The HIV Envelope but Not VSV Glycoprotein Is Capable of Mediating HIV Latent Infection of Resting CD4 T Cells

The HIV Envelope but Not VSV Glycoprotein Is Capable of Mediating HIV Latent Infection of Resting CD4 T Cells TheHIVEnvelopebutNotVSVGlycoproteinIsCapable ofMediatingHIVLatentInfectionofRestingCD4TCells DongyangYu,WeifengWang,AlysonYoder,MarkSpear,YuntaoWu* DepartmentofMolecularandMicrobiology,GeorgeMasonUniversity,Manassas,Virginia,UnitedStatesofAmerica Abstract HIVfusionandentryintoCD4Tcellsaremediatedbytworeceptors,CD4andCXCR4.Thisreceptorrequirementcanbe abrogated by pseudotyping the virion with the vesicular stomatitis virus glycoprotein (VSV-G) that mediates viral entry throughendocytosis.TheVSV-G-pseudotypedHIVishighlyinfectiousfortransformedcells,althoughtheviruscircumvents theviralreceptorsandtheactincortex.InHIVinfection,gp120bindingtothereceptorsalsotransducessignals.Recently, wedemonstratedauniquerequirementforCXCR4signalinginHIVlatentinfectionofbloodrestingCD4Tcells.Thus,we performedparallelstudiesinwhichtheVSV-G-pseudotypedHIVwasusedtoinfectbothtransformedandrestingTcellsin the absence of coreceptor signaling. Our results indicate that in transformed T cells, the VSV-G-pseudotyping results in lowerviralDNAsynthesisbutahigherrateofnuclearmigration.However,inrestingCD4Tcells,onlytheHIVenvelope- mediatedentry,butnottheVSV-G-mediatedendocytosis,canleadtoviralDNAsynthesisandnuclearmigration.Theviral particlesenteringthroughtheendocytoticpathwayweredestroyedwithin1–2days.TheseresultsindicatethattheVSV-G- mediatedendocytoticpathway,althoughactiveintransformedcells,isdefectiveandisnotapathwaythatcanestablishHIV latentinfectionofprimaryrestingTcells.OurresultshighlighttheimportanceofthegenuineHIVenvelopeanditssignaling capacityinthelatentinfectionofbloodrestingTcells.Theseresultsalsocallforcautionontheendocytoticentrymodelof HIV-1, and on data interpretation where the VSV-G-pseudotyped HIV was used for identifying HIV restriction factors in restingTcells. Citation:YuD,WangW,YoderA,SpearM,WuY(2009)TheHIVEnvelopebutNotVSVGlycoproteinIsCapableofMediatingHIVLatentInfectionofRestingCD4 TCells.PLoSPathog5(10):e1000633.doi:10.1371/journal.ppat.1000633 Editor:MichaelFarzan,HarvardMedicalSchool,UnitedStatesofAmerica ReceivedJuly7,2009;AcceptedSeptember25,2009;PublishedOctober23,2009 Copyright:ß2009Yuetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricted use,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding:ThestudywassupportedbyNIHPublicHealthServiceGrantAI069981and1R01AI081568fromNIAIDtoY.Wu,andbytheIntramuralProgramof NIMH/NIH.W.WangandM.Spearweresupportedinpartbythegenerousdonationofthe2008NYCDCAIDSRideorganizedbyM.RosenandDay2Inc.The fundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript. CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:ywu8@gmu.edu factor of 4 to 40 [21,22]. This positive effect of Nef on viral Introduction infectivity appears to be at an early step post entry, such as Binding of the HIV envelope to its receptors, CD4 and the uncoating or reverse transcription [23,24,25]. Nef itself does not directly affect reverse transcription, since Nef-defective virions chemokine coreceptor, CCR5 or CXCR4, triggers sequential displaynormallevelsofendogenousreversetranscriptaseactivity fusion and entry events [1,2,3,4]. Fusion is believed to occur [25]. It is likely that this early activity of Nef is connected to directly at the plasma membrane [5,6,7,8,9], but fusion in corticalactininsomeway.Forexample,whencellsweretreated endosomes has also been proposed recently [10]. It has been withactininhibitors,theeffectofNefonviralreplicationwaslost knownthatHIVcanentercellsthroughendocytosis,butthevirion particlesenteringthroughthispathwayappeartobetrappedand [26]. This is also consistent with the fact that the VSV-G- subsequentlydestroyed[11,12].Thisendosomaldegradationcan pseudotypedviruscircumventsthecorticalactin;thus,theimpact be rescued either by blocking the acidification of the endosomal ofNefonviralinfectivityisforfeitedmostlikelybecauseofthelack compartments[11,12]orbypseudotypingtheHIVvirionwiththe ofinteractionwiththeactincortex[15]. TheVSV-G-pseudotypedHIValsodoesnotengageCD4and vesicularstomatitisvirusglycoprotein(VSV-G)[13,14].TheVSV- G-pseudotyped HIV escapes from endosomes and is highly CCR5 or CXCR4, and is deprived of the ability to transduce infectious, giving the virus 20- to 130-fold higher infectivity signals through these receptors [27,28,29]. These intracellular signaling cascades, particularly those transduced from the [15,16]. The ease of producing high-titer virus through VSV-G chemokine coreceptors, have been suggested to be unnecessary pseudotypinghasmadethemethodverypopularformanufactur- ing viral stock used for gene delivery, drug screening, and the forviralfusion,entry,orthesubsequentstepsofviralreplicationin identification of cellular genes and factors involved in HIV transformed cell lines [30,31,32,33,34,35,36,37,38,39]. However, replication[13,17,18,19]. recently, several reports have suggested a requirement for CD4 Nevertheless,theVSV-G-pseudotypedvirusesarenotidentical receptorsignalingtomediateviralfusionandentry[40,41,42,43]. tothegenuineHIVparticles.Forexample,theHIVNefprotein,a We have also observed an absolute requirement for CXCR4 criticalfactorinvolvedinviralpathogenesis[20],nolongerplays signalinginHIV-1latentinfectionofrestingCD4Tcells[44]and