CTTCCAGGGAATTCGTCTAGGATCTACTGG39)usingpNL4-3HIV-1(1)
as a template. Because vpr contains an internal EcoRI recognition site, the
(VLPs)containingunprocessedGagandVif.Wereporthere
constructionintopCITEHA/Vprwascarriedoutintwosteps,usingBamHIand
that compared to Vpr, Vif is packaged inefficiently in Gag
EcoRIrestrictionenzymes.
VLPs,butcertaindeletionsinthecapsiddomain(CA)ofGag Thesequencesofgagdeletionmutantswereconfirmedbysequencingfrom
increased the amount of encapsidated Vif about 10-fold in primersCITE(Novagen),PS20(59GGTCCAAAATGCGAACCCAG39),and
bothGagVLPsandvirions.Ourdatasuggestthattheassoci- PS21 (59 TGGTTGGGGCTGTTTGGCTC 39). The pCITEHA/Vif sequence
wasconfirmedfromprimerCITE.
ation of Vif with virions is tightly regulated and indicate that
Cells and viruses. The COS-1 cell line was obtained from American Type
Vifmaybeactivelyexcludedfromvirusduringparticlerelease
CultureCollection(ATCC;Rockville,Md.),and293Tcellswereobtainedfrom D
andmaturation. D.Littman.CellculturesweremaintainedinDulbeccomodifiedEaglemedium o
(GIBCO/BRL,GrandIsland,N.Y.)supplementedwith10%fetalbovineserum w
MATERIALSANDMETHODS andantibiotics.H9andMT-2cellswereobtainedfromATCCandmaintainedin n
RPMI1640(GIBCO/BRL)supplementedwith10%fetalcalfserumandanti- lo
Plasmids.VectorsusedforexpressionofGagandVifwerebasedonpCITE- biotics.TheHIV-1NL4-3molecularclone(1)wasobtainedfromM.Martin a
4a(1)(Novagen,Madison,Wis.).Allgagandvifconstructswerebasedonthe throughtheNationalInstitutesofHealth(NIH)AIDSResearchReferenceand de
HIV-1 NL4-3 molecular clone (1). The Gag Pr55 intermediate construct ReagentProgram(Rockville,Md.).ConstructionoftheHIV-1molecularclone d
[apnCdIXThEo4Ias(i1te)s/GinapgC]IwTaEs-4crae(a1te)dafbteyrcalmonpilnifigctahtieonGoafgthcoedgianggorpeegniorneainditnogEfrcaomReI KvTSF278-33weaxpsredsessicnrgibtehdepbraecvtieoruioslpyha(5g4e).T7ThReNvAaccpionliyamveirrausser(e2c0o)mwbainsaonbttacilnonede fro
in a chain reaction using Pfu polymerase (Stratagene, La Jolla, Calif.) and fromT.FuerstandB.MossthroughtheNIHAIDSResearchReferenceand m
primers jGMA5 (49) and p6R (59 GCCCCCCTCGAGTTATTGTGACG 39). ReagentProgram. h
Theresultingconstruct,expressingGagwithanN-terminallyattachedS-tag(33), Transfection and expression using the vaccinia virus/T7 RNA polymerase tt
was further modified to express wild-type Gag by removing the pCITE4a(1) expressionsystem.COS-1(33106to43106)or293Tcells(53106to83106) p:
vPTeCCcRCtoGrenCsezCyqmAueTenGscyeGst5Ge1mT9G(tCoClGo6n2A9teGcbAhy,GfPuCalllG-ovTeAcClttoAor,GCre3aa9lm)if.ap)nlaidfincCdaItpiTorEinmLuers(si5n9CgGItTGhEeTRAG(dG5v9TaGnGtGGagACe pmSliaigxmtteuadr,eSinto.1fL0o0cua-imtsi,omMniocp.e)tlirapinidddipsdhhieomsspewhtheayrtlieddiyotlrecattnahsdafenecocytlaleamdmiwnmeioth(nSiuipgmlmasamb)riadosmdDiedNsecAri(bDuesdDinpAgrBea-; //jvi.a
CATGGTATCATCGTG39),followedbytreatmentwithDpnI,NcoI,andT4 s
DNAligasetoobtainaconstructdesignatedpCITED/Gag.Essentially,pCITED/ viously(52).Priortotransfection,cellswereinfectedwithvacciniavirusvTF7-3 m
(10PFU/cell).Cellsandsupernatantswereharvestedapproximately24hafter .
GagisavectorcontainingabacteriophageT7RNApolymerasepromoterand o
transfection/infection.
CITEsequence(16,17),followedbyagagopenreadingframeinnativeconfig- Productionofviruses.293TcellsweretransfectedwithproviralplasmidDNA rg
uPrCaRtionam.ApllilfiscuabtsioenquoefntthgaegpdCelIeTtEioDn/Gmaugtantetms(psleaeteFiugs.in7gB)thweerAedcvoannsttarugcetePdCbRy usingLipofectamine(GIBCO/BRL)andcocultivatedwithtargetT-lymphoblas- o/
toidcells;Tcellswerecultureduntildevelopmentofcytopathiceffects,andthen n
enzymesystemandasetofprimersencompassingadeletionandcarryingan cell-free supernatants were harvested (wild-type HIV-1). Mutant gag HIV-1 J
NheIrecognitionsite,allowingsubsequentvectorrecircularizationbytreatment recombinantswereproduceddirectlybytransfectionof293T. a
ofaPCRproductwithNheIandDpnIandligationwithT4DNAligase.Muta- n
genicprimerswereasfollows:(i)forGagDMHR,inwhichAla-SerreplacedGag Preparationofvirusandvirus-likeparticlesforanalysis.Harvestedcellswere u
lysedinsodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE) a
amino acid residues 284 to 304 composing the Gag major homology region r
(3M9)HanRd),forrewvaerrdsepprirmimererMCHADDRLF(529C(5T9ACTTATAGATGCTCCTGAAGTCCAGGCAAGGCACAGGACGTCT3T9C); sbaymlopwle-sbpuefefedrc.eCnutlrtiufuregastuiopnerannadtafinlttsraotfiotrnanthsrfeocutgehdaor0.i4n5femctmed-pcoerlels-swizeerefilctlearr,ifianedd y 5
(ii)forGagD350-362,inwhich13CAproteinC-terminalaminoacidresidues virionorVIPsweresubsequentlysedimentedbycentrifugationat100,0003gfor , 2
werereplacedbySer,reverseCSLF(59CCACTCCCTGGCTAGCTGTCATC 1hthrougha20%sucrosecushion.Insomeexperiments,virionsorVLPswere 0
39)andforwardCADRG(59AAAGCAGCTAGCTTGGCTGAAGCAATG39); treatedwithsubtilisintodigestcontaminatingcelldebrisandmembranevesicles 1
(iii)forGagD350-377,inwhichCAproteinC-terminalaminoacidresiduesand (44). Briefly, concentrated virus suspension was supplemented with an equal 9
volumeof2mgofsubtilisin(Sigma)/mlin40mMTris-HCl(pH8.0)–2mM b
thespacerpeptidep2werereplacedbySer,reverseCSLFandforwardP2DRG y
(e5n9tiGreCnTuAclCeCocGaCpsTidApGrCotAeiTnA(CNACG)wAaAsAreGpGlaCce3d9)b;y(iAv)lafo-SreGr,argeDveNrCse,iNnCwDhiLcFht(h5e9 CphaeCnly2lmanedthiynlscuulbfaotneydlflouvoerrnidigehttoa5tm3g7/°mCl.,DanigdetshtieonvirwuassosrtoVpLpPesdwbeyreadredcitoivoenreodf gu
TTGCCTTTGCTAGCCATTATGGTAGC39)andforwardNCDRG(59GAC bycentrifugationat100,0003gthrougha20%sucrosecushion,resuspendedin e
AGGCTAGCTTTTTAGGGAAG39);(v)forGagDNC1-14andDNC1-14p,in TNE(20mMTrisHCl,pH7.5,150mMNaCl,1mMEDTA),andrecentrifuged. st
whichthefirst14aminoacidresiduesinNCwerereplacedbyAla-Ser,reverse Insomeexperiments,VLPsorvirionsweretreatedwith1%TritonX-100in
NCDLFandforwardZN1DRG(59GACTGTTGCTAGCTTCAATTGTGGC TNE for 10 min at a specified temperature, followed by recentrifugation at
39);(vi)forGagDp2,inwhichtheGagsequence363to377includingap2spacer 100,0003gthrougha30%sucrosecushionsupplementedwith1%TritonX-100.
peptidewasreplacedbyAla-Ser,reverseP2DLF(59TGCTTCGCTAGCAAC ThepelletedmaterialwaslysedbyaddingSDS-PAGEsamplebuffer.
TCTTGCTTTATGG 39) and forward P2DRG. Gag mutant DNC1-14p also Equilibriumsucrosegradientcentrifugation.VLPsorvirionscollectedfrom
carried,besidesthedeletion,aterminationcodoninposition27withinthefirst supernatantswithorwithoutfurthertreatmentwereplacedontopof10mlofa
Cys-HisboxofNC,effectivelyremovingapartofNCandtheentirep1andp6 20to60%lineargradientofsucroseinphosphate-bufferedsalineorTNEand
Gagdomains.TheGagmutantsD350-362,D350-377,andDNCwereinsertedin centrifugedfor18hat100,0003gat4°C.Fractionsof1mlwerecollectedfrom
afunctionalHIV-1NL4-3proviralclonebyusingSphandBglIIrestrictionsites. thebottom,andparticlesineachfractionwererecoveredbycentrifugationat
The vif gene was cloned in pCITE4(1) plasmid modification pCITEHA. 100,0003g,washed,andlysedinSDS-PAGEsamplebuffer.
pCITEHA/VifexpressedN-terminallytaggedVifwithasequencecontainingthe Velocity Optiprep gradient centrifugation. Optiprep (Iodixanol; GIBCO/
influenza virus hemagglutinin epitope (HA; AYPYDVPDYA [27]). The con- BRL)gradientcentrifugationwasperformedessentiallyasdescribedbyDetten-
struction of pCITEHA was carried out by amplifying pCITE4a(1) using Pfu hoferandYu(15).VLPsorvirionswerecollectedfromsupernatantsthrougha
polymerasefromprimersHACITER(59GGTTCCATGGCTTATCCTTATGA sucrosecushionandwashedfreeofsucrose.Thepelletedparticleswereresus-
CGTTCTGACTATGCTGGATCCGAATTCGAGCTCCGTCG 39) and CITEL; pendedbyrepeatedpipetting,andlargeaggregateswereremovedbycentrifu-
theproductoftheamplificationwasdigestedbyDpnIandNcoIandligated. gationat1,0003gfor2min.Particlesuspension(100to250ml)wasplacedon
pCITEHA/VifwasconstructedbyinsertingthevifgenefrompGEX-2TVif(34) topof11mlof6to18%linearOptiprepgradientinTNEandcentrifugedfor1.5
inBamHIandEcoRIcloningsites.pCITE/Vifexpressingwild-typeVifwithout hat200,0003g.Sixteenfractionsofgradientwerecollected,andparticlesin
5506 SOVA ET AL. J.VIROL.
D
o
FIG. 1. AssociationofVifwithprotease-treatedvirions.(A)NL4-3viruswasproducedincellsasindicatedandtreatedwithsubtilisin.Two w
aliquots(thesecondaliquotisatwofoldamountofthefirst)ofviruslysatesfromeachproducinglinewereresolvedonSDS-PAGE.(B)KS283 n
virusproducedfromH9andMT-2cellswasresolvedonSDS-PAGEatasimilarp24CAcontent.Vifwasdetectedinimmunoblottingwithanti-Vif lo
a
rabbitantiserum(bottompanels),andGagwasdetectedbyreprobingofstrippedmembraneswithanti-CAmonoclonalantibody(toppanels). d
e
d

