An Alteration of Human Immunodeficiency Virus gp41 Leads to

JOURNAL OF VIROLOGY, June 2008, p. 5460–5471 Vol. 82, No. 110022-538X/08/$08.00�0 doi:10.1128/JVI.01049-07Copyright © 2008, American Society for Microbiology. All Rights Reserved.

An Alteration of Human Immunodeficiency Virus gp41 Leads toReduced CCR5 Dependence and CD4 Independence�

Brian M. Taylor,1,2 J. Scott Foulke,1 Robin Flinko,1 Alonso Heredia,1Anthony DeVico,1 and Marvin Reitz1,2,3*

Institute of Human Virology1 and Department of Medicine,2 School of Medicine, and Department of Microbiology andImmunology,3 University of Maryland Baltimore, 725 W. Lombard St., Baltimore, Maryland 21201

Received 14 May 2007/Accepted 11 March 2008

Human immunodeficiency virus (HIV) type 1 infection requires functional interactions of the viral surface(gp120) glycoprotein with cell surface CD4 and a chemokine coreceptor (usually CCR5 or CXCR4) and of theviral transmembrane (gp41) glycoprotein with the target cell membrane. Extensive genetic variability, gener-ally in gp120 and the gp41 ectodomain, can result in altered coreceptor use, fusion kinetics, and neutralizationsensitivity. Here we describe an R5 HIV variant that, in contrast to its parental virus, infects T-cell linesexpressing low levels of cell surface CCR5. This correlated with an ability to infect cells in the absence of CD4,increased sensitivity to a neutralizing antibody recognizing the coreceptor binding site of gp120, and increasedresistance to the fusion inhibitor T-20. Surprisingly, these properties were determined by alterations in gp41,including the cytoplasmic tail, a region not previously shown to influence coreceptor use. These data indicatethat HIV infection of cells with limiting levels of cell surface CCR5 can be facilitated by gp41 sequences thatare not exposed on the envelope ectodomain yet induce allosteric changes in gp120 that facilitate exposure ofthe CCR5 binding site.

Human immunodeficiency virus type 1 (HIV-1) enters cellsby membrane fusion mediated by its envelope (Env) glycopro-teins (51). The Env proteins are synthesized as a 160-kDaprecursor that is cleaved by a host protease to yield the surfacegp120 (SU) and the transmembrane gp41 (TM) glycoproteinsubunits. The functional Env structure is a trimer, with thegp120 subunits anchored on the virion surface by noncovalentinteractions with the gp41 trimer. The gp120 binds first to CD4and subsequently to a chemokine receptor/coreceptor (gener-ally CCR5 or CXCR4). The gp41 then interacts with the targetcell membrane through its N-terminal fusion domain, promot-ing lipid mixing and viral entry. An unusual feature of gp41 isits long cytoplasmic domain (CD) or tail of approximately 150amino acids (aa), in contrast to the TM proteins of otherretroviruses, such as avian and murine oncoretroviruses, whichhave a shorter CD (typically 20 to 30 aa).

The HIV gp41 CD region includes a number of domains, theexact functions of which are not well understood. The CDincludes one or more palmitoylated cysteines, which may me-diate localization of the Env to lipid rafts (4, 55). A tyrosine-based (Yxx�) motif in the membrane-proximal region of theCD mediates binding to components of clathrin-associatedadaptor complexes, which are involved in trafficking and en-docytosis (3, 5, 7, 48, 56), and also targets Env to the basolat-eral membrane in polarized cells, resulting in basolateralbudding (38, 49). The CD forms three highly conserved am-phipathic �-helices, termed lentiviral lytic peptides (LLPs),that have been implicated in interactions that decrease thestability of lipid bilayers, causing pore formation and mediating

T-cell death (11, 12, 22, 33, 42, 43, 62). The CD also containstwo regions that closely resemble those found in calmodulin-activated enzymes that bind calmodulin and could potentiallyinhibit calmodulin-regulated proteins (32, 44, 59, 60). Sincecalmodulin helps regulate T-cell metabolism and function,these regions may modulate T-cell signal transduction path-ways to facilitate infection.

To better understand the role of the CD in HIV infection,several investigators have introduced premature stop codons(18, 25, 66). The results do not provide a clear picture of CDfunction. Although the CD is dispensable for fusion, sometruncations significantly reduce viral infectivity. Other pointmutations and truncations of the CD, however, increase Envsurface expression (36, 71) and incorporation into virions (9,39, 69, 71), increasing the efficiency of entry. Interestingly,some truncations of CD, in combination with other env muta-tions, lead to CD4-independent infection (6, 68). A clear un-derstanding of the role of the cytoplasmic tail is complicatedfurther because truncations in the tail can have different bio-logic effects depending upon the target cell (46).

HIV-1 variants for which the initial step of CD4 binding isdispensable have been described (19, 20, 29, 30, 35). Thegp120s of these viruses are thought to be in a “pretriggered” orpartially triggered state in which the conserved coreceptorbinding site is exposed and functional. Exposure of this site,however, results in an increased sensitivity to some neutraliz-ing antibodies, such as 17b, that recognize epitopes induced byCD4 binding and overlapping the conserved coreceptor bind-ing site. Such a pretriggered state of gp120 may facilitate con-version of the gp41 trimer to its three-stranded coiled-coilfusion intermediate, leading to more rapid fusion. Faster fu-sion kinetics would have a major impact on the efficiency ofentry because fusion is likely rate limiting for entry (52).

Here we describe the isolation and characterization of

* Corresponding author. Mailing address: Institute of Human Vi-rology, 725 W. Lombard Street, Baltimore, MD 21201. Phone: (410)706-4679. Fax: (410) 706-4694. E-mail: [email protected].

� Published ahead of print on 19 March 2008.

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HXBaLm1.2, a variant derived from an R5 (Ba-L) HIV chimera,and of HXBaLm1.2-derived molecular clones pHXBaLm1.2and pHXBaLm6133. These variants infect cells expressing oth-erwise limiting levels of cell surface CCR5, in contrast to theparental virus. They also infect cells in the absence of CD4, aremore sensitive to neutralization by soluble CD4 (sCD4) and anantibody against conserved epitopes that overlap the corecep-tor binding site of gp120, and are less sensitive to inhibition bythe fusion inhibitor T-20 (65) than is the parental virus. Thegenetic determinants for these properties are within the gp41,including the CD, a region not previously shown to influencecoreceptor use. Our interpretation of the data is that the al-tered properties are due to changes in gp41 that induce allo-steric alterations in either the tertiary or quarternary gp120conformation that partly expose the chemokine receptor bind-ing site, allowing more rapid or efficient fusion with the cellmembrane.