an important role in the infection by the VSV-G-pseudotyped demonstrated that HIV-1 relies on viral envelope and the Gai- virus [15]. Nef has been known to enhance viral infectivity by adependent signaling from CXCR4 to activate a cellular actin - PLoSPathogens | www.plospathogens.org 1 October2009 | Volume 5 | Issue 10 | e1000633 Gp120versusVSV-GinHIV-1Infection AuthorSummary While receptor-mediated viral endocytosis or fusion with the cell membrane can be achieved through multiple surface molecules, the repetitious selection of two chemokine receptors, CCR5 or CXCR4, as the main HIV entry coreceptor implies an urgent viral need to exploit thechemotacticprocessintheimmunesystem.Cytoskel- etal rearrangement and cell migration are the primary consequences of chemotactic signaling. Nevertheless, previously published data demonstrated that depriving thevirusofitssignalingabilityconferredhigherinfectivity throughVSV-G-mediatedendocytoticentryintransformed cells. We revisited the issue of chemokine coreceptor signaling and the role of cortical actin in HIV-1 latent infection of resting CD4 T cells, in which the virus can establishlatencywithapotentialforproductivereplication uponTcellactivation.Ourresultsconfirmedthatonlythe genuineHIV-1envelopeprotein,butnotVSV-G,iscapable ofmediatinglatentinfectionofrestingCD4Tcells.These findingshighlighttheimportanceoftheHIVenvelopeand its signaling capacity in HIVinfection ofits naturaltarget cells. depolymerizingfactor,cofilin,toincreasethecorticalactindynamics for viral intracellular migration [44]. Given that the VSV-G- pseudotyped HIV infects cells in the absence of receptor signaling, weperformedparallelstudiesinwhichtheVSV-G-pseudotypedHIV was used to infect both transformed and resting CD4 T cells to understandpossiblealternativepathwaysthattheVSV-G-pseudotyped HIV-1mayemploytoestablishlatentinfectionofrestingCD4Tcells. Surprisingly, the VSV-G-pseudotyped HIV-1 exhibited a highly diminishedabilitytoinitiateviralDNAsynthesisandnuclearmigration inrestingTcells,whichisinstrikingcontrasttothehighefficiencyof VSV-G to mediate HIV infection of transformed cells. The viral particles entering through the endocytotic pathway were destroyed within1–2daysinrestingTcells.TheseresultsindicatethattheVSV- G-mediated endocytotic pathway, althoughactive intransformedT Figure1.ReplicationofHIV-1carryingeitherVSV-GortheHIV envelope in a transformed T cell, CEM-SS. (A) DNA constructs cells, is defective and not a pathway that can establish HIV latent used to generate VSV-G-pseudotyped and HIV-1 Env-typed HIV-1. infection of primary CD4 T cells. These results highlight the Virusesweregeneratedbycotransfectionandharvestedat48hours.(B) importance of the genuine HIV envelope and its signaling capacity Anequalp24level(260ng)ofHIV-1(VSV-G)orHIV-1(Env)wasusedto inthelatentinfectionofprimaryrestingTcells. infect16106cells.Followinginfectionfor2hours,cell-freeviruseswere washed away and cells were continuously incubated for 3 days. Viral replicationwasmonitoredbyp24release. Results doi:10.1371/journal.ppat.1000633.g001 CharacterizationoftheVSV-G-pseudotypedHIV replicationintransformedTcells Wealsocomparedviralearlyprocessesafterentrybyfollowing WecomparedtheinfectivityofHIV-1carryingeithertheHIV viralDNAsynthesisandnuclearmigration.Weinfectedcellsusing envelope (Wt) or the VSV glycoprotein (VSV-G). Both viruses anequalTCID50dosageinsteadofanequalp24.Althoughmore were produced in parallel using the same cell culture and Wt particles were used (based on p24), infection with an equal transfection conditions (Figure 1A). Following harvesting of viral TCID50 ensured thattheproductive viral processes suchas viral particles, an equal p24 level of both viruses was used to infect a DNAsynthesisandnuclearmigrationwouldoccuratcomparable transformedTcellline,CEM-SS.Viralreplicationwasmonitored levels within the viral population in each case. The TCIDof50 by p24 release. As shown in Figure 1B, we observed faster andboth vi ruses was measured on a Rev-dependent indicator cell, stronger replication of the VSV-G- pseudotyped virus, which Rev-CEM,aspreviouslydescribed[45].AsshowninFigure2,at reachedalevelapproximately30foldhigher(at48hours)thanthe 2hourspostinfection,viralDNAsynthesiswasmeasured,andthe wild-typeHIV-1.Ourresultwasconsistentwithpreviousreports VSV-G-pseudotypedHIVsynthesizedonlyapproximately20%of showing that the VSV-G-pseudotyped virus was20to 130times viral DNA in comparison with the wild-type virus (Figure 2A), more infectious than wild-type HIV-1 [15,16]. It is likely that probably either because fewer of the VSV-G-pseudotyped without the limitation of HIV receptors, much more productive particlesenterthecellsorbecausetheseparticlesarelessefficient viralentrymayoccurthroughtheVSV-G-mediatedendocytosis, atmediatingviralDNAsynthesis.Wealsofollowedviralnuclear resulting in a much higher level of viral replication. In addition,migration at later time points using viral 2 -LTR circles as a the faster replication kinetics of the VSV-G-pseudotyped virus is surrogate.AsshowninFigure2B,theVSV-G-pseudotypedvirus likelyaresultoffasterentryandnuclearmigration. produced slightly more2-LTR circlesthanthewild-typeHIV-1, PLoSPathogens | www.plospathogens.org 2 October2009 | Volume 5 | Issue 10 | e1000633 Gp120versusVSV-GinHIV-1Infection