f
each fraction were recovered by centrifugation at 100,000 3 g and lysed in WefurthertestedtheassociationofVifwithvirionspurified ro
SDS-PAGEsamplebuffer. through Optiprep gradients, the method that was shown to m
Electrophoresisandimmunoblotting.Lysedcellsorpelletedmaterialwere separate nonvirion particles containing Vif from bona fide h
resolvedbySDS-PAGEandtransferredontoa0.2-mmTrans-Blotnitrocellulose t
membrane(Bio-Rad,Hercules,Calif.)followedbyimmunoblottingasdescribed virions(15).HIV-1KS283wasproducedbyinfectionofMT-2 tp
:
elsewhere(37).Boundantibodieswerevisualizedbyusinganenhancedchemi- cells, and NL4-3 mutant in Gag lacking the NC domain were //
luminescence (ECL) kit (Amersham, Arlington Heights, Ill.). When needed, madebytransfectionof293TcellswithproviralplasmidDNA. jv
antibodieswerestrippedfromnitrocellulosemembranesbyincubationin0.2M TheNCdeletionmutantwasusedbecauseNCwasreportedto i.a
NaOHfor5min.Antibodiesusedwereasfollows:anti-HAmonoclonalantibody s
mediatetheinteractionofViftoGagandtheinclusionofVif m
HA.11, clone 16B12 (BabCo, Berkeley, Calif.); anti-CA monoclonal antibody
producedfromHybridoma183,cloneH12-5C(9),kindlyprovidedbyB.Chese- inVLPs(5,31).Virusescollectedfromsupernatantsbyultra- .o
bro and H. Chen through the NIH AIDS Research Reference and Reagent centrifugationwereresolvedinOptiprepvelocitygradientcen- rg
Program;andanti-Vifantibody(24),kindlyprovidedbyD.Gabuzdathroughthe trifugation essentially as described previously (15). Individual /
NIHAIDSResearchReferenceandReagentProgram.Densitometrywasper- o
gradient fractions were centrifuged to pellet the virions, and n
formedusingtheKodakDigitalSciencesystem(Kodak,Rochester,N.Y.).
theGagandVifcontentofthepelletedmaterialwasanalyzed J
a
byelectrophoresisandimmunoblotting(Fig.2).Wefoundthe n
RESULTS u
bulk of Gag in the bottom fraction (number 1) and the rest a
Vif is specifically associated with HIV-1 virions made in distributed through the lower half of gradients with a slight ry