(Much of this work was performed by Brian M. Taylor inpartial satisfaction of his Ph.D. thesis requirements.)

MATERIALS AND METHODS

Cell lines. COS-1 cells were from the ATCC. HOS cells expressing CD4 andCCR5, CXCR4, CCR1, CCR2b, or CCR3 were from the NIH AIDS ReagentRepository (Bethesda, MD). COS-1 and HOS cells were maintained in 90%Dulbecco’s modified Eagle’s medium and 10% heat-inactivated fetal bovineserum (FBS). The T-cell lines CEM-SS, H9, Sup-T1, and PM1 were obtainedfrom the NIH AIDS Reagent Repository and were maintained in 90% RPMI1640 and 10% heat-inactivated FBS. U87MG cells (a human astroglioma line),U87.CD4.CCR5 cells (expressing CD4 and CCR5) and U373/CD4/MAGI cells(63) (human astroglioma lines that express chemokine receptors and have a�-galactosidase [�-gal] indicator gene regulated by the HIV long terminal re-peat), and Cf2Th/synCCR5 cells (an adherent canine thymocyte line expressing0.5 to 1.0 �106 human CCR5 molecules per cell) were from the NIH AIDSReagent Repository. The JC10 and JC20 cell lines expressing defined levels ofcell surface CCR5 (64) were a gift from David Kabat (Oregon Health SciencesUniversity, Portland, OR). BC7/CCR5, a SupT1-derived T-cell line expressingCCR5 but not CD4 (40), was a gift from James Hoxie (University of Pennsyl-vania, Philadelphia, PA).

Reagents and antibodies. The chemokines RANTES, macrophage inflamma-tory protein 1� (MIP-1�), and stromal cell-derived factor 1 (SDF-1) were pur-chased from R&D Systems (Minneapolis, MN). The CCR5-specific inhibitorTAK-779 (2) was obtained from the NIH AIDS Research and Reference Re-agent Program. T-20 and �2 RANTES were gifts from Lai Xi Wang andAnthony DeVico, respectively, from the Institute of Human Virology (IHV),Baltimore, MD. The HIV-1 gp120 monoclonal antibody (MAb) immunoglobulinG1 (IgG1) b12 was obtained from the NIH AIDS Research and ReferenceReagent Program. Monoclonal antibody 2G12 was purchased from PolymunScientific Inc., Vienna, Austria. MAbs A32, ED47, C11, 19E, and 17B, as well assCD4, were obtained from the �QUANT Core Facility, IHV. Human IgG waspurchased from Sigma.

Infectious HIV-1 molecular clones. The plasmids pHXB2-MCS and pHXB2-MCS�env were generously provided by Andrew Leigh Brown (University ofEdinburgh, Scotland). pHXB2-MCS is a derivative of the infectious molecularclone pHXB2 with two cloning sites added by site-directed mutagenesis, a BstEIIsite 5 aa downstream of the gp160 signal sequence and an XbaI site 110 aa fromthe amino terminus of gp41. The addition of these sites does not alter the codingcapacity of the clone. pHXB2-MCS�env is a derivative of pHXB2-MCS fromwhich a NdeI-StuI fragment containing the V1 and V2 domains of gp120 wasexcised, and it does not produce viable virus. The proviral DNA sequences frompHXB2-MCS and pHXB2-MCS�env were excised with PacI, which cuts in the5and 3 host cellular flanking regions, and spliced into the cloning vectorpSP65gpt(PacI), containing a PacI site in place of the XbaI site of pSP65gpt.pHXBaL was constructed by substituting the coding regions for gp120 and theamino-terminal 110 aa of the gp41 coding regions of the R5 HIV-1(Ba-L) intothe homologous region in pHXB2-MCS. This region was amplified from theplasmid pBaL-C3 using primers 5-TGT-GGG-TCA-CCG-TCT-ATT-AT-3 and5-CTC-ATC-TAG-AGA-TTT-ATT-ACT-CC-3 and spliced into the BstEII/XbaI site of pHXB2-MCS�env.

To construct pHXBaLm1.2, a plasmid encoding a virus expressing the mutantEnv proteins, the entire env gene from HXBaLm1.2 was PCR amplified usingprimers 5-TGG-CAA-TGA-GAG-TGA-AGG-AG-3 and 5-CTA-AGA-TCT-ACA-GCT-GCC-TTG-TAA-3 and spliced into the BstEII/Bpu1102 site ofpHXB2-MCS�env. pHXBaLm1.2 includes the mutations in the gp120 of HXBaLm1.2 as well as its altered gp41 region. To make pHXBaLm3.4, the V3-V4region of HXBaLm1.2 was PCR amplified and spliced into the BstEII/XbaI siteof the parental plasmid, pHXBaL, using primers 5-TGT-GGG-TCA-CCG-TCT-ATT-AT-3 and 5-CTT-ATC-TAG-AGA-TTT-ATT-ACT-CCA-ACT-AG-3. pHXBaLm3.4 is identical to the parental virus except for the mutations in V3and C3. To make pHXBaLm6133, the region of the gp41 of HXBaLm1.2 fromheptad repeat 1 (HR1) to LLP1 was PCR amplified with primers 5-GGC-AGT-CTA-GCA-GAA-GAA-GAG-GT-3 and 5-CTA-AGA-TCT-ACA-GCT-GCC-TTG-TAA-3 and substituted for the reciprocal region in the parental vector,HXBaL. A polylinker that includes BstEII and XhoI sites was added to thevector, pcDNA3.1�/Zeo (Invitrogen). The BstEII/XhoI region from HXBaLwas ligated into this vector to create a new vector, pcDNA/HXBaL. The PCR-amplified product from HXBaLm1.2 was spliced into the PshAI/Bpu1102 site ofpcDNA/HXBaL. The BstEII/BlpI fragment from the resulting vector, pcDNA/BaLm6133, was cut and ligated into the same region in pHXBaL to yieldpHXBaLm6133. pHXBaLm6133 contains the gp41 of the original mutant butlacks the gp120 mutations. All constructs were verified by DNA sequence anal-yses. To generate infectious virus from plasmids, 2 � 105 COS-1 cells weretransfected with 2 �g of plasmid DNA using FuGene6 (Roche Diagnostics,Indianapolis, IN) under conditions specified by the manufacturer. At 72 h aftertransfection, supernatants were collected and stored at �80°C.