Figure2.ComparisonofviralDNAsynthesisinCEM-SScellsinfectedwitheitherHIV-1(VSV-G)orHIV-1NL4-3.Cellswereinfectedwith anequalTCID50 doseofHIV-1(VSV-G)orHIV-1NL4-3 (Wt)(1.276105TCID50/Rev-CEM permillioncells).Followinginfectionfor2hours,cell-freeviruses werewashedaway.TotalcellularDNAwasextractedfromcells,andthenamplifiedbyreal-timePCRtomeasurethesynthesisoffull-lengthHIV-1 DNA (2hours post infection) (A) and 2-LTR circles at later time points (6 and 12hours post infection) (B). The relative ratios of 2-LTR circles at 12hoursandHIV-1DNAat2hourswereplotted(C). doi:10.1371/journal.ppat.1000633.g002 and the relative ratio of 2-LTR circle to total viral DNA was activated T cells, productive viral replication occurred, and the approximately 7 times higher in the VSV-G-mediated infection VSV-G-pseudotyped viral replication was approximately 10-fold (Figure 2C). These data suggest that in transformed T cells, thegreater(48h.p.i)thanthatofthewild -typevirus.Thisissimilarto VSV-G-mediated endocytotic entry is much more efficient in the VSV-G-pseudotyped viral replication in transformed T cells deliveringviralDNAintothenucleus.Evenwithaloweramount (Figure1).However,inlatentlyinfectedrestingCD4Tcells,when of viral DNA synthesized initially, a higher percentage of these cellswereactivatedatday5postinfection,onlythewild-typevirus DNA molecules entered the nucleus. On the other hand, in thebutnot the VSV -G-pseudotyped HIV-1wasinduced toreplicate (Figure 3D). This was strikingly different from the 10 to 30-fold wild-typeinfection,evenwithmoreviralDNAsynthesis,alower higherreplicationcapacityoftheVSV-G-pseudotypedvirusinpre- percentageofviralDNAmoleculescanenterthenucleus.These resultsareconsistentwithamodel[46]inwhichthecorticalactin activatedandtransformedTcells(Figure1andFigure3C).These playsanimportantroleinviralreversetranscription[47],butthe resultswererepeatedusingCD4Tcellsfromanotherdonorwith actin cortexalso serves as anatural barrier forviral intracellular AZTaddedtolimitviralreplicationtoasinglecycle(Figure3Eand 3F). Reproducibly, only the wild-type virus but not the VSV-G- migration[44]. pseudotyped virus was able to replicate following activation of resting T cells at day 5 (Figure 3F), even though the VSV-G- InabilityoftheVSV-G-pseudotypedHIV-1toestablish pseudotyped virus replicated to a 30-fold higher level (48h.p.i) in latentinfectionofrestingCD4Tcells pre-activatedTcells(Figure3E). IncontrasttotheVSV-G-mediatedendocytoticentry,theHIV envelope-mediated fusion and entry requires specific interaction InabilityoftheVSV-G-pseudotypedHIV-1tosupportviral withCD4andthechemokinecoreceptor,CCR5orCXCR4.These DNAsynthesisandnuclearmigrationinrestingCD4T receptorsnotonlymediatefusionbutalsotransducesignalsupon gp120binding[27,48,49].Inparticular,signalstransducedfromthe cells chemokine coreceptor CXCR4 have recently been shown to be WefollowedthestepsforviralinfectionofrestingTcells.Using essential for HIV-1 latent infection of resting CD4 T cells [44]. a sensitive Nef-luciferase-based entry assay [50], we detected Wt Thus, we examined the ability of VSV-G-pseudotyped HIV-1 to viral entry into both resting and activated T cells (Figure 4A), establishlatentinfectionofrestingCD4Tcellsintheabsenceof although the entry into resting T cells was significantly lower. HIVcoreceptorsignaling.RestingCD4Tcellswerepurifiedfrom However, we could not detect viral entry into both resting and the peripheral blood of healthy donors by negative depletion activated T cells in the VSV-G-pseudotyped virus infection, (Figure3A).Cellswererestedovernight,andtheninfectedwithan although wedetected theentryofthe VSV-G-pseudotyped virus equalp24leveloftheVSV-G-pseudotypedHIV-1orthewild-type into CEM-SS cells using the identical infection condition HIV-1.Followinginfection,cellswerewashedandincubatedfor5 (Figure 4A). Since the VSV-G-pseudotyped HIV-1 can replicate days in the absence of T cell activation. During this incubation, inactivatedTcells(Figure4Cand4E),theseresultssuggestedthat productive viral replication does not occur. However, viral theNef-luciferase-basedentryassaymaynothavethesensitivityto replication remains inducible upon T cell activation [44]. As a measure the VSV-G-mediated entry in primary T cells, either control, cells were also pre-activated for 1 day with antibody restingoractivated.Thus,weusedanalternativemethodtodetect stimulation of the CD3/CD28 receptors (Figure 3B) and then viralentrybymeasuringintracellularp24followinginfection.Cells identically infected. As shown in Figure 3C, in CD3/CD28 pre- wereinfectedfor2hours,trypsinized,washed,andthenlysedfor PLoSPathogens | www.plospathogens.org 3 October2009 | Volume 5 | Issue 10 | e1000633 Gp120versusVSV-GinHIV-1Infection Figure3.TheHIVenvelopebutnotVSV-GiscapableofmediatinglatentinfectionofrestingCD4Tcells.RestingCD4Tcellswere purifiedfromtheperipheralbloodbynegativedepletion.Cellswereunstimulated(A)oractivatedwithmagneticbeadsconjugatedwithantibodies againstthehumanCD3andCD28receptors(twobeadspercell)for1day(B),andthenanalyzedforcellcycleprogressionusing7-AAD,PYstainingto confirmsufficientTcellactivationfollowingstimulation.(C)InCD3/CD28pre-stimulatedCD4Tcells,ahigherthanWtlevelofviralreplicationwas observedincellsinfectedwithHIV-1(VSV-G).Onemillioncellswereinfectedwith25ng(p24)ofHIV-1NL4-3 (wt)ortheVSV-Gpseudotypedvirus(VSV- G).