The Vif and Vpr accessory proteins independentlycause HIV-1-induced T cell cytopathicity and cellcycle arrestKeiko Sakai, Joseph Dimas*, and Michael J. Lenardo†

Laboratory of Immunology, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892-1892

Edited by Robert A. Lamb, Northwestern University, Evanston, IL, and approved January 6, 2006 (received for review October 28, 2005)

HIV type I (HIV-1) can cause G2 cell cycle arrest and death of CD4�

T lymphocytes in vitro and inexorable depletion of these cells invivo. However, the molecular mechanism of viral cytopathicity hasnot been satisfactorily elucidated. Previously, we showed thatHIV-1 kills T cells by a necrotic form of cell death that requires highlevel expression of an integrated provirus but not the env or nefgenes. To determine which viral protein(s) are required for celldeath, we systematically mutated, alone and in combination, theORFs of the NL4-3 strain of HIV-1. We found that the elimination ofthe viral functions encoded by gag-pol and vpu, tat, and rev did notmitigate cytopathicity. However, elimination of the vif and vpraccessory genes together, but not individually, renders the virusincapable of causing cell death and G2 cell cycle blockade. We thusidentify vif and vpr as necessary for T cell cytopathic effectsinduced by HIV-1. These findings may provide an important insightinto the molecular mechanism of viral pathogenesis in AIDS.

AIDS � apoptosis � lymphocytes � necrosis � NL4-3

In the absence of antiviral drug therapy, infection with HIV-1leads to a progressive decline in CD4� T cell number. This cell

depletion produces an immunodeficiency state resulting in theopportunistic infections characteristic of AIDS (reviewed in ref.1). Mathematical modeling suggested that the majority of HIV-infected cells die with a half-life of �2 days (2–4). However, thenumber of infected cells is reported to be approximately constant(3), suggesting that large numbers of infected cells are producedand destroyed each day. T cell production does not fully balancedestruction, leading to the eventual depletion of CD4� T cells.Recent studies suggest that direct viral infection causes themassive destruction of memory CD4� T cells in various tissueswithin 2–3 weeks after acute infection (5–8). In macaquesinfected with simian immunodeficiency virus, the majority ofCD4� T cells are lost in a period of only 4 days (7, 8). The lossof CD4� T lymphocytes could be due to direct cytopathic effectsor possibly to CD8� cytolytic T cell responses that kill virallyinfected cells.

Viral pathogenesis is a multifactorial process, involving hostand virus factors that trigger multiple pathologic mechanisms.Cytopathic effects could derive from direct cell injury by thevirus or immunopathology as a result of the host’s immuneresponse. Accumulating evidence suggests that significant celldeath during HIV-1 infection can be attributed to direct viralkilling of infected cells in cultures and in peripheral bloodmononuclear cells from AIDS patients (9–11). However, themolecular basis of the viral cytopathic effect, including thespecific viral and host components involved, is not yet completelydefined.

To understand viral cytopathicity, we have used a single-cellanalysis system that distinguishes between infected and unin-fected cells by monitoring the expression of a mouse heat-stableantigen (HSA)�CD24 marker engineered in the NL4-3 stain ofHIV-1 (12, 13). Our previous quantitative measurements of celldeath in infected versus uninfected cells with the NL4-3HSAstrain by using flow cytometry showed that only infected cells

were killed (14). Moreover, the death of T cells was necroticrather than apoptotic in nature (12–14). Previous work hassuggested that the nef and env genes may have important rolesin viral pathogenesis (15–17, reviewed in refs. 18–20). However,direct CD4� T cell death induced by HIV-1 infection is inde-pendent of Env and Nef proteins (12, 13). Thus, the viral genesthat account for direct CD4� T cell necrotic death have not beenconclusively determined.

Vpr, an accessory protein of uncertain function in the HIV-1life cycle, has been implicated in cytopathicity and G2 cell cyclearrest (21–25). Mutational analyses indicated a correlation be-tween cell-cycle arrest and virally induced cell death (25–27).However, abrogation of the G2-phase arrest did not preventVpr-induced cell death in other studies (24, 28–31), raising thepossibility that cell cycle arrest is not the cause of Vpr-mediatedcell death. It is important to ascertain the role Vpr plays in theprocesses of cell cycle arrest and cell death, vis-a-vis other HIV-1genes, during infection in CD4� T lymphocytes.