Infectivity assays. Infectivity assays with HOS cells were performed in six-wellplates containing 100,000 cells/well; 200 �l of transfected cell supernatant wasadded to 2 ml of medium. After 24 h, cells were washed three times withDulbecco’s phosphate-buffered saline (DPBS) and the medium replaced. Super-natant was collected every 3 days and tested for HIV-1 core antigen p24 byenzyme-linked immunosorbent assay (ELISA) (Perkin-Elmer Life Science, Inc.,Boston, MA) per the directions of the manufacturer. Infected cells were de-tached, counted, and replated in 2 ml of fresh medium on six-well dishes at200,000 cells per well. Stocks of resultant viruses were propagated in PM1 cells,and the 50% tissue culture infectious dose (TCID50) per milliliter of viruses inthe supernatants determined by the �QUANT Core Facility, IHV, by limitingdilution on phytohemagglutinin-stimulated PBMCs using the Spearman-Karbermethod (25).

For infection of T-cell lines, 100,000 cells were treated with 50 TCID50s ofvirus in 1 ml of 90% RPMI–10% FBS, incubated for 3 h at 37°C, washed threetimes with DPBS, and resuspended in 1 ml of medium. The cultures were split ata ratio of 1:5 every 3 days and medium p24 antigen measured by ELISA. Forchemokine inhibition assays, cells were incubated with 1 �g/ml of RANTES,SDF-1, MIP-1�, or no chemokine for 1 hour at 37°C. Virus was then added at amultiplicity of infection of 0.01 and incubated for 3 h. Cultures were then washedthree times with DPBS, and new medium and chemokines were added. Cultureswere monitored for p24 and chemokines added with each medium change.

Optiprep gradients. Virions were prepared from supernatants from chroni-cally infected PM1 cells by centrifugation to remove cellular debris (1,800 � g, 10min) and pelleting the virus (141,000 � g, 2 h) through a 32% (wt/vol) sucrosecushion in a Beckman SW28 rotor. Virus was resuspended in 250 �l of PBS andcentrifuged in an Optiprep (60% iodixanol [wt/vol]; Gibco Life Technologies)velocity gradient as described previously (17). Briefly, iodixanol gradients wereprepared in PBS in 11 increments of 1.2%, ranging from 6 to 18%. Virions werelayered on top of the gradient and centrifuged (200,000 � g, 1.5 h) in a BeckmanSW41 Ti rotor. The bottom four fractions were collected and precipitated with20% trichloroacetic acid, except that 50 �l from each pool of fractions was savedand used to infect PM1 cells. Acid-precipitated virions were resuspended in 50 �lPBS, with 1 �l used to determine p24 concentration prior to Western blotanalyses.

Immunoblot analyses. Samples from the Optiprep gradient were normalizedby p24 concentration and electrophoresed on sodium dodecyl sulfate-polyacryl-amide gels (Novex) in 4 to 20% Tris-glycine. An equivalent of 14 ng of p24 wasloaded in each well. Envelope glycoprotein was detected by Western immuno-blotting using a pool of gp120-specific antibodies (provided by George Lewis,IHV) and a secondary anti-mouse IgG, horseradish peroxidase-linked antibody(Cell Signaling Technology, Beverly, MA). Purified HIV-1 Ba-L gp120 protein(�QUANT Core Facility, IHV) was used as a positive control. p24 was measuredwith MAbs 13B6 and 13G4 (�QUANT Core Facility, IHV). Western blots werevisualized by chemifluorescence using ECL-Plus (Amersham Biosciences Corp,Piscataway, NJ) and bands quantified densitometrically using ImageQuant 5.0

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(Molecular Dynamics, Sunnyvale, CA) and a Storm Fluor-Imager (MolecularDynamics).

Doxycycline-regulated expression of CCR5. U87MG cells were sequentiallytransfected with three plasmids (pcDNA-CD4, pBI-EGFP-CCR5, and pEF1prtTA) using FuGene6 (Roche) and stable clones selected to create theU87.DOX.CCR5 cell line. pcDNA-CD4 was constructed by inserting a HindIII-BamHI fragment from the plasmid plck-CD4 A�, containing human CD4 cDNA(8) (a gift from Harris Goldstein, Albert Einstein College of Medicine, Bronx,NY), into the cognate sites of pcDNA 3.1�/Zeo (Invitrogen). pBI-EGFP-CCR5was constructed by inserting a polylinker containing BclI and SalI sites into aplasmid containing a bidirectional tetracycline-inducible promoter (pBI-EGFP;Clontech) and then ligating the BamHI/SalI fragment from pBABE.CCR-5,obtained from the NIH AIDS Research and Reference Reagent Program, tocreate pBI-EGFP-CCR5. pEF1prtTA, encoding the transactivator for the tetra-cycline-inducible promoter (26), was a gift from P. B. Fisher (Columbia Univer-sity, New York, NY). As a negative control, U87MG cells were transfected withpcDNA-CD4 alone. Cells were maintained in Dulbecco’s modified Eagle’s me-dium–10% FBS supplemented with 250 �g/ml zeocin and 250 �g/ml geneticin tomaintain the CD4 and “reverse” Tet repressor genes, respectively. Variablelevels of CCR5 expression were induced by addition of different concentrations(0.1 to 1,000 ng/ml) of doxycycline (Sigma) to the culture medium. Infectivityassays in U87.DOX.CCR5 cells were performed in six-well plates containing50,000 cells per well. The cells were treated with the indicated amount ofdoxycycline and incubated at 37°C overnight to induce CCR5 expression. Thenext day, 100 TCID50s of virus was added and the culture incubated at 37°C.After 3 h, cells were thrice washed with DPBS, and 2 ml of culture medium andantibiotics were added. Supernatants were collected every 3 days and tested formedium p24 by ELISA.

Determination of absolute surface levels of CCR5 and CD4. A total of 100,000cells were washed twice and resuspended in DPBS plus 2% heat-inactivated FBS,2% heat-inactivated human AB serum (Sigma), and 0.1% sodium azide (wash/stain buffer) and then incubated for 15 min at room temperature with purifiedanti-CD16 and anti-CD32 (both from BD Pharmingen) diluted 1:20 in wash/stainbuffer to block nonspecific binding. Then, either phycoerythrin (PE)-conjugatedanti-human CD195 and allophycocyanin-conjugated anti-human CD4 or PE-conjugated anti-human CD4 (all from BD Pharmingen) alone was added and thecells incubated at room temperature for 30 min, washed twice in wash/stainbuffer, and fixed in 1% paraformaldehyde. A minimum of 50,000 events wereacquired with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA)and analyzed with FlowJo batch analysis software (Treestar, San Carlos, CA).Quantibrite PE flow cytometry beads (Becton Dickinson) containing a knownnumber of PE molecules per bead were used to prepare a calibration curve toquantify the number of CD4 and CCR5 receptors per cell. The calibration curvewas used to estimate the number of molecules of PE-labeled reagent bound percell from the median fluorescence intensity of the samples.