(D)InrestingCD4Tcellsthatwerenotpre-stimulated,onlytheWtbutnottheVSV-GpseudotypedHIV-1replicatedfollowingTcellactivationat day5.Cellswereinfectedwith25ng(p24)ofbothviruses,incubatedfor5days,andthenactivatedwithanti-CD3/CD28beads.(E)and(F)werea repeatof(C)and(D)inanotherdonor,withAZT(50mM)addedatday1andday5postinfection,respectively,tolimitviralreplicationtoasingle cycle. doi:10.1371/journal.ppat.1000633.g003 intracellular p24. As shown in Figure 4B, we observed a WethenfollowedthecourseofviralDNAsynthesisandnuclear comparable level of intracellular p24 in resting T cells infected migration in resting CD4 T cells after infection. Unstimulated with the wild-type or the VSV-G-pseudotyped HIV-1. We also resting CD4 T cells from another donor were infected with an observed a higher level of intracellular p24 in HIV-1(VSV-G)- equal TCID50 dose of the VSV-G-pseudotyped HIV-1 or the infected active T cells (Figure 4B). These results suggested that wild-type virus. After washing away free viruses at 2hours post entryofvirionparticleswassimilarinrestingTcellsinfectedwith infection,cellswerecontinuouslyincubatedwithoutactivationfor thewild-typeHIV-1orHIV-1(VSV-G). 5days,andthenactivatedatday5withCD3/CD28stimulation PLoSPathogens | www.plospathogens.org 4 October2009 | Volume 5 | Issue 10 | e1000633 Gp120versusVSV-GinHIV-1Infection Figure4.MeasurementofviralentryandDNAsynthesisfollowingHIV-1(VSV-G)infectionofrestingCD4Tcells.(A)Restingorpre- activated(CD3/CD28beads,twobeadspercell)CD4Tcells(16106)wereinfectedwith200ngofNef-luciferase-taggedHIV-1NL4-3 (Wt)orHIV-1(VSV- G) for 2hours. Infected cells were washed three times and then used to measureluciferase activity in live cells. As a control,CEM-SS cells were identicallyinfectedwiththeNef-luciferase-taggedHIV-1(VSV-G).Uninfectedcellswereidenticallytreatedandmeasuredforluciferaseactivities.(B) Restingor pre-activated (overnightPHAplusIL-2 treatment) CD4Tcells (16106)wereinfectedwith200ng ofHIV-1NL4-3 (Wt)or HIV-1(VSV-G)in 0.5mlfor2hours.Followinginfection,cellsweretreatedwithTrypLE(Invitrogen)for2minutesat37uCandthenwashedanadditionalthreetimes withmedium.Cellswerepelletedandsubsequentlylysedforp24ELISA.(CtoE)RestingCD4TcellswereinfectedwithanequalTCID50doseofHIV- 1NL4-3 (Wt)orHIV-1(VSV-G)(2.536105TCID50/Rev-CEM permillioncells).Followinginfectionfor2hours,cell-freeviruseswerewashedaway.Cellswere culturedfor5daysandthenactivatedwithanti-CD3/CD28beads.(C)Ameasurementofp24releaseconfirmedthatonlytheWtvirusbutnotHIV- 1(VSV-G)replicatedfollowingCD3/CD28stimulation.(D,E)TotalcellularDNAfrominfectedcellswasextractedatdifferenttimepoints,andthen amplifiedwithreal-timePCRforHIVlateDNA(D)or2-LTR-circles(E).(F,G)isarepeatof(D,E)onanotherdonorusinganequalp24levelofWtand HIV-1(VSV-G)toinfectrestingTcells.TotalcellularDNAwasextractedfrominfectedcellsatdifferenttimepointsandPCR-amplifiedforHIVlateDNA (F)or1-LTRcircles(G),alongwiththeb-actinpseudogeneasacontrol. doi:10.1371/journal.ppat.1000633.g004 PLoSPathogens | www.plospathogens.org 5 October2009 | Volume 5 | Issue 10 | e1000633 Gp120versusVSV-GinHIV-1Infection toinitiateviralreplication(Figure4C).WhenviralDNAsynthesis This capacity has been attributed to the synthesis of Nef, which wasanalyzed,wedidnotobserveviralDNAsynthesisabovethe lowersthethresholdrequiredfortheactivationofrestingCD4T initial background (Figure 4D)in cellsinfected with theVSV-G- cells[52,53,54,55,56].Certainly,ourdataconfirmedtheseprevious pseudotypedvirusatanytimepointpostinfection,whereasincells findingsandfurtherindicatedthatonlythegenuineHIVenvelope infectedwiththewild-typevirus,weobservedthetypicalcourseof proteinbutnottheVSV-Gcandeliverthevirusintothenucleus, viralDNAsynthesisinwhichviralDNAsynthesisproceedsslowly wherethesubsequentactionofNefcanoccur. and usually peaks at day 2, recedes at day 3, and then increases again following T cell activation [44]. When viral 2-LTR circles DifferentialinhibitionofHIV-1andtheVSV-G- were measured, in cells infected with the VSV-G-pseudotyped pseudotypedHIV-1bydynasore virus, we also could not detect 2-LTR circles at any time point, IncontrasttorestingTcells,intransformedcelllines,theVSV- even after CD3/CD28 stimulation at day 5, whereas in cells G-mediated entry is very efficient in mediating HIV infection. infectedwiththewild-typevirus,2-LTRcirclesweredetectedand This fact has prompted a major argument that HIV may the copy number increased with time (Figure 4E). We repeated predominately fuse in the endosome rather than at the plasma theseexperimentsusingrestingCD4Tcellsfromanotherdonor. membrane [10]. Microscopic imaging tracking the behaviors of This time, resting cells were infected with an equal p24 level of themajorityofMLVparticlessuggestedthattheHIV-1envelope- bothviruses.Weobservedsimilarresults.AsshowninFigure4F, pseudotypedvirusenteredcellspredominantlythroughendocyto- in cells infected with the VSV-G-pseudotyped virus, the initial sis [10]. Indeed, dynasore, a dynamin-dependent endosomal viralDNAdetected(0.1to0.5dayinFigure4F)diminishedwith scission inhibitor, was shown to inhibit viral replication [10], time,andno1-LTRcirclescanbedetectedatanytimepointpost supportingthemodelthattheendosomalfusionisassociatedwith infection,whereasinthewild-typeinfectedcells,thesynthesesof a productive pathway. Nevertheless, this mode of entry is in both viral DNAand1-LTR circleswereobvious (Figure 4Fand conflict with numerous previous observations suggesting that 4G).Basedontheseresults,weconcludedthatinrestingCD4T