permissive and nonpermissive cells. We first examined peakinhigherfractions4to7.Ourinterpretationofthisresult 5
,
whether the level of Vif incorporation in HIV-1 virions de- is that the bottom fraction contained virion aggregates while 2
pendsontheabilityofhostcellstosupportreplicationofvif2 fractions4to7containedvirionparticlesinsuspension.Most 01
HIV-1. We used permissive SupT1 cells (22), nonpermissive importantly,Vif-specificsignalsalignedpreciselywithGagsig- 9

H9 and MT-2 cells (22, 37, 64), and 293T human embryonic nals, confirming that Vif copurifies with virions in Optiprep b
y
kidney cells, which in our hands produce vif2 HIV-1 virions gradients.DeletionoftheNCdomaindidnotaffectVifincor- g
withapproximatelytwofoldlowerinfectivitythanthatofwild- poration. Similar results were obtained using HIV-1 KS283 u
e
type HIV-1 (data not shown) and thus can be considered produced from H9 cells (data not shown). We conclude that s
t
semipermissive.Virusfromsupernatantsofinfectedortrans- VifisspecificallyassociatedwithhighlypurifiedHIV-1virions.
fected cells (see Materials and Methods) was sedimented by Vif resides predominantly in virions composed of Pr55Gag
centrifugationat100,0003gthrougha20%sucrosecushion. that are insoluble in detergent. The virion samples from dif-
Resuspended virions were treated with subtilisin to remove ferentcelllinesusedherecontainedrelativelyhighamountsof
membrane vesicles and proteinaceous debris (44) and resedi- unprocessedPr55Gaginadditiontop24CA(Fig.1and2).The
mented through a 20% sucrose cushion (Fig. 1). As shown in presenceofbothprocessedandunprocessedGagobservedin
Fig. 1A, virions produced in either permissive or nonpermis- these experiments can be explained in two ways. Either indi-
sivecellscontainedclearlydetectableVifaftersubtilisintreat- vidualvirionscontainbothprocessedandunprocessedGag,or
ment,andMT-2cellscarriedthehighestamountofVifamong virions consist of mixed populations of distinct particle types,
the cell lines tested (compare Vif signals in lanes 1, 4, and 5, onecontainingmaturep24CAandnounprocessedGagandthe
which contain an approximately equivalent amount of Gag). other containing Pr55Gag and no p24CA. To distinguish be-
VirionsproducedinnonpermissiveH9cellscontainedVifata tween these two alternatives, we treated purified virions with
levelsimilartothatfoundinMT-2-producedvirions(Fig.1B). TritonX-100andanalyzedtheircompositioncomparedtothat
ThusVifassociateswithprotease-treatedvirionsmadeinper- ofuntreatedvirions(Fig.3).Ithasbeenshownthatmaturebut
missiveornonpermissivecells. not immature retroviral cores dissolve in nonionic detergents
VOL.75,2001 HIV-1 Vif, VIRIONS, AND VIRUS-LIKE PARTICLES 5507
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a
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FIG. 2. AssociationofVifwithvirionspurifiedbyOptiprepgradientvelocitycentrifugation.Wild-typeHIV-1NL4-3(topsetofpanels)and
GagDNCNL4-3(middleset)producedin293TcellsorHIV-1KS283producedinMT-2(bottomset)wereresolvedbyvelocityOptiprepgradient 5,