Inhibition of HIV-1 infection. PM1 cells were cultured in RPMI 1640 mediumwith 10% heat-inactivated FBS, and 100,000 cells were resuspended in 100-�laliquots of TAK-779, MIP-1�, or �2 RANTES serially diluted in culture me-dium and incubated for 1 h at 37°C. Next, 100-�l aliquots of culture mediumcontaining 50 TCID50s of virus were added. The cultures were incubated at 37°Cfor 3 h, washed three times with PBS, and resuspended in fresh culture mediumcontaining the appropriate concentration of inhibitor. For studies with T-20(which binds to the virus, not the target cell), 50-�l aliquots of serially dilutedinhibitor were combined with 50-�l aliquots of culture medium containing 50TCID50s of virus for 1 h at 37°C. The mixtures of T-20 with virus were combinedwith PM1 cells (100,000 cells resuspended in 100 �l of culture medium) for 3 hat 37°C. The cultures were washed three times with PBS and resuspended infresh culture medium containing the appropriate concentration of inhibitor.Three days later, half the medium was replaced with the corresponding mediumor medium plus inhibitor. Tests under all conditions were carried out in tripli-cate. HIV-1 replication was quantified by medium p24 at days 3 and 6 postin-fection. The percent inhibition of HIV-1 was calculated from p24 concentrationsobtained in the presence of the inhibitors divided by the p24 concentration in theabsence of any inhibitors.

Antibody neutralization assays. A total of 5 � 103 U373/CD4/MAGI cellsexpressing CCR5 were added to wells in a 96-well microtiter plate and incubatedovernight at 37°C in 100 �l of complete medium. The medium was removed andreplaced with 50 �l of fresh medium plus 50 �l of medium containing 50 TCID50sof virus preincubated for 1 h at 37°C with the indicated antibodies. Followingincubation at 37°C for 18 h, the cells were washed in PBS and 100 �l of freshmedium with antibodies. The cultures were incubated at 37°C for 4 days andtreated with a �-gal chemiluminescent reagent (Galactostar; Tropix, Bedford,MA) according to the manufacturer’s protocol. Infection was quantified by

chemiluminescence using a Victor2 (EG&G Wallac, Gaithersburg, MD) fluo-rescence plate reader. �-gal expression from the long terminal repeat is depen-dent upon infection and expression of Tat. Values were corrected by subtractingthe background value determined in the absence of virus. The percent infectionwas calculated by dividing the corrected relative light units for each experimentalwell by the corrected relative light units for control wells containing only cells andvirus. Inhibition data were analyzed by linear regression to calculate the proteinconcentrations that gave 50% reductions in infection using GraphPad Prismversion 4.00 for Windows (GraphPad Software, San Diego, CA). Tests under allconditions were carried out in triplicate.

Selection of a CD4-independent HIV-1. The method of Kolchinsky et al. (35)was modified to isolate a CD4-independent HIV(Ba-L) variant (BaL10001) fromU87MG cell mixtures expressing CCR5 plus CD4 or CCR5 alone. A heteroge-neous population of HIV(Ba-L) (from the �QUANT Facility, IHV) was used toinfect a 10:1 mixture of U87.CCR5 and U87.CD4.CCR5 cells. At day 7 postin-fection, virus-containing supernatants were collected and used to infect a 20:1mixture of U87.CCR5 and U87.CD4.CCR5 cells. This process was repeatedevery 7 days at increasing ratios of CD4� to CD4� cells of 50:1, 100:1, 200:1,500:1, and 1,000:1.

To verify CD4 independence, viruses were tested on two CD4� T-cell lines. Atotal of 100,000 BC7/CCR5 cells were infected with 50 TCID50s of virus in 1 mlof 90% RPMI–10% FBS. The cultures were incubated for 3 h at 37°C, washedthree times with DPBS, and resuspended in 1 ml of medium. The cultures weresplit at a ratio of 1:5 every three days, and supernatant p24 was measured byELISA. Cf2TH/synCCR5 cells were plated at 100,000 cells in a 12-well plate andincubated at 37°C overnight. The next day, 50 TCID50s of virus was added andthe culture incubated at 37°C. After 3 h, the cells were washed three times withDPBS and 1 ml of culture medium replaced. Cultures were monitored by p24 asdescribed above.

RESULTS

Generation of a variant R5 HIV able to infect T-cell lines.HXBaL is a chimeric infectious molecular clone that encodesmost of the R5 HIV-1(Ba-L) gp120 and the first 110 aa ofHIV-1(Ba-L) gp41 as well as the first five amino-terminalamino acids of gp120 and the 237 carboxy-terminal aminoacids of gp41 of the X4 HIV-1(HXB2). All other genes areHXB2 derived. To assess the coreceptor usage of HXBaL, aninfection assay was performed using HOS.CD4 cells that ex-press either CCR5 or CXCR4. Surface coreceptor expressionwas verified by flow cytometry (data not shown). As deter-mined by extracellular p24 Gag protein, HXBaL infected cellsexpressing CCR5, but not CXCR4, as did an R5 control virus,uncloned HIV-1(Ba-L) (data not shown). HXB2-MCS, theinfectious molecular clone from which HXBaL was con-structed, infected cells expressing either CXCR4 or CCR5,confirming an X4 phenotype (HOS.CD4.CCR5 cells are leakyfor CXCR4 expression).

CCR5 antagonists are the most recent class of drugs fortreating HIV infection. One concern is that the use of suchcompounds might lead to the generation of more virulent HIVvariants that are able to use CXCR4 as a coreceptor. Wewanted to determine the kinds of genetic changes that wouldresult in a switch in coreceptor usage from CCR5 to CXCR4.To this end, a library of mutated HIV-1(Ba-L) env genes wasgenerated from a 1.75-kb env fragment encoding gp120 and theN-terminal region of gp41by PCR-based in vitro saturationmutagenesis (53) and ligated into pHXB2-MCS�env, whichlacks a functional env gene. The resultant plasmid library wasexpanded in Escherichia coli. DNA sequence analyses of theV3 regions of four individual clones indicated a point mutationfrequency of 0.6% (four mutations in 655 aa). Four libraries ofmutagenized env genes were transfected into COS-1 cells. Thesupernatants were collected at 72 h and extracellular p24 mea-

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sured to monitor virus production. Concentrations of p24ranged from 57 to 1,120 ng/ml.