Vif is an accessory protein that can promote HIV-1 replicationin certain restrictive cell types but whose role in viral pathogen-esis is controversial (reviewed in ref. 32; refs. 33–38). The firstwell-described function of Vif came from recent studies thatrevealed an interaction between Vif and an innate antiviralfactor, APOBEC 3G (39). Vif prevents packaging ofAPOBEC3G, a cytidine deaminase and viral DNA modifier, intovirus particles by targeting it for proteasomal degradation (re-viewed in ref. 32; 33–35). APOBEC3G expression is limited toprimary cells and certain cell lines, including H9 and CEM, inwhich Vif is required for productive infection by HIV (39). Thisobservation solved the long-standing mystery of why Vif functionis cell-type-dependent. However, a possible contribution of Vifto viral pathogenesis and cytopathicity, besides its role in pro-ductive infection, has not been clearly delineated.

To identify a viral genes essential for cytopathicity, we createda null mutation in each HIV-1 gene of a laboratory strain,NL4-3, and examined the ability of each mutant virus to killCD4� T cells during a single round of infection. We observed theloss in cytopathic capacity if both Vif and Vpr were absent fromthe virus. Our results indicate that these accessory proteins playcentral roles in the HIV-1 direct killing of T cells. Intriguingly,proliferation of highly infected cells was observed only in theabsence of both Vif and Vpr. Detailed analyses of DNA contentin infected cells indicated that Vif and Vpr exclusively andindependently cause cell cycle arrest at the G2 phase. Our resultssuggest a link between HIV-1 cytopathicity and cell cycle arrestand identify two HIV-1 genes responsible for these effects.

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: HSA, heat-stable antigen; MOI, multiplicity of infection; PI, propidiumiodide.

*Present address: University of Texas Health Science Center, San Antonio, TX 78229.

†To whom correspondence should be addressed. E-mail: lenardo@nih.gov.

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ResultsTo identify the HIV-1 gene or genes essential for cytopathicity ininfected CD4� T cells, we created various mutant versions of thelaboratory strain, NL4-3HSA possessing combinations of ‘‘knock-out’’ mutations in different coding regions of the viral genome (Fig.1A; see also Table 1, which is published as supporting informationon the PNAS web site). The mutations were nonsense substitutionsdesigned to generate premature stops in the ORFs (Table 1). Weintroduced stop codons and microdeletions to inactivate ORFs,rather than the deletions of entire genes to prevent the destabili-zation of viral genomes due to large deletions.

For a Gag-deficient strain, elimination of the start codonresulted in internal translation starting at M142 (data notshown). Therefore, we generated two additional stop codons intandem at R150 and L152 to eliminate spurious internal trans-lation initiations. A Western blot of lysates from 293T cellstransfected with a plasmid encoding the Gag-Pol-deficient virusconfirmed the expected absence of the Pr160 Gag-Pro-Polpolyprotein and Pr55 Gag (Fig. 1B). Similarly, stop codons wereused to eliminate Vif and Vpr, which was also confirmed byWestern blotting (Fig. 1C). A virus containing a Vpu deletionwas produced by using a construct generated and validated inrefs. 40 and 41. All of the mutant strains were constructed on aEnv�Nef�HSA� version of NL4-3 described in refs. 12, 13, and21. This construct harbors a frameshift mutation in the env geneto restrict replication to a single round of infection by usingVSV-G-pseudotyped virus (12, 13, 21). Moreover, the nef geneis replaced by the mouse heat stable antigen (HSA�CD24),which serves as a cell surface marker for infection that is readilydetected by flow cytometry (12, 13, 21).