centrifugation.Thegradientfractionswerecollectedfromthebottom(left,startingwithnumber1),andpelletedvirionsfromeachfractionwere 2
resolvedonSDS-PAGE.IntheHIVKS283gradient,virionswerealsocollectedfromthebottomofthecentrifugationtube(B).Vifwasdetected 0
1
by immunoblotting with anti-Vif rabbit antiserum (lower panel in each set), and Gag was detected by reprobing of stripped membranes with 9
anti-CAmonoclonalantibody(toppanelineachset). b
y

g
u
e
suchasTritonX-100orNonidetP-40(7,46,47,50,65,67,70, result suggests that virions contain either mature p24CA or s
t
71). We reasoned that particles composed exclusively of unprocessed Pr55Gag but not both. Upon Triton X-100 treat-
Pr55Gag should remain intact and thus able to sediment after ment,mostofVifpresentinvirionpreparationscosedimented
detergenttreatment,whilematureparticlescomposedsolelyof withPr55Gag(Fig.3A;comparelanes1to2and3to4inthe
processedGagandpresumablyalsoparticlescontainingamix- bottom panels), indicating that Vif associates primarily with
ture of mature and unprocessed Gag should dissolve upon particles containing unprocessed Gag and not with mature
detergent treatment and fail to sediment. We treated KS283 virions. The slight decrease in the Vif signal following deter-
and NL4-3 virions produced in H9 and MT2 cells with 1% genttreatmentisconsistentwithremovalofmembranevesicles
Triton X-100 and centrifuged treated and untreated virions orcelldebriscontainingVifassuggestedbyDettenhoferand
through20%sucrosecushions(Fig.3).Asexpected,thep24CA Yu (15) and our experiments with expression of Vif in the
signal was almost entirely eliminated by Triton X-100 treat- absenceofGag(seebelow).
ment (Fig. 3A; compare lanes 1 to 2 and 3 to 4 in the top EffectofHIV-1PRinhibitoronVifincorporationinvirions.
panels), consistent with the solubilization of mature virions TheresultshowninFig.3Aindicatedthatmostofthevirion-
containingp24CA.Incontrast,thesignalofPr55Gagwasessen- associated Vif resides in virus particles containing Pr55Gag,
tially unchanged, suggesting that detergent-stable particles someofVifispresentinmembranevesiclesorcelldebris,but
containing unprocessed Gag contain no mature p24CA. This verylittleornoVifbecomesincorporatedintomaturevirions.
5508 SOVA ET AL. J.VIROL.
D
FIG. 3. AssociationofVifwithvirionstreatedwithdetergentorviralproteaseinhibitor.(A)KS283viruswasharvestedfromH9orMT-2cells o
w
andtreatedwith1%TritonX-100for10minatroomtemperature.Virionswereresedimentedthrougha20%sucrosecushionandresolvedon
n
SDS-PAGE.(B)KS-283-infectedMT-2cellsweretreatedwiththeviralproteaseinhibitorsaquinavir2daysafterinfection.Viruswascollected lo
24 h later and centrifuged through a 20% sucrose cushion. Cell (left) or virus lysates were resolved on SDS-PAGE. Vif was detected in a
immunoblottingwithanti-Vifrabbitantiserum(bottom),andGagwasdetectedbyreprobingofstrippedmembraneswithanti-CAmonoclonal d
antibody(top). e
d