genuine HIV envelope-associated endocytotic entry, although cells,onlytheHIVenvelope-mediatedentrybutnottheVSV-G- occurring at a significant scale, does not naturally lead to mediatedendocytosiscanleadtoviralDNAsynthesisandnuclear productive infection [5,6,7,9,11,12]. For example, inhibition of migration,whichareaprerequisitefortheestablishmentofHIV the endosomal/lysosomal functionality can spare HIV from latentinfectionofrestingCD4Tcells[44]. degradationandenhanceviralreplication[11,12],demonstrating thattheendosomalvirusesarenormallydirectedfordegradation. DecayoftheVSV-G-pseudotypedHIV-1inrestingCD4T Inaddition,therateofCD4orCCR5endocytosisdoesnotappear cells to affect viral entry or replication [6,8,9], supporting direct viral We also measured the decay kinetics of the VSV-G-pseudo- fusion at the plasma membrane. Nevertheless, the endocytosis typedHIV-1inrestingCD4Tcells.UnstimulatedrestingCD4T entry as proposed [10] is an attractive alternative pathway. If cells were infected with an equal p24 level of both viruses. provenbiologically,itwouldrequiresignificantremodelingofthe Following infection for 2hours, cell-free viruses were washed roleofthecorticalactininviralentryandearlypost-entrysteps. away.Infectedcellswerethenactivatedimmediatelyoractivated Theinvolvementofthecorticalactininearlyendocytosisislargely atday1,3,or5postinfection.Asacontrol,restingcellswerealso limited to membrane scission of clathrin-coated pits [57]. This pre-activated with CD3/CD28 for 1hour and then identically processdoesnotinvolvedirectcontactbetweenthecorticalactin infected.AsshowninFigure5,inCD3/CD28pre-activatedCD4 and the viral particles. If there is any viral contact with actin, it T cells, both the VSV-G-pseudotyped HIV-1 and the wild-type would be in the cytoplasm following endosomal fusion. This virusreplicatedafterinfection(Figure5A1and5B1).InrestingT interaction may also affect reverse transcription and nuclear cells, when cells were activated immediately after infection and migration, but such effects would occur at different levels. The washing(2hourspostinfection),viralreplicationwasalsoinitiated issueofentryissocriticalintheunderstandingoftheroleofthe from both the VSV-G- pseudotyped HIV-1 and the wild-type corticalactininHIVbiologythatwefeltcompelledtorevisitsome virus(Figure5A2and5B2).Theseresultssuggestthatentryofthe ofthekeybiologicalevidence-inparticular,theinhibitionofHIV VSV-G-pseudotyped virus into resting T cells occurs, and viral replicationbythedynamin-dependentendosomalfusioninhibitor, replication can be rescued if cells are activated immediately. dynasore.Dynaminsareagroupoffundamentalproteinsinvolved However,whenrestingcellswereleftunactivated,after1day,the inmultiplecellularprocessessuchasvesicletransport,cytokinesis, replicationoftheVSV-G-pseudotypedvirusfollowingactivation organelle division and cell signaling (for a review, see [58]). To wasgreatlydiminished(Figure5A3),andnoviralreplicationcould minimize possible cytotoxicity from prolonged inhibition of beinitiatedafter3days(Figure5A4and5A5).Thiswasingreat fundamental cellular proteins, we treated cells only briefly with contrasttothewild-typeHIVinfectionofrestingTcells,inwhich dynasore during viral infection. Viruses that failed to enter were the capacity of HIV to replicate following activation increased subsequentlywashedawayalongwiththedrug.Wealsousedthe with time (Figure 5B1 to 5B5). The highest viral replication Rev-dependent indicator cell, Rev-CEM [45], to measure occurred after 5 days of incubation. These data, in combinationdynasore inhibition, instead of simply using p24 ELIS A, which withtheresultsinFigure4,suggestthatinrestingCD4Tcells,the by itself is not capable of distinguishing between HIV-specific VSV-G-mediatedendocytoticentrydoesnotleadtoaproductive inhibitionandgeneraldrugcytotoxicity.Additionaladvantagesof pathway, and the viral particles are trapped in cells and using Rev-CEM are its high specificity and the ability to subsequently destroyed within 1–2 days. Our data are also distinguish subpopulations of cells by flow cytometry so that consistent with a previous study demonstrating that the VSV-G- non-specific cytotoxicity can be excluded [59]. As shown in pseudotypedHIV-1hasahalf-lifeofonly1–2daysinrestingCD4T Figure6A,wefirsttesteddynasoreintheinhibitionoftheVSV-G- cells[51].Theincreasingabilityofthewild-typeHIV-1toreplicate pseudotyped HIV-1 replication and observed dosage-dependent followingincubationhasalsobeenobservedpreviously[52,53,54]. inhibition of viral replication. At 80mM, dynasore moderately AlthoughHIVdoesnotdirectlyreplicateinrestingCD4Tcells,the decreasedtheGFP+populationfrom16.1%to11.5%;at8mM, viral envelope-mediated entry establishes an active process that dynasorealsoslightlydecreasedtheGFP+population;at0.8mM, enhancestheabilityofHIVtoreplicatefollowingTcellactivation. dynasore minimally affected viral infection. However, when PLoSPathogens | www.plospathogens.org 6 October2009 | Volume 5 | Issue 10 | e1000633 Gp120versusVSV-GinHIV-1Infection Figure5.DecayoftheVSV-G-pseudotypedHIV-1inrestingCD4Tcells.(A)RestingCD4Tcellswerepurifiedbynegativedepletion,rested overnight,andthenpre-stimulatedwithanti-CD3/CD28beadsfor1hourandinfectedwithHIV-1(VSV-G)(552ngp24permillioncells)(A1).Cells werealsoinfectedwithoutpre-stimulation(A2toA5)andthenstimulatedwithanti-CD3/CD28beadsat2hours,day1,day3,orday5postinfection toinitiateviralreplication.Thep24releasewasmeasuredfollowinganti-CD3/CD28stimulation(markedasday‘‘0’’ontheX-axisofeachpanel).