HOS cells expressing CD4 and either CCR5 or CXCR4were infected by viruses from the four libraries and monitoredfor virus expression. In no case did we observe virus from cellsexpressing CXCR4. Virus was detected, however, from oneculture of cells expressing CCR5 at 31 days postinfection.Since HOS.CD4.CCR5 cells do express CXCR4 at low levels,we wondered whether the emergent virus, designated HXBaLm1.2, had adapted to use CXCR4. Indeed, HXBaLm1.2 in-fected the T-cell line CEM-SS, which is often considered to benegative for cell surface CCR5 (not shown). To determinewhether HXBaLm1.2 was using CXCR4 as a coreceptor,CEM-SS cells were pretreated with 1 �g/ml RANTES orSDF-1 or with no chemokines. HXBaLm1.2 infected SDF-1-treated CEM cells and weakly infected untreated cells, butRANTES blocked infection (not shown). In contrast, infectionof CEM-SS cells by HXB2, the X4 control virus, was blockedby SDF-1, but not by RANTES. These results suggested that

HXBaLm1.2 was not using CXCR4 as a coreceptor. HXBaL(the parental clone) did not detectably infect CEM-SS cellsunder any conditions, indicating that HXBaLm1.2 had an al-tered cell host range. HXBaLm1.2 from supernatants ofCEM-SS cells treated with SDF-1 was serially passaged inCEM-SS cells not treated with SDF-1. The serially passagedvirus was highly infectious (500 ng/ml supernatant p24) forCEM-SS cells in the absence or presence of added SDF-1 (notshown).

To determine whether HXBaLm1.2 could infect other T-celllines, we compared its infectivity for CEM-SS, H9, and SupT-1cells, all of which are often considered to not express surfaceCCR5. HXBaLm1.2 infected all three cell lines, in contrast toR5 viruses Ba-L and HXBaL (the parental cloned virus) (Fig.1A and B and data not shown). As expected, HXB2-MCS alsoinfected all three cell lines. These data confirmed an alterationin cell host range.

Chemokine blocking studies were performed to confirm thatHXBaLm1.2 remained R5 dependent. H9 and SupT-1 cells

FIG. 1. HXBaLm1.2 infects T-cell lines. CEM-SS cells (A) or H9 cells (B) were infected with the indicated viruses or not infected (control).H9 cells (C) or Sup-T1 cells (D) were infected with the indicated viruses without added chemokines or in the presence of CCR5 ligands (MIP-1�or RANTES) or the CXCR4 ligand SDF. p24 antigen in supernatants was measured by ELISA on the indicated days after infection. Results arefrom representative experiments. Error bars show standard deviations determined on replicate wells.



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were treated with 1 �g/ml SDF-1, RANTES, MIP-1�, or nochemokine and challenged with either HXBaLm1.2 or theparental virus, HXBaL (Fig. 1C and D). The R5 ligandsRANTES and MIP-1� blocked infection by HXBaLm1.2,while the X4 ligand SDF-1 had no effect. The parental virus didnot infect H9 or SupT-1 cells, whether treated or not. Consis-tent with the idea that HXBaLm1.2 was infecting these T-celllines through CCR5, we could detect CCR5 mRNA by reversetranscription-PCR of RNA from each cell line (data notshown). CCR5 surface protein expression was confirmed byflow cytometry (see below).

The range of coreceptors that HXBaLm1.2 could use wastested using HOS-CD4 cell lines expressing CCR1, CCR2b,CCR3, CCR4, CCR5, or CXCR4. In repeated experiments,HXBaLm1.2 only infected HOS-CD4 cells expressing CCR5(data not shown). These results, taken together with thechemokine blocking experiments, confirm that althoughHXBaLm1.2 is able to infect most T-cell lines, it remainsstrictly R5 dependent.

Sequence analysis of HXBaLm1.2. To identify mutations inHXBaLm1.2, we sequenced env from cDNA prepared frominfected cultures of CEM-SS and H9 cells and from virionspelleted from culture supernatants. Identical sequences wereobtained from all sources. There were three point mutations ingp120, His to Arg at aa 313 in the V3 loop, Ile to Val at aa 349,and Lys to Arg at aa 352. In addition, the comparison of HXBaLm1.2 with the parental virus, HXBaL, revealed an unex-pected change. The original construct contained only first 108aa of the Ba-L gp41. HXBaLm1.2, however, contained anadditional Ba-L-related substitution, encoding approximately

an additional140 aa of the Ba-L gp41, in place of the HXB2-derived sequence of the parental virus (Fig. 2). Both HXBaLm1.2 and the parental virus have the fusion domain and HR1of Ba-L. However, HXBaLm1.2 also has the HR2, the TMdomain, and 60 aa of the membrane-proximal CD of Ba-Lrather than that of HXB2, which the parental virus has. Thereason for this change is not clear, but it may have occurred byrecombination with Ba-L, which we speculate could have beenpresent as a contaminant at some point during the long timeperiod before viral production became evident. Figure 2 com-pares variant and parental gp41 sequences.

Identifying genetic determinants for altered tropism. Toidentify which mutations in HXBaLm1.2 determine the alteredtropism, smaller segments of env were subcloned and insertedback into the parental vector, pHXBaL. The constructs in-cluded pHXBaLm1.2 (which contains all the mutations), pHXBaLm3.4 (which contains only the three mutations in V3 andC3), and pHXBaLm6133 (which contains only the alteration ingp41) (Fig. 3). The clones were tested for their ability to infectT-cell lines. As summarized in the bottom panel of Fig. 3, allthe mutant viruses productively infected PM1 cells, indicatingthat they were replication competent. HXBaLm1.2 retainedthe ability to infect CEM-SS, H9, and Sup-T1 cells. HXBaLm3.4, with only the gp120 mutations, did not infect these T-celllines. In contrast, HXBaLm6133, which contains only the al-tered gp41, infected all three cell lines efficiently (consistentlyreaching supernatant p24 levels of 0.1 ng/ml). Thus, the al-terations in gp41 determine the altered host of HXBaLm1.2.

HXBaLm1.2 infects cells with low surface CCR5 expression.R5 viruses do not infect most T-cell lines. Since HXBaLm1.2

FIG. 2. HXBaLm1.2 gp41 DNA sequence. Shown is a comparison of the gp41-coding regions of HXBaLm1.2 and its T-cell-line-competentderived clone HXBaLm6133 with those of the parental virus and wild-type HIV-1(Ba-L). Functional domains are outlined with boxes. FD, fusiondomain. Arrows denote predicted sites of palmitoylation. Dashes indicate amino acid identity; letters indicate differences. The numbers above thealignment are the amino acid residue numbers based on HIV-1(Ba-L) gp160.