f
r
o
m
TheapparentselectiveincorporationofVifintoPr55Gag-con- independent translational enhancer derived from encephalo-
h
taining particles and, conversely, its exclusion from mature myocarditisvirusincis(CITE[16,17]).TheT7polymeraseis t
t
p
virionswassurprising.OnepossibleexplanationforVifexclu- suppliedintransbyinfectionofcellswithrecombinantvaccinia :
/
sion is that it may be degraded by viral protease in mature virusvTF7-3(20).VifandVprwereN-terminallytaggedwith /jv
virions.However,thisisunlikelybecauseVifwasshowntobe anHAtag(27)tofacilitatedetectionandtodirectlycompare i.
a
resistant to PR-mediated cleavage in bacteria, in mammalian theefficiencyofincorporationofVifandVprinVLPs,andthe s
m
cells,andincell-freeproteolysisassays(34,49).Alternatively, Stag(33)wasusedasacontroltotheHAtag.Similarsystems
.
Vifmaybeexcludedfromvirionsbysomemechanismduring havebeenusedpreviouslytoanalyzetheassociationofHIV-1 o
r
buddingandmaturationwhileafractionoftheproteinremains VprorHIV-2VpxwithGagandVLPs(40,41,45,62).Inthe g
/
associatedwithdefectiveGagassemblycomplexesthatarenot firstsetofexperiments,wecomparedtherelativeefficiencyof o
n
destinedformaturationintoinfectiousvirionsbutarereleased Gag and Vif expression from pCITE with the expression of
J
asPr55Gag-containingparticles.Toinvestigatethispossibility, these proteins during HIV-1 infection (Fig. 4). As shown in a
n
wetreatedHIV-1-infectedMT-2withtheHIV-1proteasein- Fig.4A,theoverallexpressionofGagandViffromprovirusin
u
hibitor saquinavir (51) under conditions in which processing 293T cells was slightly more efficient than the expression of a
r
and infectivity are blocked (Fig. 3B). If Gag and Gag-Pol these proteins from pCITE (Fig. 4A; compare Gag and Vif y

processingbyviralproteaseisresponsibleforexclusionofVif signals in lanes 3 and 6). To interpret obtained data, it was 5
,

from virions or Vif is degraded by protease, its inhibition crucial to maintain the ratio of Gag and Vif expressed from 2
0
shouldincreasethecontentofVifinvirions.AsshowninFig. pCITE similar to that of the proviral expression. This was 1
3B,inhibitionofprotease-mediatedproteolysiseliminatedthe achieved when the pCITED/Gag and pCITEHA/Vif DNAs 9

p24CA signal in both virions and cell extracts and slightly re- weretransfectedata1:1or1:2weightratio(Fig.4B;compare b
y
duced—rather than increased—the amount of Vif associated Gag and Vif signals in lanes 1, 2, and 5), and we therefore g
withvirions(Fig.3B;comparelanes3and4,bottompanels). typicallycotransfected5mgofpCITED/Gagand2.5to5mgof u
e
This indicates that Gag processing does not lead to exclusion pCITEHA/Vifperpetridish. s
t
ofViffrommaturevirionsandthatVifisnotdegradedbyviral VifispackagedintoVLPslessefficientlythanVpr.Wenext
protease. Rather, this result suggests that Vif may associate examinedwhethercoexpressionofGagandVifwouldleadto
withputativedefectiveparticleslackingotherviralproteinsor theproductionofVLPscontainingVif.AsshowninFig.5,the
assuming a defective structure and subsequently be unable to expressionofGaginCOS-1or293Tcellsdirectedtheassem-
processGagandpassthroughmaturationofthevirioncore. bly and release of VLPs, which could be sedimented from
Analysis of Vif encapsidation in Gag and Vif coexpression culture supernatants by ultracentrifugation. This is shown by
system. The association of Vif with Pr55Gag containing viral detection of a CA-specific 55-kDa band corresponding to the
coressuggestedaspecificinteractionbetweenthetwoproteins. Gag precursor polyprotein in culture supernatant sediments
TodeterminetherequirementsfortheassociationofVifwith (Fig.5A,lanes3and5;Fig.5B,lanes6and8to12;toppanels).
immature virions, we adopted a versatile system for Gag and VifcoexpressedwithGagcosedimentedwithVLPsasattested
Vif mutagenesis and coexpression to produce Gag-containing by the presence of a Vif-specific protein of 24 kDa in Gag-
VLPs that closely resemble immature virions (21). Gag, Vif, containinglanes(Fig.5A,lanes3and5;Fig.5B,lane6;bottom
andVprwereexpressedfromthepCITEvector,whichpermits panels).Wild-typeVifcosedimentedwithGagequallyaswell
efficient transcription of inserted genes from the T7 bacterio- as the N-terminally HA- or S-tagged Vif (Fig. 6B), indicating
phage promoter, further facilitated by the presence of a cap- thatVifassociationwithVLPswasnotduetothepresenceof
VOL.75,2001 HIV-1 Vif, VIRIONS, AND VIRUS-LIKE PARTICLES 5509
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FIG. 4. ComparisonofGagandVif(HA-Vif)expressionin293TcellstransfectedwithpCITEorwithanNL4-3proviralclone.(A)293Tcells
r
cotransfectedwithpCITED/GagandpCITEHA/Vifor293TcellstransfectedwithpNL4-3wereloadedintheamountsindicated.(B)293Tcells g
were cotransfected with pCITED/Gag and pCITEHA/Vif as indicated (in mg DNA per petri dish) or with pNL4-3. Gag and Vif in 17,000 o/
pCITE-transfected or 8,300 pNL4-3-transfected cells were detected in immunoblotting with anti-Vif rabbit antiserum (bottom), and Gag was n
detectedbyreprobingofstrippedmembraneswithanti-CAmonoclonalantibody(top).NotethatexposureofVifinpanelBwaslongerthanin J
panelAtodetectweaksignalsinlanes3and4. a
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oligopeptide tags. To exclude the possibility that Vif itself collectedfromsupernatantsofcellscoexpressingGagandHA- 5
,

formsextracellularsedimentableaggregates,wecollectedand Vpr (Fig. 5B, lanes 9 to 12), we estimated that HA-Vpr is 2
0
analyzed particulate material present in the supernatants of approximately10-to20-foldmoreabundantinGagVLPsthan 1
cells expressing Vif in the absence of Gag. Although a small is HA-Vif. Thus, Vif is packaged into Gag VLPs much less 9