We found that infection of Jurkat cells with Env� strains ofNL4-3HSA with an multiplicity of infection (MOI) of 5 led todeath of infected cells within 4 days (Fig. 2Ac). Substantial death

also occurred in an infected population at an MOI of 1 or 3,manifesting 3% and 13% viability, respectively, although therewas greater cell survival at each time point as the MOI decreased(Fig. 2B). These results demonstrated the profound cytopath-icity of the NL4-3HSAEnv� strain in these culture conditions asshown in refs. 12–14. In a typical experiment, very few HSAhi,i.e., infected, cells remained alive after 4 and 8 days at a MOI of5 and 3, respectively. As a control, Jurkat cells were infected withvirus particles carrying a GFP-encoding genome devoid of allHIV proteins except for Tat and Rev. We found that even at ahigh MOI, this virus produced no significant decrease in cellviability (Fig. 2 Ab). Thus, proviral expression of NL4-3 genes arenecessary for cytopathicity under these culture conditions asshown in refs. 12 and 14.

Because of their well described biochemical functions, weinitially hypothesized that one of the enzymatic or structuralproteins, such as protease, would be responsible for the cyto-pathic effect of HIV-1. We, therefore, initially disrupted thegag-pol reading frame (Fig. 1 A and Table 1). We preparedinfectious virions harboring HIV-1 genomes lacking structuraland�or enzymatic genes by cotransfecting a complementinghelper plasmid, �R8.2�Vpr into the 293T producer cells (23).This encapsidation-deficient NL4-3 helper plasmid provides intrans the wild-type proteins necessary for packaging and virusproduction. Surprisingly, we found that the Gag�Pol� strain stillkilled infected Jurkat cells at a rate comparable to that of theNL4-3HSA Env� parental strain (Fig. 3A). We therefore testedcombinations of mutations in accessory genes together withthose in structural and enzymatic genes. A frameshift mutationin vif gene in the Gag�Pol� background retarded the onset of celldeath by 2 days; however, it did not reduce the eventual overalllevel of cell death. Removal of Vpr in the Gag�Pol� backbonealso diminished direct cell killing such that �25% of HSAhi cellswere still alive after 8 days of infection (Fig. 3 A and B). Bycontrast, little change in cytopathicity was detected withGag�Pol� and with Vpu� strain of NL4-3HSA. The higherviability in cultures exposed to virus with mutations in Gag, Pol,and Vpr reflected outgrowth of uninfected cells and the survivalof infected cells. The fraction of HSA-expressing cells becamesmaller at day 8 compared to earlier days because of uninfected,rather than infected, cells increasing in the culture (Fig. 3B). Theoutgrowth of uninfected cells was usually observed with Env�

strains because a single round of infection was never 100%efficient.

Next, we investigated the properties of viruses with multiplemutations in accessory genes and to Gag and Pol mutations.

Fig. 1. Locations of mutations in HIV-1NL4-3 and corresponding loss of proteinexpression due to mutations. (A) NL4-3HSA strains contained various individualor multiple mutations in the ORFs that alter the indicated amino acids shownin the standard single letter code. The mutations either prevent (ATG muta-tions) or prematurely terminate (stop codons) protein translation. Mouse heatstable antigen (HSA�CD24) replaced the nef gene coding sequence (21). Toverify the expected loss of protein expression, lysates from 293T cells trans-fected with mutant NL4-3HSA strains were analyzed by Western blotting. Mocktransfected 293T cells served as a negative control, and the expression of�-actin was used as a loading control. (B and C) The Western blot was probedwith HIV-1-infected patient sera to examine Gag and Pol expression (B) orantibodies specifically directed against Vif or Vpr (C).

Fig. 2. The NL4-3HSA strain of HIV-1 causes cytopathicity in Jurkat cells. (A)Jurkat cells were mock infected (a) or infected with NL-EGFP (b) or NL4-3HSA

Env� (c) at a MOI of 5. The viability of infected cells was analyzed by flowcytometry by using forward light scatter (FSC; x axis) and staining with PI (yaxis). Each panel represents 10,000 total events on day 4 after the start ofinfection. The polygonal gate indicates the percent of viable, PI-negative cells.(B) Jurkat cells were infected with the NL4-3HSA Env� strain at various MOI, andviability was monitored by flow cytometry over time. Mock-infected Jurkatcells were at least 95% viable on day 4 based on PI staining. Viability ofinfected (HSA-positive) cells over time was plotted on the graph.