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remains R5 dependent yet can infect CEM-SS, H9, and Sup-T1cells, we wanted to confirm that these cells express CCR5 ontheir surface and determine their expression levels. As shownin the bottom panel of Fig. 3, CCR5 could be detected onT-cell lines by flow cytometry using Quantibrite PE beads andthe mean number of molecules of CD4 and CCR5 per cellestimated. PM1, CEM-SS, and H9 cells expressed similaramounts of cell surface CCR5, even though only PM-1 cells arepermissive for the parental virus. These results confirm previ-

ous findings by Lee et al. (37) that T-cell lines express surfaceCCR5, and they are in approximate agreement with the re-ported expression levels except for that of CCR5 by Sup-T1cells. Differences in Sup-T1 sublines could explain this discrep-ancy. The absolute levels of CCR5 expressed on these cell linesthus do not appear to be the sole determinants for permissivityfor the mutants, nor are they explained by absolute CD4 levels.Other factors, such as production of inhibitory chemokines, thedegree of CD4-CCR5 colocalization, or posttranslational mod-ifications of CCR5 or CD4, may account for these differences.

To test whether HXBaLm1.2 has a reduced dependence onhigh CCR5 levels, we constructed a cell line with regulatedCCR5 expression. U87 cells stably expressing CD4 were trans-fected with the plasmid pBI-EGFP-CCR5, which coexpressesCCR5 and enhanced green fluorescent protein from a tetracy-cline-sensitive bidirectional promoter. Increasing the doxycy-cline concentration increases expression of both enhancedgreen fluorescent protein and CCR5. As determined by flowcytometry using Quantibrite PE beads, increasing doxycyclinefrom 0.1 to 1,000 ng/ml increased CCR5 surface expressionfrom 5,000 to 80,000 molecules/cell (Fig. 4A). CCR5 expres-sion was leaky; the lower limit of expression was 4,500 mole-cules of CCR5 per cell. All cells expressed equivalent levelsof CD4. Viruses were tested for the ability to infect U87.DOX.CCR5 cells expressing different levels of CCR5. A re-duction from 80,000 CCR5 molecules/cell to 16,000 molecules/cell or lower caused a 50 to 60% decrease in the infectivity ofBa-L and the parental virus HXBaL (Fig. 4B). In contrast,there was no reduction of infection of cells expressing even thelowest levels of CCR5 per cell by HXBaLm1.2 or HXBaLm6133. None of the viruses could infect control cells thatexpressed no CCR5 (not shown).

To confirm that HXBaLm1.2 and HXBaLm6133 could in-fect cells expressing too few CCR5 molecules to support in-fection with the parental virus, infections were also carried outin JC.10 and JC.20 cells. JC.10 cells express only 2,000 mole-cules of CCR5/cell, and JC.20 cells express fewer than 700molecules/cell (64). We confirmed these numbers using Quan-tibrite PE beads. All viruses tested were able to infect JC.10cells (Fig. 4C). However, only HXBaLm1.2 and HXBaLm6133were able to infect JC.20 cells (Fig. 4D), confirming that HXBaLm1.2 can infect cells expressing reduced levels of CCR5.

HXBaLm1.2 has wild-type levels of virion gp120. The onlydifferences between HXBaLm6133 and the parental virusesBa-L and HXBaL are in the CD and the region of HR2 to justupstream from the LLP2/3 region (including the TM domain),respectively. In light of previous studies showing that the CD ofsimian immunodeficiency virus (SIV) gp41 can affect the den-sity of virion gp120 (36, 57, 71), we thought it possible that themutant viruses could infect cells with low levels of CCR5 be-cause of an increase in virion gp120, resulting in a higherprobability of gp120-CCR5 interactions. To examine this pos-sibility, virions were purified from chronically infected PM1cells and analyzed by Western immunoblotting. We adapted aprotocol that uses velocity rather than density gradients toisolate highly purified HIV-1 virions without disrupting theinfectivity of the virus (17). Viral protein inputs were normal-ized for p24. Densitometric analyses showed no consistentdifferences in gp120 content that could explain the increasedinfectivity of HXBaLm1.2 and HXBaLm 6133 (Fig. 5). Indeed,

FIG. 3. T-cell line competence is determined by gp41. The toppanel shows a linear representation of env genes of HXBaLm1.2 andderived viruses. Shaded boxes denote HXB2 sequence; white boxesdenote Ba-L sequence. gp120 and gp41 are not to scale. FD, fusiondomain. The bottom panel shows the ability of these viruses to repli-cate on the indicated cell lines. (A) Surface expression levels of CCR5and CD4 on the indicated T-cell lines were determined by flow cytom-etry using Quantibrite PE microbeads as described in Materials andMethods. (B) Relative levels of infection were judged by medium p24at 6 days after infection. Infections were performed in triplicate. �,p24 not detectable; �, 0. 050 to 0.500 ng/ml p24; ��, 0.501 to 0.750ng/ml p24; ��, �0.750 ng/ml p24.



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Ba-L generally contained the largest amount of gp120, and weconclude that the reduced dependence on CCR5 levels forentry is not due to elevated levels of gp120.

HXBaLm1.2 is not more resistant to CCR5 blocking agents.HIV-1 envelope gp120s can differ in their affinity for CCR5.We wondered whether the alteration in gp41 caused an in-creased affinity of gp120 for CCR5, thereby making the virusesless sensitive to entry inhibitors. To test this possibility, PM1cells were infected with mutant or parental virus in the pres-ence of serially diluted concentrations of the CCR5 bindinginhibitor TAK-779 or the CCR5 ligands �2 RANTES andMIP-1�. As seen in Fig. 6A and B (and data not shown), HXBaLm1.2 had sensitivities similar to those of the parental virusHXBaL, suggesting that the mutant gp120 does not have anincreased affinity for CCR5.

HXBaLm1.2 is more resistant to a fusion inhibitor. Trun-cations of the gp41 CD can decrease sensitivity to the fusionentry inhibitor T-20 (Enfuvirtide) (1, 68). We therefore askedwhether the altered HXBaLm1.2 gp41 would have a similareffect. Infectivity assays were performed with PM1 cells in thepresence of increasing T-20 concentrations. HXBaLm1.2 wasmuch less sensitive to T-20 than was the parental virus (Fig.6C). HXBaLm6133, which lacks the gp120 mutations of HXB

aLm1.2 but has the altered gp41, was likewise less sensitivethan the parental virus, indicating that the phenotype is deter-mined by the gp41. The gp41 of HXBaLm1.2 contains anadditional Ba-L-derived region compared to the parental virus.However, this alone cannot explain the increase in T-20 resis-tance, as pure Ba-L virus was even more sensitive to T-20 thanwas HXBaL. The decreased sensitivity to T-20 suggests thatthe gp41 alteration is causing accelerated fusion kinetics orincreased fusion efficiency, determined partially by the Ba-L-related substitutions within HR2 and the transmembrane re-gion and partially by the HXB2-related substitutions in the CD(Fig. 2 and 3).