(B)is arepeatof(A)inthesamedonor,usingHIV-1NL4-3 (Wt)(552ngp24permillioncells). doi:10.1371/journal.ppat.1000633.g005 dynasore was used on identically treated cells that were infected infection(Figure6B).Theseresultsdemonstrateacleardistinction with HIV-1, we did not observe similar dosage-dependent betweentheVSV-G-mediatedendocytoticentryandtheHIV-1- inhibition. Even at 80mM, dynasore minimally affected HIV-1 envelope-mediatedentryinmediatingproductiveviralreplication. PLoSPathogens | www.plospathogens.org 7 October2009 | Volume 5 | Issue 10 | e1000633 Gp120versusVSV-GinHIV-1Infection Figure6.DifferenteffectsofdynasoreonthereplicationofHIV-1(VSV-G)andWtinahumanTcell,Rev-CEM.(A)Rev-CEM,aRev- dependentGFPindicatorcell,waspretreatedfor30minuteswith80mM,8mM,or0.8mMdynasore,respectively,ortreatedwith0.1%DMSOasa control.CellsweresubsequentlyinfectedwithHIV-1(VSV-G)inthepresenceofdynasorefor2hours.Followinginfection,cellswerewashedthree timeswithmedium,andthenculturedintheabsenceofdynasore.ViralreplicationwasmonitoredbyflowcytometryanalysisofHIV-dependentGFP expressionat48hours(20,000cellsanalyzedpersample).Propidumiodide(PI)wasaddedintothecellsuspensionpriortoflowcytometry.Viable cellsweregated(R1)basedonlowPIstainingandcellsize(FSC).GFPexpressionwithintheviablecellpopulation(R1)wasmeasured.BoththeGFP percentage(%)andmeanintensity(M)wereshown.(B)isanidenticalexperimentusingHIV-1NL4-3 (Wt).Forthe80mMdynasoretreatment,another three-independent infections with each virus were performed. The averages from the three-independent experiments are: 15.05%60.21 (VSV-G), 10.11%60.06(VSV-Gplus80mMdynasore),p=0.0003;2.94%60.39(Wt),2.84%60.30(Wtplus80mMdynasore),p =0.38. doi:10.1371/journal.ppat.1000633.g006 1–2 days in resting T cells. These results demonstrated the Discussion importance of the genuine HIV envelope in mediating latent In this report, we demonstrated a fundamental difference infectionofrestingTcells. between the HIV-1 envelope and VSV-G in mediating HIV-1 Previously, we demonstrated a critical function of the HIV-1 latentinfectionofprimaryrestingCD4Tcells,namelythatonly envelope in mediating CXCR4 signaling and promoting the theHIV-1envelopebutnotVSV-GiscapableofsupportingHIV cortical actin dynamics necessary for HIV latent infection of latent infection of resting T cells. The block to the VSV-G- restingTcells[44].WealsoproposedadualfunctionofF-actinin pseudotypedvirusinrestingTcellswasmostobviousatpost-entry which the actin cortex serves as an anchorage for reverse steps such as viral DNA synthesis and nuclear migration. The transcriptionandasavehicleforthedeliveryofthepreintegration virionparticlestrappedincellsweresubsequentlydestroyedwithin complex across the cortical actin through actin treadmilling PLoSPathogens | www.plospathogens.org 8 October2009 | Volume 5 | Issue 10 | e1000633 Gp120versusVSV-GinHIV-1Infection [44,46].AtleastfourHIVproteinsinthepreintegrationcomplex factorsforuncoating[71]DNAsynthesis,ornuclearlocalization. OurattemptstorescuetheVSV-G-pseudotypedvirusbychanging are known to interact with actin; the viral nuclear capsid [60,61,62,63], thelarge subunit ofthereverse transcriptase[64], the intracellular PH were not successful (data not shown). Pre- stimulation of the CD4 and CXCR4 receptors with gp120 or theintegrase[65],andNef[66].Wehavealsoshownthatblocking actinpolymerizationwithJasplakinolid(120nM)orLatrunculinA antibodiesalsocouldnotrescuetheVSV-G-pseudotypedvirusin resting T cells (data not shown), although these pre-stimulations (2.5mM) inhibits viral DNA synthesis or HIV latent infection. Conversely, triggering actin polymerization through cofilin enhanced the wild-type HIV replication several fold following T shRNA knockdown enhances viral DNA synthesis [44]. These cellactivation[44].Theseresultsareconsistentwiththefactthat thepositivebenefitsofviralreceptorsignalingareonlyassociated previous results and other studies [47] are consistent with the with gp120-mediated entry but not with the VSV-G-mediated findings in this study, in which the VSV-G-mediated entry that endocytosisthatcircumventsthecorticalactin. bypasses the cortical actin led to less viral DNA synthesis in ThehighefficiencyofVSV-Gtomediateendosomalescapeand transformed cells (Figure 2). The VSV-G-pseudotyping also HIVreplicationintransformedcellshasledtothemisconception resultedinalackoftheslowviralDNAsynthesisthatisnormally seen in HIV-1 latent infection of resting T cells (Figure 4D andthat the VSV -G-pseudotyped HIV should be as effective as the wild-type HIV for latent infection of resting T cells [72,73]. 