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Cells infected with a Gag�Pol�Vpr�Vpu� strain showed 68%viability and 37% HSAhi cells at 8 days after infection (Fig. 3 Aand B). However, the most impressive results were obtained withGag�Pol�Vif�Vpr� and Gag�Pol�Vif�Vpr�Vpu� mutantstrains at MOI of 5, which showed 67% and 78% viability at 8days of infection, respectively, with 65–85% of these cells beingHSAhi (Fig. 3 A and B). Hence, cell death was clearly reducedwhen both Vif and Vpr were absent. No other combination ofgene mutations produced a comparable attenuation of cytopath-icity and survival of highly infected cells.

To directly examine the contribution of Vif and Vpr tocytopathicity, we created the same mutations in the Gag�Pol�background of NL4-3HSAEnv�. We first investigated the phe-notypes of mutant NL4-3 strains lacking only one of the acces-sory genes, vif, vpr, or vpu. We found these mutant strains to beas potently cytopathic as NL4-3HSAEnv� at an MOI of 5, despiteminor variations in the initial killing rate (Fig. 4 A and B). Thecombined loss of Vif and Vpu or Vpr and Vpu expression did notsignificantly alter cytopathicity. By contrast, when both Vif andVpr were disrupted, killing was clearly diminished (Fig. 4 A andB; see also Fig. 7, which is published as supporting informationon the PNAS web site). The effect was even more prominent at

an MOI of 3, at which 80% of the cells survived 8 days afterinfection with 71% HSAhi cells (Fig. 4A; see also Fig. 8, whichis published as supporting information on the PNAS web site).The HSAhi cells were still viable and proliferating 25 days afterinfection (Fig. 7B). These results indicated important roles forthe Vif and Vpr accessory proteins in HIV-1 cytopathicity.

We found that the expression of vpu contributes modestly todirect cell killing when infection is performed at a higher MOI(Fig. 4B and 8B). In cells infected with Vif�Vpr� strain, highHSA expression was detected at day 8 in 72% and 78% of livecells at MOI of 3 and 5, respectively (Fig. 8). However, thecytopathicity of this Vpu expressing virus was severely compro-mised at an MOI of 3, indicating that Vif and Vpr play thedominant cytopathic role. Additionally, cell death due toVif�Vpr� and Vif�Vpr�Vpu� strains was the same when infec-tion was performed at an MOI of 3 (Fig. 4A). Together, thesedata indicate that the effect of Vpu on direct cell killing is notsubstantial, especially at low levels of infection.

We also examined the phenotypes of the mutant strains inperipheral CD4� T cells, a MOLT-4 T cell line, and a SupT1 Tcell line to extend our results to other transformed and non-transformed T cells. Peripheral CD4� T cells were infected with

Fig. 3. Cytopathicity of mutant NL4-3HSA strains in the absence of Gag and Pol. Mutant virus stocks were generated in the presence of �R8.2�Vpr helper plasmidin 293T producer cells and used to infect Jurkat cells at a MOI of 5. Cytopathicity of the total population (A) and fractions of infected cells (B) were monitoredby flow cytometry. Cell viability was analyzed as a function of time after the initiation of infection by propidium iodide exclusion staining as shown in Fig. 2A.The fraction of infected cells was monitored by HSA staining. Viability associated with virus expression is shown on the y axis, and time in days is shown for eachrow of samples.

Fig. 4. Cytopathicity of mutant HIV-1NL4-3 strains. Jurkat T cells were mock-infected or infected with various strains of NL4-3HSA at a MOI of 3 (A) and 5 (B). Theviability of the total population was measured as described in Fig. 2A. The unpaired Student’s t test for mutant vs. the parental NL4-3HSA Env� strain on day 7indicated P � 0.05 for the viability of cells infected with Vif�Vpr� and Vif�Vpr�Vpu� strains.