Infection of T-cell lines is dependent upon CCR5. Inhibitionof infection of SupT1 and H9 cells by RANTES and MIP-1�(Fig. 1) strongly suggested that infection of these T-cell linesdepended on CCR5 as a coreceptor. To confirm this, we car-ried out infection of both cell lines in the presence of 500 pMand 500 nM TAK-779, a specific CCR5 antagonist. As shownin Fig. 7, infection was strongly inhibited by TAK-779, con-firming a dependence upon CCR5.

HXBaLm1.2 is CD4 independent. HIV variants that are ableto infect in the absence of CD4 have been described (19, 20, 29,

FIG. 4. HXBaLm1.2 infects cells expressing low levels of CCR5. (A) Surface expression of CD4 and CCR5 on U87.DOX.CCR5 cells wasmeasured as a function of doxycycline (Dox) concentration by flow cytometry using Quantibrite PE microbeads as described in Materials andMethods. Also shown are the mean channel fluorescence and percent positive cells for CCR5. (B) Infection of U87.DOX.CCR5 cells was measuredas a function of cell surface CCR5 expression. Medium p24 antigen was measured at day 3 after infection. Results are from a representativeexperiment. Standard deviations were determined from triplicate wells. (C) Infection of JC cells expressing different CCR5 levels was measuredby p24 at 7 and 10 days after infection. Results are from a representative experiment. Standard deviations were determined from triplicate wells.



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30, 35). Several studies have reported that truncations of theHIV or SIV gp41 CD can cause such a CD4-independent viralphenotype (6, 68). We tested the ability of HXBaLm1.2 toinfect CD4-negative cells. For a positive control, we selected aculture of HIV-1(Ba-L) with a decreasing ratio of CD4� toCD4� T cells and obtained a variant, BaL10001, that couldproductively infect CD4� cells (not shown). BaL10001 has sixmutations in the V1/V2 region, two in the C2 region, and threein the CD compared with published sequences of the parentalvirus Ba-L (not shown). To assess CD4 independence, infec-tions were carried out in Cf2th/synCCR5 canine thymus cellsand BC7/CCR5 cells, a CD4-CCR5� derivative of SupT1.HXBaLm1.2 and its derivative HXBaLm6133 infected bothcell types, similarly to BaL10001 but in contrast to the parentalvirus and to Ba-L itself (Fig. 8A and B). Thus, the changes ingp41 result in both a more efficient utilization of CCR5 and theability to infect cells lacking CD4.

HXBaLm1.2 has an altered neutralization profile. Severalstudies have shown CD4-independent viruses to be highly sen-sitive to sCD4 and to neutralizing antibodies that recognizebinding sites exposed after CD4 binding (15, 20, 21, 29, 34). Ifthe coreceptor binding site in HXBaLm1.2 gp120 was indeedmore exposed, HXBaLm1.2 might be similarly more neutral-ization sensitive. U373/CD4/MAGI cells expressing CCR5were used as target cells. Infection was quantified after 4 daysby chemiluminescent detection of �-gal. HXBaL and HXBaLm6133 were compared for neutralization sensitivity to severalMAbs and sCD4. HXBaL was neutralized by 2G12, a broadlyneutralizing MAb that binds a carbohydrate-dependent

FIG. 5. HXBaLm1.2 virions do not incorporate excess gp120. Viri-ons were purified by sucrose cushion and Optiprep (6 to 18% iodixa-nol) velocity gradient. Protein profiles were analyzed by immunoblot-ting with anti-gp120 and anti-p24 antibodies. PM1, uninfected negativecontrol; rgp120, purified recombinant gp120 protein. Bands werequantified by densitometric analysis. Shown is the ratio of gp120 to p24expression for the indicated viruses.

FIG. 6. HXBaLm1.2 is resistant to a fusion inhibitor but not entry inhibitors. PM1 cells were infected by HXBaL or HXBaLm1.2 and infectionmeasured 3 days later by medium p24 levels as described in Materials and Methods. Cells were treated with the indicated concentrations ofTAK-779 (A), �2 RANTES (B), or T-20 (C) as described in Materials and Methods. Results are from representative experiments. Error bars showstandard deviations determined from triplicate wells.



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epitope on gp120; by the 17B antibody, which recognizes con-served, discontinuous structures on the HIV-1 gp120 that areexposed upon CD4 binding; and by sCD4. HXBaLm6133 wasneutralized more efficiently by 17B and sCD4 (Fig. 9A and B),

similar to neutralization data reported for other CD4-indepen-dent viruses. In contrast, HXBaLm6133 was neutralized lessefficiently by 2G12 (Fig. 9C). The data confirm a conforma-tional change in gp120.

DISCUSSION

We characterized a variant of an R5 HXB2/Ba-L chimera(HXBaLm1.2) that is able to grow in T-cell lines to betterunderstand how structural changes in Env can lead to alter-ations in functional interactions with its coreceptors. HXBaLm1.2 and its clonal derivative HXBaLm6133 were able to in-fect cells expressing �700 molecules of surface CCR5/cell, incontrast to the parental viruses. Several lines of evidence sug-gested that the CCR5 binding site of gp120 of the mutantviruses is partially exposed without prior binding to CD4,thereby reducing the threshold level of CCR5 required totrigger fusion. First, the variant viruses could infect cells ex-pressing CCR5 and no CD4, while the parental viruses werestrictly CD4 dependent. Second, the variants were more sen-sitive than the parental viruses to neutralization by sCD4 or aMAb against gp120 sites exposed after binding to CD4.

A substitution within the coding region for gp41 was suffi-cient to cause the observed phenotypic changes. The pheno-type clearly requires the interaction of several regions withinthe gp41, however. The gp41 of HXBaLm1.2 differs from thoseof both the parental virus HXBaL and Ba-L itself, neither ofwhich is CD4 independent or able to infect cells expressinglimiting CCR5. The gp41 differs from the HXBaL gp41 inresidues 619 to 757, which are derived from Ba-L rather thanHXB2. This encompasses HR2 in the ectodomain and the TM

FIG. 7. Infection of T-cell lines is CCR5 dependent. T-cell lines were infected with HXB2 or HXBaLm6133 as described in Materials andMethods in the absence or presence of 500 pM or 500 nM TAK-779. Six days after infection, virus production was determined by ELISA of mediump24. (A) SupT1 cells were infected with BaLm6133; (B) SupT1 cells were infected with HXB2; (C) H9 cells were infected with BaLm6133; (D) H9cells were infected with HXB2. All samples were run in triplicate; error bars show standard deviations.