b
amountofVifwaspelletable,itdissolvedupontreatmentwith efficientlythanVpr.
y
detergent,indicatingthatVifreleasedfromcellsintheabsence Vif is specifically incorporated in Gag VLPs resolved by g
ofGagmostlyassociateswithmembranevesiclesormembra- gradientcentrifugation.Wesoughttoobtainmoreconclusive u
e
nous debris (Fig. 5A, lanes 4 and 6). The amount of Vif in evidenceforthespecificityofVifpackaginginVLPsbypuri- s
t
particles released from Gag and Vif coexpressing cells also fyingparticlesthroughsucrosedensitygradients.Extracellular
slightly diminished after detergent treatment (Fig. 5A; com- VLPswereisolatedfromsupernatantsofCOS-1cellsexpress-
pare lanes 3 and 5), confirming that a small portion of Vif ingwild-typeGagandN-terminallytaggedVifandresolvedby
sedimentable from supernatants of these cells associates with densityequilibriumcentrifugationthrougha20to60%linear
membranevesicleswhilemostofitrepresentsVifincorporated sucrosegradient.AsshowninFig.6A,leftpanels,GagVLPs
inVLPs.WeconcludethatVifispackagedintoGagVLPs. were most abundant in fractions 4 to 6, corresponding to the
Thiscoexpressionsystemallowedustodirectlycomparethe density of 1.16 to 1.20 g/cm3 typical for retroviral particles.
efficiencyoftheincorporationofVifintoGagVLPswiththat DetergenttreatmentofVLPsshiftedtheGagsignaltofraction
ofVprwhenbothproteinswereN-terminallytaggedwithHA 3withadensityof1.26g/cm3(Fig.6A,rightpanels),consistent
epitope(Fig.5B).Vprisknowntobeefficientlyencapsidated with the removal of lipid envelopes. The peak signals of Vif
in HIV-1 virions or Gag VLPs (11, 41, 42, 62). Although the alignedpreciselywiththepeakGagsignals,indicatingthatVif
expressionlevelsofHA-ViforHA-Vprweresimilar(Fig.5B, specifically associates as an integral component with Gag
lanes2and4),extracellularGagVLPscontainedmuchhigher VLPs.VLPscomposedofGagaloneorGagandVifmigrated
levelsofHA-VprthanHA-Vif(comparelanes6and8inFig. to fractions of similar density, indicating that the presence of
5B, bottom panel). By loading decreasing amounts of VLPs VifdoesnotaffectthedensityofVLPs(datanotshown).
5510 SOVA ET AL. J.VIROL.
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FIG. 5. AssociationofViforVprwithGagVLPs.LysatesofcellscoexpressingGagwithHA-ViforHA-Vprorlysatesofsupernatantsediments .
o
wereresolvedonSDS-PAGE.(A)S-taggedVifwascoexpressedwiththewild-typeGaginCOS-1cells.AnaliquotofsedimentedVLPswastreated r
g
with1%TritonX-100for15minatroomtemperatureandresedimented.S-Vifwasdetectedinimmunoblottingwithanti-Vifrabbitantiserum
/
(bottom), and Gag was detected by reprobing of stripped membranes with anti-CA monoclonal antibody (top). (B) HA-Vif or HA-Vpr were o
coexpressedwithwild-typeGagfromthepCITEvectorin293Tcells.Lanes8to12:decreasingfractionsofsupernatantpelletlysatewereloaded n

intheorder1/2,1/5,1/10,1/20,and1/50.HA-VifandHA-Vprweredetectedinimmunoblottingwithanti-HAmonoclonalantibody(bottomand J
a
middlepanels),andGagwasdetectedbyreprobingofstrippedmembraneswithanti-CAmonoclonalantibody(top).Inset:longerexposureof
n
lanes5to8indicatesbackgroundlevelsofsedimentableVifandVpr. u
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5
,

Using Optiprep velocity gradients, we demonstrated that have been shown to mediate interaction with Vif as well as 2
0
highlypurifiedHIV-1virionscontainVif(Fig.2).Toconfirm packaging of Vif in Gag VLPs isolated from insect cells (31). 1
thatVLPsreproducethisassociation,weexaminedthecontent We sought to examine whether this C-terminal region of the 9