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mutant NL4-3HSA strains expressing Env to increase infectivity.In infected cultures, 80–90% of the cells were HSA-positive(data not shown). The removal of both Vif and Vpr significantlyalleviated HIV killing among these infected cells, permitting57% viability in HSAhi cells as opposed to 15–21% viability in Vifand�or Vpr-expressing infected cells (Fig. 5A). Vif killing wasalso observed in the peripheral CD4� T cells, which expressesAPOBEC3G. Therefore, Vif-induced cytopathicity in Jurkatcells is unlikely to be a result of overexpression�hyperactivationof Vif in the absence of its natural substrate APOBEC3G.

MOLT-4 and SupT1 cells were similarly infected with themutant NL4-3HSA Env� strains used for Jurkat infections (Fig.5 B and C). MOLT-4 showed the highest viability of all infectedcells examined. However, only 30–40% of MOLT-4 cells wereHSAhi compared to 80–90% for the other cell types, whichprobably accounts for the apparent increase in viability amongthese cells (Fig. 5B). Nevertheless, infection of either MOLT-4or SupT1 cells with Vif�Vpr� and Vif�Vpr�Vpu� virusesresulted in viability comparable to that seen among uninfectedcells, in contrast to the 40% reduction in viability observed whenvif and vpr genes were intact. Hence, the data from multiple Tcell lines and normal nontransformed T cells reveal a principalrole for both the Vpr and Vif accessory proteins, but not otherviral proteins, in HIV-1 mediated cytopathicity.

During these experiments, we noticed that cells infected withVif�Vpr� strains underwent cell division and maintained high HSAexpression for extended periods of time. However, the proliferationof infected cells was not observed with the other strains, even thoselacking Vpr, if Vif was intact. The HSAhi fraction of these cellsappeared to diminish as a consequence of a higher rate of prolif-eration among uninfected cells as well as cytopathicity (Fig. 7). Thisobservation was surprising because cell cycle arrest is typicallybelieved to require the presence of Vpr. An intriguing possibilitywas that Vif also caused cell cycle arrest. We therefore examined theDNA content of cells infected with various mutant viruses. Themajority of uninfected Jurkat cells or those infected with theGFP-only virus were at the G1 phase of the cell cycle (Fig. 6; seealso Fig. 9 a and b, which is published as supporting information onthe PNAS web site). As expected, the DNA profile shifted from theG1 phase to the G2 phase in cells infected with Env� virus, whichcontained intact Vpr, indicating cell cycle arrest (Figs. 6 and 9c).Surprisingly, we found that viruses lacking Vpr also showed arrestat the G2 phase (Fig. 6 and 9 f and j). However, G2 blockade wasabolished if Vif was eliminated from the Vpr� virus (Figs. 6 and 9d, h, and k). Infected peripheral CD4� T cells also showed similarcell cycle profiles (Fig. 10, which is published as supporting infor-mation on the PNAS web site), suggesting that APOBEC3G doesnot affect Vif-induced cell cycle arrest.

To confirm Vif-induced cell cycle arrest, we infected Jurkat cellsthat were synchronized at the border of G1 and S phases andexamined the progression of cell cycle in infected cells (42). We

pretreated cells with G1�S blocker aphidicolin for 16 h, and after a10-h release, we infected the cells in the presence of the drug. Then,infected cells were again released from G1�S phase blockade after16 h of the drug treatment. Cells were analyzed for HSA expressionand DNA content at 0, 10, 20, and 40 h after the release by flowcytometry. At the time of release (16 h after infection), infectedcells were at G1 phase (Fig. 11, which is published as supportinginformation on the PNAS web site). Ten hours after release,infected cells clearly accumulated at the G2 phase in the presenceof Vif and Vpr either alone or in combination (Fig 11 A, C, and D),confirming cycle arrest by Vif and Vpr. However, in the absence ofVif and Vpr, infected cells did not show the characteristic G2 arrestprofile even at 40 h after infection (Fig. 11B), supporting G2 cellcycle blockade independently by Vif and Vpr.