4F).ViralnuclearDNAwasalsocompletelymissingintheVSV- G-mediatedentryinrestingcells(Figure4Eand4G).Theseresults Several previous studies have also used the VSV-G-pseudotyped virustoidentifyrestrictionfactorsinrestingTcells[17,74].Our suggestthattheVSV-G-pseudotypedparticlesmaybedeliveredto results suggest that these data need to be interpreted cautiously. a different cytoplasmic location and enter the nucleus by a Apparently, the VSV-G-mediated entry does not experience the differentroute,onethatisnormallyhighlyeffectiveintransformed sameintracellularenvironmentasHIVdoes,andcannotleadto ormetabolicallyactivecellsbutdefectiveinrestingTcells. Our results are consistent with a recent independent study theestablishmentoflatentinfectioninrestingTcells.Thus,those previously identified cytoplasmic restriction factors may or may demonstrating that only the CXCR4-tropic HIV-1 envelope but not directly affect HIV infection. Interestingly, a recent imaging not VSV-G can support lentiviral vectors to deliver genes into study demonstrated a direct dependence of active viral nuclear restingCD4Tcells[67].Inthisstudy,Agostoandco-authorsalso migrationonF-actin,sinceactininhibitorsdiminishedthenuclear foundthatviralDNAsynthesiswasgreatlydiminishedinresting concentration of the preintegration complex (PIC) (Dr. Thomas CD4 T cells infected with the VSV-G-pseudotyped lentiviral Hope, personal communication). This study raises the possibility particles. Nevertheless, the limitation on viral infection was thatPICmaybeassociatedwithF-actinuptothenucleus[75,76]. specifically attributed to the lack of viral entry and fusion in the GiventhatvirusesusuallyuseF-actinforshort-distancetravel,and VSV-G-mediated infection of resting T cells [67]. Our results thecytoplasmicspacebetweenthecorticalactinandthenucleusis suggestedthattherestrictionwaslikelyatanunknownpost-entry relativelythininTcells,itispossiblethatthecytosolicexposureof stepsuchasendosomalfusion,uncoating,orreversetranscription. PICinTcellsisminimal. Thediscrepancyinconclusionsarisesfromdifferentexplanations of the data acquired from entry and fusion assays. Both Agosto and co-authors [67] and we observed an absolute lack of entry MaterialsandMethods signals in HIV-1(VSV-G)-infected resting T cells, using two Ethicsstatement different entry assays. However, these assays, particularly the All protocols involving human subjects were reviewed and BlaM-Vpr-basedfusionassay[68]maynotbeappropriateforthe approvedbytheGMUIRB.Informedwrittenconsentsfromthe measurement of VSV-G-mediated fusion in resting T cells. It is humansubjectswereobtainedinthisstudy. possible that if the VSV-G-pseudotyped virus is trapped in a compartment, or is going through a degradation process with a PlasmidsandDNAcloning half-lifeofonly1day[51],theBlaMsubstratewhichtakesabout Plasmid pNL4-3 was kindly provided by Dr. Malcolm Martin 12–18hourstoloadmaynotbeabletoaccessorsufficientlyreact [77].Theenvmutant,pNL4-3(KFS),waskindlyprovidedbyDr. with the enzyme. Given this lack of mechanistic clarity of how EricFreed[78].pHCMV-Gthatexpressesthevesicularstomatitis these enzyme-tagged particles are delivered through VSV-G in virusglycoproteinhasbeendescribedpreviously[79].pNLDYEnv restingTcells,wedidnotfeelconfidentthatconclusionscanbe wasconstructedbyinsertingtheenvgeneofHIV-1NL4-3 intothe drawn based on a fusion assay. Thus, we drew our conclusions lentiviral vector pNL-RRE-SA [80]. The packaging signal was relying on multiple results. Firstly, we detected a comparable furtherdeletedbycuttingwithKasIplusBssHIIandre-ligating. intracellularp24levelinrestingTcellsinfectedwithWtorHIV- 1(VSV-G) (Figure 4B). Secondly, the VSV-G-pseudotyped virus canbepartiallyrescuedifrestingTcellswereactivatedwithin1 Virusesandcells day of infection, indicating some viral entry into the cells HIV-1NL4-3 was generated by transfection of plasmid pNL4-3 (Figure5A2).Thirdly,lowlevelsofviralDNAwerealsodetected into HEK293T cells using lipofectamine 2000 (Invitrogen) as atearlytime(2hours,Figure4F),indicatingagainthattherewere described previously [80]. The VSV-G-pseudotyped virus, HIV- some levels ofentry. Giventhat theVSV-G-pseudotyped viruses 1(VSV-G), was produced by cotransfection of HEK393T cells (36106) with 10mg of pHCMV-G and 10mg of plasmid pNL4- are20to130-foldmoreinfectiousthanthewild-typeHIV-1,these initialviralactivitiesshouldgiverisetoameasurablelevelofviral 3(KFS). The HIV-1 envelope-typed virus, HIV-1(Env), was replication,buttheydidnot. produced by cotransfection of HEK293T cells with 10mg of The failure of the VSV-G-mediated entry to establish latent pNLDYEnv and 10mg of pNL4-3(KFS). Viral supernatant was harvested at 48hours post cotransfection, centrifuged for infectionofrestingTcellsisnotcurrentlyunderstood.Itispossible that the cellular environment in resting T cells may not permit 15minutes at 5006g to remove cellular debris, filtered through viral fusion in endosomes. Alternatively, successful endosomal a0.45mmfilter,treatedwithBenzonase(Novagen)(250U/ml)at fusionmayoccur,butthequickdeliveryofviralparticlesintothe 37uCfor15minutes,andthenstored at-80uC.Levelsofp24in cytosol may be detrimental [69], likely due to the possible viralsupernatantweremeasuredusingthePerkinElmerAlliance restrictiveenvironmentofrestingcells[17,70]oralackofcytosolic p24antigenELISAKit(PerkinElmer).Plateswerekineticallyread PLoSPathogens | www.plospathogens.org 9 October2009 | Volume 5 | Issue 10 | e1000633 Gp120versusVSV-GinHIV-1Infection using an ELx808 automatic microplate reader (Bio-Tek Instru- 2000(Invitrogen)asrecommended bythemanufacturer.Viruses ments) at 630nm. Viral titer (TCID50) was determined on the were harvested at 48hours post cotransfection and filtered Rev-dependent GFP indicator cell, Rev-CEM [45,81]. CEM-SS through a 0.45mM filter. For entry assays, cells (16106 ) were cellsfromDr.PeterL.Nara[82]wereobtainedthroughtheAIDS infectedwith200ngofNef-luciferasecontainingvirusesat37uC Research and Reference Reagent Program, Division of AIDS, for2hours,andthenwashedthreetimeswithmedium.Cellswere NIAID, NIH. All