b
ofVifinVLPspurifiedthroughOptiprepgradients(Fig.6B). GagprecursorpolyproteinisrequiredforVifparticlepackag-
y
Gag and wild-type Vif were coexpressed in 293T cells, and ing in animal cells, anticipating that coexpression of Vif with g
particlesfromculturesupernatantsedimentedthrougha20% Gag precursor polyprotein carrying C-terminal deletions will u
e
sucrosecushionandwashedfreeofsucrosewereresolvedina leadtosecretionofVLPslackingVif.Weconstructedasetof s
t
6 to 18% Optiprep gradient. The pelletable material in indi- deletionortruncationmutantsspanningtheC-terminalpartof
vidualfractionswasscreenedforGagandVifbyimmunoblot- Gag including the CA and NC domains as well as the spacer
ting (Fig. 6B). Strong signals of both Gag and Vif were de- peptide p2 (see Materials and Methods and Fig. 7B). Figure
tected in the bottom fraction (number 1), and Gag and Vif 7AshowstheGagandVifcontentinVLPsobtainedinthree
werefurtherdispersedinidenticalfractions2through10with experimentsinwhichGagmutantswerecoexpressedwithHA-
a slight increase in fractions 7 and 8. We conclude that coex- Vifin293TorCOS-1cells.SampleswhereVifwasexpressed
pression of wild-type Gag with wild-type or N-terminally togetherwithwild-typeGag(lanes1,9,and12)oralone(lanes
tagged Vif results in the assembly and release of VLPs that 2, 10, and 13) served as controls. As shown in the VLP Gag
specifically incorporate Vif. A detectable amount of particu- panelsinFig.7A(thirdrowfromtop)andsummarizedinthe
late Vif is also secreted, in addition to that present in Gag table in Fig. 7B, most constructs produced VLPs at levels
VLPs,butitcanbedistinguishedfromVifwithinVLPsonthe similar to that of the wild-type Gag. Vif was present in VLPs
basisofitsdetergentsensitivity(Fig.5A)butnotbyitsdensity formed of all Gag mutants but its amount strongly depended
orshapeasrevealedbymigrationindensitygradients(Fig.6). on the Gag structure. Significantly, Vif was present in VLPs
PatternofVifincorporationinVLPsandvirionsconsisting formed of GagDNC, GagDNC1-14, and GagDNC1-14p, indi-
of Gag with deletions. Sequences in the NC domain of Gag catingthatthenucleocapsidregionanddownstreamC-termi-
VOL.75,2001 HIV-1 Vif, VIRIONS, AND VIRUS-LIKE PARTICLES 5511
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FIG. 6. AssociationofVifwithVLPspurifiedbygradientcentrifugation.(A)GagwascoexpressedwithS-ViffrompCITEinCOS-1cells. n
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SedimentedVLPsweretreatedwithTritonX-100for15minoniceorleftuntreatedandcentrifugedthrougha20to60%sucrosegradientto
a
equilibrium.Gradientfractionscollectedfromthebottom(fractionnumber1)werepelletedandprobedforVifwithanti-Vifrabbitantiserum r
y
(bottompanels)andforGagwithanti-CAmonoclonalantibodyafterstripping(toppanels).Thesucrosedensityofindividualfractionsorthe
5
intensityofbandsobtainedbydensitometryareplottedbeloweachpanel:thesolidlinewithsquaresymbolsrepresentsVifsignalintensity;the ,
solidlinewithcirclesymbolsrepresentsGagsignalintensity;thedashedlinewithcrossedsymbolsrepresentssucrosedensity.(B)Wild-typeGag 2
VLPs containing wild-type Vif were produced in 293T cells upon cotransfection with pCITED/Gag and pCITE/Vif and resolved by velocity 0
1
Optiprepgradientcentrifugation.Thegradientfractionswerecollectedfromthebottom(left,startingwithnumber1),andpelletedVLPsfrom 9
eachfractionwereresolvedonSDS-PAGEandimmunoblottedwithanti-Vif(bottom)andanti-CA(top)antibodiesasinpanelA.
b
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g
nal areas in Gag are not required for Vif packaging in VLPs subtilisin,andtestedforVifcontent(Fig.7C).GagD350-377 u
e
(Fig. 7A, lanes 6, 7, 14, and 15, and the table in Fig. 7B). virus showed a higher ratio of Vif to Gag than the wild-type s
Surprisingly, VLPs consisting of Gag containing short dele- virus.Similarly,HIV-1NL4-3GagmutantD350-362carriedan t
tions in the C-terminal region of CA protein (DMHR, D350- increased amount of Vif (data not shown), verifying that CA
362,andD350-377)incorporatedasignificantlylargeramount deletionscauseincreasedpackagingofVifinbonafidevirions.
ofVifthanthewild-typeGagVLPs(Fig.7A,lanes3,4,8,and ToconfirmthattheincreasedcontentofVifinVLPsmade
11), an approximately 10-fold excess (table in Fig. 7B for ofGagcontainingCAdeletionswasindeedspecific,VLPfrac-
GagD350-362andGagD350-377).Incontrasttootherdeletion tions were subjected to overnight treatment with subtilisin to
mutants,VLPsconsistingofDp2Gagcontainedapproximately degrade vesicles and cellular debris (44). The treatment with
30to60%oftheVifpresentinwild-typeGagVLPs(Fig.7A, subtilisin removed approximately 50% of Gag and Vif from
lane5,andthetableinFig.7B). sedimentable material (data not shown) but the relative
TodeterminewhethertheeffectofGagstructureuponVif amount of HA-Vif in VLPs treated with subtilisin was equiv-
incorporation into VLPs applied also to its encapsidation in alenttothatinuntreatedVLPsandthemarkedincreaseofVif
virions, we constructed an NL4-3 recombinant clone contain- content in VLPs consisting of Gag with a deletion in the CA
ing a deletion of the extreme CA C terminus and p2, (D350- domainwaspreserved(Fig.8A;comparelanes6to8tolanes
377) and expressed it by transfection in 293T cells. Wild-type 10to12).
NL4-3 and Gag D350-377 virions were isolated, treated with TofurtherverifytheassociationofincreasedamountsofVif
5512 SOVA ET AL. J.VIROL.
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FIG. 7. AssociationofVifwithmutantGagVLPsandvirions.(A)GagwascoexpressedwithHA-Vifin293T(lanes1to8)orCOS-1(lanes rg
9to15)cells.VLPssedimentedfromsupernatantsbyultracentrifugationweretreatedwith1%TritonX-100toremovebackgroundVifsignal, /
o
resedimented,andresolvedinSDS-PAGEtogetherwithafractionofcelllysates.Vifwasdetectedinimmunoblottingwithanti-HAmonoclonal
n
antibody(secondandfourthpanelsfromthetop)andGagwasdetectedbyreprobingofstrippedmembraneswithanti-CAmonoclonalantibody
(top panel and third panel from the top). Gag DMHR in lane 8 was detected with AIDS patient sera. Differences in signal intensity among Ja
individualpanelsdonotreflectdifferencesinexpressionlevelsbutratherthevariabilityinfilmexposure.(B)SchematicrepresentationofGag n
deletionmutantsusedinthestudyandrelativecontentofVifinVLPs,normalizedtoGagcontent.(C)Wild-typeNL4-3(wt)andGagD350-377 u
a
NL4-3 (D7) were produced in 293T cells upon transfection with proviral plasmid DNA and treated with subtilisin. Vif was detected in r
y
immunoblotting with anti-Vif rabbit antiserum (bottom panel), and Gag was detected by reprobing of stripped membranes with anti-CA
5
monoclonalantibody(top).
,