Because flow cytometric analyses of DNA content do notdistinguish between G2 and M phases, we also examined thepresence or absence of a mitotic marker by Western blotting.Lysates from infected Jurkat cells were analyzed for the presenceor absence of phosphorylation of histone H3 at serine 28. Thephosphorylation of histone H3 at Ser-28 occurs during mitoticchromosomal condensation and, thus, serves as an early mitoticmarker (43, 44). Ser-28 phosphorylation of histone H3 wasdetected in mock-infected cells, indicating the presence ofmitotic cells in culture (Fig. 12, lane 1, which is published as

Fig. 5. Time course measurements of the viability of CD4� T cell lines infected with NL4-3HSAEnv� strains carrying mutations in accessory genes. (A) PeripheralCD4� T cells after 3 days of stimulation and infection with VSV-G pseudotyped viruses at a MOI of 30. The extent of infection and viability were monitored byflow cytometry by staining for the HSA marker and with PI staining, respectively, as described in Fig. 2. The percentage of PI-negative infected cells is shown inthe graph. (B and C) MOLT-4 (B) and SupT1 (C) T cell lines were infected with mutant strains of NL4-3HSA, and the viability of infected cells was monitored by flowcytometry.

Fig. 6. Cell cycle profiles of Jurkat cells infected with various mutantNL4-3HSA strains. Infected Jurkat cells were examined for DNA content (x axis)vs. cell number (y axis) 2 days after infection to assess their position in the cellcycle.

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supporting information on the PNAS web site). The amount ofSer-28-phosphorylated H3 was greatly diminished in cells in-fected with Vif or Vpr expressing viruses (Fig. 12, lanes 2, 4, and5), suggesting that cell cycle arrest occurs at the G2 rather thanthe M phase of cell cycle. By contrast, phosphorylated H3 atSer-28 was restored in cells infected with Vif�Vpr� virus (Fig.12, lane 3), suggesting that the mutant virus does not cause G2cell cycle arrest. Thus, Vif, in addition to Vpr, independentlycauses cell cycle arrest at the G2 phase in infected cells. Hence,both genes that play a major role in cytopathicity share theproperty of cell cycle inhibition at the G2 stage.

DiscussionSince discovery of the in vitro cytopathic effect of poliovirus byEnders and coworkers (45), viral cytopathicity has been believed tobe a key element of viral pathogenesis in vivo. In vitro, HIV-1 is ahighly cytopathic virus that causes CD4� T cell death shortly afterinfection. Nevertheless, the importance of this event for AIDSpathogenesis and the mechanism(s) by which it comes about havebeen unresolved after extensive investigation. Recent work hasemphasized that direct viral killing of infected T cells, especiallymemory T cells, makes an important contribution to AIDS patho-genesis during acute infection (7, 8). We previously demonstratedthat necrotic death occurs in CD4� T cells during a single round ofinfection with the NL4-3HSA laboratory strain of HIV-1 (12–14).We also observed that this cytopathic effect could be attributed todirect killing rather than killing of bystander cells (14). Therefore,in the present study, we sought to define the viral moleculesresponsible for the direct killing of CD4� T cells. In particular, weattempted to attribute a lethal effect to specific viral moleculesindependent of any role in viral infectivity. We systematicallycreated various combinations of mutations that prevented theexpression of various viral proteins in the NL4-3HSA Env� strainand examined cytopathicity. Although we initially hypothesized thatone or more of the structural proteins or enzymes encoded in theHIV genome would cause cell death, this conjecture was notcorrect. The cytopathicity of the Gag�Pol� strain of NL4-3HSA wasunchanged from that of the Env� strain, resulting in �80% celldeath in 6 days (Fig. 3).

By contrast, disruption of both the Vif and Vpr coding regionssubstantially ameliorated virus-induced cell death, indicatingthat either of these gene products could independently render aviral cytopathic effect. The abrogation of cell death did notdiminish and, therefore, was independent of infectivity at leastin this in vitro culture system. Jurkat cells highly infected with theNL4-3HSAVif�Vpr� or NL4-3HSAVif�Vpr�Vpu� strain werestill viable 25 days after infection (Fig. 7B). Viability of periph-eral blood lymphocytes and other T cell lines was sustained ifthey were infected with the NL4-3HSAVif�Vpr� or NL4-3HSAVif�Vpr�Vpu� strain (Fig. 5). Although all of these resultswere derived from in vitro tissue culture with an MOI �1, therequirement for either Vif and Vpr for cytopathic effectssuggests that these genes could be important in HIV pathogen-esis beyond their role in virus infectivity.