FIG. 8. HXBaLm1.2 infects cells not expressing CD4. Cells were in-fected with HXBaL, HXBaLm1.2, HXBaLm6133, or BaL10001 [derivedfrom HIV(BaL) by passage on CD4� cells cocultured with progressivelydecreasing levels of CD4� cells as described in Materials and Methods].Infection was monitored by medium p24 at the indicated times after infection.Target cells were BC7/CCR5 CD4-negative cells (A) or Cf2th/synCCR5CD4-negative cells (B). Results are from representative experiments. Errorbars show standard deviations determined from triplicate wells.



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region. Residue 758 to the carboxy terminus, including the CD,is derived from HXB2 rather than from Ba-L. Thus, bothchanges are necessary, suggesting that the two regions actcooperatively to affect gp120 and allow infection of cells ex-pressing low surface CCR5 levels.

The ability to infect cells expressing limiting CCR5 does notappear to be due to an increased affinity for CCR5, since bothmutants and the parental viruses are equally sensitive to theCCR5 blockers TAK-779, MIP-1�, and �2 RANTES. Further-more, although several studies have shown that some gp41mutations increase gp120 density on virions (9, 39, 69, 71), thisis not the case for HXBaLm1.2. This suggests that the changesin gp41 cause alterations of the tertiary or quarternary struc-ture of gp120. As judged by 50% inhibitory concentrations(IC50), HXBaLm6133 is approximately sevenfold more sensi-tive than the parental virus to neutralization by MAb 17B(which recognizes sites on gp120 exposed after binding toCD4), suggesting that these sites are more exposed on HXBaL

m1.2 gp120. Greater exposure of similar epitopes in the ab-sence of CD4 has been reported for several CD4-independentHIV variants, compared to their CD4-dependent counterparts(20, 21, 29, 34). Primary HIV-1 variants selected in vitro thatcan enter cells expressing reduced levels of CD4 are also neu-tralized more readily by sCD4 (34), similar to HXBaLm1.2.

HXBaLm1.2 has a sharply decreased sensitivity to the fusioninhibitor T-20, a peptide based on the gp41 HR2 sequence thatblocks the association of HR2 with HR1 during formation ofthe gp41 six-helix bundle (41, 65). In other studies, T-20 resis-tance of CD4-independent viruses has been attributed to morerapid fusion kinetics leading to decreased duration of exposureof the gp41 fusion intermediate (1, 52), making this likely toalso be the case for HXBaLm1.2 and HXBaLm6133. Theseresults are thus consistent with the increased sensitivity toneutralization by the 17B antibody and sCD4, to suggest pre-exposure of the coreceptor binding site.

Lentiviral envelope transmembrane glycoproteins, includingHIV-1 and SIV gp41, have an unusually long CD. There is nota clear understanding of the functions of this domain, butalterations in the CD can affect Env levels on the cell surfaceand incorporation into viral particles (9, 23, 25, 39, 52, 66, 71).Truncations of the CD can also affect fusogenicity and depen-dence on CD4 for viral entry (6, 68). Similarly, alterations inthe HXBaLm1.2 gp41 allow CD4-independent infection, butto our knowledge, the observation that determinants in the CDcan influence coreceptor use is novel. Several previous variantsthat can infect cells expressing low CCR5 levels have beenselected in vitro. Using the R5 entry inhibitor AD101, Trkolaet al. (61) selected a variant of a primary R5 isolate that wasable to use low CCR5 levels for entry. Dejucq et al. (16)described a JRCSF-C3 variant selected by in vitro passage inperipheral blood mononuclear cells that can infect the T-celllines Molt4 and Sup-T1, which have low cell surface CCR5. Inboth instances, determinants for the altered tropism werewithin gp120, altering its conformation to allow more efficientutilization of CCR5. Variants that can use low CCR5 levelsalso occur in vivo; determinants in env of a brain-derived iso-late apparently increase affinity for CCR5 and reduce depen-dence on CCR5 and CD4 (27).

In a study of the energetics of HIV gp120-CD4 binding,Myszka et al. demonstrated that there is considerable confor-mational flexibility within gp120 (47). However, once bound toCD4, gp120 gains conformational rigidity, leading to a meta-stable association with gp41 (28, 45) and a predisposition to-ward fusion (10, 24). Thus, we speculate that the gp41 changesdescribed here influence gp120 to adapt a more stable confor-mation in which preexposure of the coreceptor binding siteresults in more rapid formation of the six-helix bundle in gp41,resulting in more efficient membrane fusion.

Several lines of evidence underscore the importance ofCCR5 in HIV-1 transmission and indicate that CCR5 may bea promising target for therapeutic intervention in HIV infec-tion. First, primary infection with HIV-1 principally involvesR5 viruses (54, 58, 70). Second, individuals who are homozy-gous for a mutant CCR5 gene containing a deletion (�32) inthe coding region are strongly resistant to infection (50, 67),and those who do become infected display lower rates of dis-ease progression (14, 31). Finally, cross-sectional studies havecorrelated a natural capacity to produce high levels of MIP-1�

FIG. 9. HXBaLm1.2 is more sensitive to sCD4 and the neutralizingMAb 17b. U373/CD4/MAGI cells expressing CCR5 were infected withHXBaL or HXBaLm6133. Virus was treated with sCD4 (A), 17B (B),2G12 (C), or control IgG (D) as described in Materials and Methods.�-gal activity was measured at 4 days postinfection. Results are nor-malized for values from control samples with no antibodies or sCD4,and IC50 were determined. Results are from representative experi-ments. Error bars show standard deviations determined from triplicatewells. The table shows the calculated IC50, with the 95% confidenceintervals for CD4, 17B, and 2G12 indicated in parentheses.



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and MIP-1� with better clinical outcome in HIV infection (13).However, therapies that target CCR5 put HIV under selectivepressures. Although our isolation of a CD4-independent vari-ant of HIV-1 that uses low levels of CCR5 for entry occurredin vitro, it is likely that similar adaptations will occur in vivo,especially under adaptive pressure from entry or fusion inhib-itors, and could affect their efficacy. An ability to infect cellsexpressing low levels of CCR5 in vivo would expand the cel-lular host range in infected people and could make the virusmore pathogenic.

ACKNOWLEDGMENTS

Part of this work was supported by NIH grants R01 HL59796 andR01 AI060481-03 to A.D.

We thank Gregory Melikian for a critical reading of the manuscript.

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An Alteration of Human Immunodeficiency Virus gp41 Leads to