2
0
1
within mutant Gag VLPs, GagD350-362 and GagD350-377 right set of panels, fractions 6 and 7), although some Gag 9

b
VLPs containing HA-Vif were purified by centrifugation signal was also detected in bottom fractions with a density of
y
throughasucrosedensitygradient(Fig.8B);VLPswereeither 1.27to1.3g/cm3.Itisnotclearwhythedensityofdetergent- g
treatedwithTritonX-100(rightpanels)orleftuntreatedand treated GagD350-377 VLPs decreased upon detergent treat- u
e
evaluatedforthedistributionofVifandGag.Similartodata ment that invariably increases the density of wild-type Gag s
t
shown in Fig. 6, the Vif signal colocalized with Gag, demon- VLPs(Fig.6).WeconsiderthatthedeletioninGag(position
strating that Vif was specifically incorporated in VLPs. For 350to377)grosslyaffectsthestructureoftheresultingVLPs
GagD350-362,thedensityofuntreatedVLPsrangedbetween and their conformation after detergent treatment. The pres-
1.15and1.17g/cm3(Fig.8B,topleftsetofpanels,fractions6 enceofVifdidnotaffectthedensityofVLPsformedbyGag
and7),whilethedetergent-treatedVLPsmigratedtofractions mutants D350-362 and D350-377 as these mutant Gag VLPs
with a density of 1.25 to 1.27 g/cm3 (Fig. 8B, top right set of displayedsimilardensitiesandresponsetoTritonX-100treat-
panels,fractions2and3),identicaltothewild-typeGagVLPs ment in the absence of Vif (data not shown). Specific comi-
(compare with Fig. 6). VLPs composed of GagD350-377 dis- gration of HA-Vif with GagD350-362 was also seen in the
playedmuchbroaderdensitydistribution,withapeakbetween Optiprepvelocitygradient(datanotshown).Weconcludethat
1.13and1.24g/cm3,indicatingthatthelargerdeletionoftheC two short deletions in the CA domain of the Gag precursor
terminus of CA protein plus spacer peptide p2 disturbed the polyproteinthatotherwisearenotdisruptivetoassemblyand
structureofVLPs(Fig.8B,bottomleftsetofpanels,fractions releaseofGagVLPsleadtoa10-foldincreaseinthepackaging
4 to 8). The detergent treatment of GagD350-377 VLPs, un- ofVifinVLPs.
expectedly, concentrated the peak Gag and Vif signals in ComparisonofVifandVprincorporationinCAmutantand
lighter 1.14 to 1.16 g/cm3 density fractions (Fig. 8B, bottom wild-type Gag VLPs. In the next experiment, we sought to
VOL.75,2001 HIV-1 Vif, VIRIONS, AND VIRUS-LIKE PARTICLES 5513
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FIG. 8. AssociationofVifwithmutantGagVLPpurifiedbyproteasetreatmentorthroughasucrosegradient.(A)HA-Vifwascoexpressed
withwild-typeGag(wt),GagD350-362(D3),orGagDMHR(DM)in293Tcells.VLPscollectedfromsupernatantsweretreatedwithsubtilisinand
resolvedtogetherwithcelllysatesonSDS-PAGE.(B)GagwascoexpressedwithHA-Vifin293Tcells,andVLPspelletedfromsupernatantwere
treatedwithTritonX-100(rightpanels)orleftuntreatedandresedimentedthrougha20to60%densitysucrosegradientovernight.Tenfractions
ofgradient,startingfromthebottom(fractionnumber1),aswellasthesedimentfromthebottomofcentrifugationtubesrepresentingmaterial
denserthan60%sucrose(fractions11),werecollected,resedimented,andanalyzedforVifcontentinimmunoblottingwithanti-HAmonoclonal
antibody(bottompanels)andforGagbyreprobingofstrippedmembraneswithanti-CAmonoclonalantibody(toppanels).Thesucrosedensity
ofindividualfractionsobtainedbyrefractometryortheintensityofVifandGagbandsobtainedbydensitometryareplottedbeloweachpanel.
ThesolidlinewithsquaresymbolsrepresentsVifsignalintensity;thesolidlinewithcirclesymbolsrepresentsGagsignalintensity;andthedashed
linewithcrossedsymbolsrepresentssucrosedensity.
determine whether increased incorporation of Vif in VLPs in the presence of wild-type Gag or GagD350-362 and deter-
formed by CA deletion mutants is a result of the general minedtheefficiencyofincorporationoftheseproteinsinVLPs
tendencyofthesemutantGagVLPstoincorporateincreased and the stability of incorporation after detergent treatment
amountsofviralproteins.WecoexpressedHA-ViforHA-Vpr (Fig. 9). Intracellularly, HA-Vif and HA-Vpr were expressed