Could there be viral components besides Vif and Vpr involved?It was evident that a low level of cell death could be instigated byviruses devoid of Vif or Vpr. However, it was difficult to ascribe thiseffect to any particular viral molecule, and, therefore, the questionremains open. Interestingly, although Gag and Pol themselves didnot contribute significantly to cytopathicity, their removal en-hanced the protective effect of eliminating Vpr. For example, aVpr� strain caused �88% death in the presence of Gag and Pol, butGag�Pol�Vpr� mutant strain led to only 55% cell death (Figs. 3and 4). Furthermore, strains lacking Vpr and Vpu killed 90% ofcells in 8 days, whereas cells infected with a Gag�Pol�Vpr�Vpu�

strain killed only 32% of the cells (Figs. 3 and 4). The decrease incytopathicity in Gag�Pol� strains was evident only when coupledwith the Vpr mutation. Thus, the Gag-Pol polyprotein apparently

enhances killing by Vif, which causes cytopathicity in the vpr mutantstrain. Vif is the least stable HIV-1 protein (46), and Gag-Pol mayact to stabilize the protein levels of Vif (K.S. and M.J.L., unpub-lished observations).

Unexpectedly, we observed cell cycle arrest at the G2 phase ininfected cells in the absence of Vpr and determined that Vif wasresponsible. It is possible that HIV-1 cytopathicity and cell cyclearrest might be correlated; both properties have been previouslyascribed to Vpr (reviewed in refs. 22, 47, and 48). Other studiesreported an increase in LTR activity at the G2 phase andsuggested that this increase contributes to viral killing (42, 49,50). However, the HSA marker was driven by the LTR in ourNL4-3HSA vector, and we observed no increased HSA expressionin the presence of Vif and Vpr.

Agents that cause G2 cell cycle blockade such as etoposide,vincristine, and nocodazole are known to cause cell death (39, 40,51). Hence, it may be that, unlike the G1 phase, arrest in G2, inwhich replication has doubled the DNA content, is not a naturalstopping point in the cell cycle that is tolerated by T cells.Increased ploidy can lead to untoward effects such as chromo-some instability (52). In conclusion, our study sheds light on thecomponents of HIV-1 required for cell cycle arrest and cell deathand sets the stage for determining the interrelationship of thetwo in viral pathogenesis.

Materials and MethodsVirus Stock Production. All virus stocks were produced in 293T cellsand pseudotyped by using VSV-G to replace Env, as described inref. 51. Briefly, 2 �g of HIV-1 plasmid was cotransfected with 1 �geach of pLVSV-G (National Institutes of Allergy and InfectiousDiseases AIDS Research and Reference Reagent Program) andpCMV�R8.2�Vpr (23) into 293T cells in a six-well plate by using13.2 �l of ExGen 500 reagent according to the manufacturer’sinstructions (Fermentas, Hanover, MD). The virus was collected48 h after transfection and stored at �80°C. Virus titers weredetermined by infecting Jurkat T cells with various volumes of virusstocks and assessing the MOI based on the Poisson distribution asdescribed in ref. 13. For cytopathicity studies, 106 Jurkat cells wereinfected at a MOI of 3 or 5 in the presence of 5 �g�ml polybrene(Sigma) in a 12-well plate, followed by 30 min of centrifugation at800 � g at room temperature.

For CD4� T cell infection, peripheral blood lymphocytes wereprepared from buffy coats by Ficoll-Paque Plus gradient sepa-ration (Amersham Pharmacia Biosciences), and CD4� T cellswere isolated by positive selection by using MACS CD4 mi-crobeads (Miltenyi Biotec, Auburn, CA). After the isolation,CD4� T cells were immediately stimulated with 3 �g�ml ofplate-bound CD3 and 2 �g�ml CD28 antibodies for 3 days beforeinfection. Infection was performed at a MOI of 30 as describedabove except that 15 �g�ml polybrene and 100 units�ml of IL-2were added at the time of infection.