cells were cultured in RPMI 1640 medium resuspended in 0.1ml of luciferase assay buffer (Promega) and supplemented with 10% heat-inactivated fetal bovine serum luciferaseactivitywasmeasuredinlivecellsusingaGloMax-Multi (Invitrogen),penicillin(50U/ml),andstreptomycin(50mg/ml). DetectionSystem(Promega). Isolation,culturing,andinfectionofrestingCD4Tcells PCRandReal-timePCR TotalcellularDNAwaspurifiedusingtheWizardSVGenomic Peripheralbloodmononuclearcells(PBMC)wereobtainedfrom healthy donors at the Student Health Center, George Mason DNA Purification System as recommended by the manufacturer (Promega).ThedetectionofvirallateDNAand1-LTR-circlesby University(GMU),Fairfax,VA.RestingCD4Tcellswerepurified by two rounds of negative selection as previously described [54]. PCRwasperformedasdescribedpreviously[83].Briefly,forviral late DNA, forward primer: 59 GGTTAGACCAGATCT- PurifiedcellswereculturedinRPMI1604mediumsupplemented with10%heat-inactivatedfetalbovineserum(Invitrogen),penicillin 39 and reverse primer: 59 TTAATACC- GAGCCTG GACGCTCTCGCACC 39 were used. PCR was carried out in (50U/ml),andstreptomycin(50mg/ml)overnightbeforeinfection ortreatment.ForactivationofrestingCD4TcellswithPHA(3mg/ 16Ambion PCR buffer, 125mM dNTP, 50pmol each primer, ml) (Sigma) plus IL-2 (100U/ml) (Roche Applied Science), cells 1U SuperTaq Plus (Ambion) with 30 cycles at 94uC for were cultured in the presence of these agents for 12hours. For 20seconds, 68uC for 40seconds. For detection of 1-LTR circle, infection,CD4Tcellswereincubatedwiththevirusfor2hoursand primers LTR-nef2 (59 TGGGTTTTCCAGTCACACCTCAG thenwashedtwicewithmediumtoremoveunboundvirus.Infected 39)andLTR-gag (59GATTAACTGCGAATCGTTCTAGC 39) cellswereresuspendedinfreshRPMI1604mediumsupplemented were used. The reaction was carried out in 16Ambion PCR + buffer,1.5nMMg2 ,125mMdNTP,50pmoleachprimer,1U with10%heat-inactivatedfetalbovineserumatadensityof106per ml and incubated for 5 days without stimulation. Cells were SuperTaq Plus (Ambion) with 35 cycles at 94uC for 20seconds, activatedatday5withanti-CD3/CD28magneticbeadsat4beads 68uC for 90seconds. Real-time PCR quantification of viral late percell.Fortheviralreplicationassay,10%ofinfectedcellswere DNA and 2-LTR circles was also performed as described takenatdays1,3,5,6,7,8,and9postinfection.Cellswerepelleted previously [44,84], using 300nM primers and 200nM probes. andthesupernatantwassavedforp24ELISA. The DNA standard used for both late DNA and 2-LTR circle quantification was constructed using a plasmid containing a complete 2 LTR region (pLTR-2C); the plasmid was cloned by CD3/CD28beadconjugationandstimulationofresting amplificationofinfected cellswith59-TGGGTTTTCCAGTCA- CD4Tcells CACCTCAG-39 and 59-GATTAACTGCGAATCGTTC- Monoclonal antibodies against human CD3 (clone UCHT1) TAGC-39. Measurement was run in triplicate ranging from 1 to andCD28(cloneCD28.2)werepurchasedfromBDPharmingen 106copiesofpLTR-2CmixedwithDNAfromuninfectedcells. (BD Biosciences). For conjugation, antibodies were conjugated with 46108 Dynal beads (Invitrogen) for 30minutes at room Confocalmicroscopy temperature. Free antibodies were washed away with PBS-0.5% staining of F-actin has been described FITC-phalloidin BSA.Theconjugatedmagneticbeadswereresuspendedin1mlof previously [44]. Stained cells were imaged using a Zeiss Laser PBS-0.5%BSA.ForstimulationofrestingCD4Tcells,antibody- ScanningMicroscope,LSM510META,witha40NA1.3or60 conjugated beads were washed twice and then added to cell NA 1.4 oil DIC Plan-Neofluar objective. Images were processed cultureandrockedfor5minutes. andanalyzedbyLSM510METAsoftware. Cellcycleanalysisby7-AADandPYstaining RestingCD4TcellsorCD3/CD28-stimulatedcells(106 )were Flowcytometry used for the analysis. Before staining, magnetic beads were Dynasore monohydrate (Sigma) was dissolved in DMSO. removed by incubating with DNase I releasing buffer as Following dynasore treatment, infection, and washing, cells were recommended by the manufacturer. Cells were suspended in incubatedfor48hours,andthen500mlcellswereremovedand 1ml of 0.03% saponin in PBS and then incubated in 20mM 7- stained with 2mg/ml propidium iodide solution (Fluka) for amino-actinomycin D (Sigma) for 30minutes at room tempera- 5minutesatroomtemperature.Followingincubation,cellswere ture in the dark. Cells were kept on ice for at least 5minutes, analyzedusingtheFACSCalibur(BDBiosciences).Dataanalysis pyronin Y (Sigma) was added to a final concentration of 5mM, wasperformedusingCellQuest(BDBiosciences). andthecellswerethenincubatedfor10minutesonice.Stained cellsweredirectlyanalyzedbyflowcytometryonaFACS(Becton Acknowledgments DickinsonFACSCalibur). WethanktheGeorgeMasonUniversity(GMU)StudentHealthCenterfor blood donations; the NIH AIDS Research and Reference Reagent ProductionofHIV-1andVSV-G-pseudotypedHIV-1 Program, NIAID, NIH for reagents; Z. Li for help on experiments; M. Martin,R.A.DaveyandE.Freedforplasmids;J.W.MarshandH.A. containingNef-luciferasefusionproteinforentryassay Nashfordiscussions;andJ.Guernseyforeditorialassistance. PlasmidpCDNA3-Nef-LucwaskindlyprovidedbyDr.Robert Davey [50]. Viruses containing Nef-luciferase was produced as AuthorContributions describedpreviously[50].Briefly,293Tcellsculturedina10cm petri dish were cotransfected with 10mg pNL4-3 plus 10m g of Conceived and designed the experiments: YW. 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