Viability and Infectivity Assay. Cell viability and infectivity wereassessed by flow cytometry on FACS Calibur (Becton Dickin-son). Cells were harvested every 24 h and stained with propidiumiodide (PI) for viability and with fluorescein-conjugated CD24antibody (HSA, BD Pharmingen) for infectivity.

Construction of Mutant NL4-3HSA Strains. Various mutations wereintroduced into pNL4-3HSA Env� by site-directed mutagenesis orsubcloning of mutant fragments. Details are described in SupportingText, which is published as supporting information on the PNASweb site. The mutations were confirmed by DNA sequencing.

Cell Cycle Analysis. To analyze cell cycle arrest induced by HIV-1,the DNA content of infected Jurkat cells and peripheral CD4� Tcells was examined by flow cytometry. Jurkat cells stained for thesurface expression of HSA (13) were first incubated in 1% para-

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formaldehyde for 10 min at room temperature, washed with 1�PBS, and fixed with 70% ethanol at �20°C. Subsequently, the fixedcells were washed and stained with DNA staining solution (4 �g/mlPI�50 �g/ml RNase�0.45 mg/ml sodium citrate in 1� PBS). Pe-ripheral CD4� T cells were stained with 20 �M cell-permeableDNA dye, DRAQ5 (Axxora, San Diego), for 5 min at 37°Caccording to the manufacturer’s instructions after HSA staining.Stained cells were examined on a FACS Calibur flow cytometer andanalyzed by FLOWJO software (Tree Star, Ashland, OR).

Cell Synchronization and Infection. Jurkat cells were synchronizedwith a G1�S phase blocker, aphidicolin, as described in ref. 42.Briefly, cells were treated with aphidicolin for 16 h and washedfor three times to remove the drug. After 10 h of release, cellswere infected with NL4-3HSA wild-type or mutant strains in thepresence or absence of aphidicolin. Cells were harvested 0, 10,20, and 40 h after the release. Viability, fractions of infected cells,and DNA content were analyzed by flow cytometry as describedabove by using PI, HSA, and DRAQ5 staining, respectively. Cellcycle status was also examined at 20 h after the release byWestern blotting as described below.

Western Blot Analyses. To verify the loss of protein expression bysite-directed mutagenesis, infected Jurkat cells were lysed with2% SDS lysis buffer (30 mM Tris�Cl, pH 8.0�150 mM NaCl�2%

SDS�1 mM EDTA), and protein concentration in lysates wasestimated by a bicinchoninic acid assay (Pierce). Equal amountof proteins were loaded onto a SDS�PAGE, and the loss ofprotein expression was examined by Western blotting. Patientsera were used to detect Gag and Pol, and Vif and Vpr wereprobed with specific antibodies. Vif monoclonal antibody (no.319) was obtained from Michael H. Malim (National Institutesof Health) (36, 53, 54). The polyclonal antibody against Vpr wasgenerously provided by K. Strebel (National Institutes of Allergyand Infectious Diseases, National Institutes of Health). For cellcycle profile, lysates were analyzed by a Western blot probed foran early mitotic marker by using anti-phopho-histone H3 (Ser-28) antibody (Upstate Biotechnology, Charlottesville, VA).

We thank Klaus Strebel for providing us a �Vpu plasmid and Vprantibody and for valuable discussions; Stephen Porcella at the RockyMountain Laboratory for DNA sequencing support; Diane Bolton, JohnCoffin, Malcolm Martin, Danny Douek, and the members of the Lenardolaboratory for helpful suggestions; Eugenia Park, Marianne Selkirk, andMelody Duvall for early studies; Nicolas Bidere, Lixin Zhang, RonaldGermain, and Danny Douek for critical reading of the manuscript;Shirley Starnes for manuscript preparation; and Anthony Fauci for thegenerous availability of his BL-3 facility. K.S. is a Japan Society for thePromotion of Science research fellow for Japanese biomedical andbehavioral research at the National Insitutes of Health. This research wassupported by the Intramural Research Program of the National Institutesof Allergy and Infectious Diseases, National Institutes of Health.

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