CD4-Chemokine Receptor Hybrids in Human ... - Journal of Virology

JOURNAL OF VIROLOGY, Sept. 1999, p. 7453–7466 0022-538X/99/$04.00⫹0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 73, No. 9

CD4-Chemokine Receptor Hybrids in Human Immunodeficiency Virus Type 1 Infection P. J. KLASSE,1* METTE M. ROSENKILDE,2 NATHALIE SIGNORET,1 ANNEGRET PELCHEN-MATTHEWS,1 THUE W. SCHWARTZ,2 AND MARK MARSH1 MRC Laboratory for Molecular Cell Biology and Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom,1 and Laboratory for Molecular Pharmacology, Department of Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen DK-2200, Denmark2 Received 14 January 1999/Accepted 28 May 1999

Most human immunodeficiency virus (HIV) strains require both CD4 and a chemokine receptor for entry into a host cell. In order to analyze how the HIV-1 envelope glycoprotein interacts with these cellular molecules, we constructed single-molecule hybrids of CD4 and chemokine receptors and expressed these constructs in the mink cell line Mv-1-lu. The two N-terminal (2D) or all four (4D) extracellular domains of CD4 were linked to the N terminus of the chemokine receptor CXCR4. The CD4(2D)CXCR4 hybrid mediated infection by HIV-1LAI to nearly the same extent as the wild-type molecules, whereas CD4(4D)CXCR4 was less efficient. Recombinant SULAI protein competed more efficiently with the CXCR4-specific monoclonal antibody 12G5 for binding to CD4(2D)CXCR4 than for binding to CD4(4D)CXCR4. Stromal cell-derived factor 1 (SDF-1) blocked HIV-1LAI infection of cells expressing CD4(2D)CXCR4 less efficiently than for cells expressing wild-type CXCR4 and CD4, whereas down-modulation of CXCR4 by SDF-1 was similar for hybrids and wild-type CXCR4. In contrast, the bicyclam AMD3100, a nonpeptide CXCR4 ligand that did not down-modulate the hybrids, blocked hybrid-mediated infection at least as potently as for wild-type CXCR4. Thus SDF-1, but not the smaller molecule AMD3100, may interfere at multiple points with the binding of the surface unit (SU)-CD4 complex to CXCR4, a mechanism that the covalent linkage of CD4 to CXCR4 impedes. Although the CD4-CXCR4 hybrids yielded enhanced SU interactions with the chemokine receptor moiety, this did not overcome the specific coreceptor requirement of different HIV-1 strains: the X4 virus HIV-1LAI and the X4R5 virus HIV-189.6, unlike the R5 strain HIV-1SF162, infected Mv-1-lu cells expressing the CD4(2D)CXCR4 hybrid, but none could use hybrids of CD4 and the chemokine receptor CCR2b, CCR5, or CXCR2. Thus single-molecule hybrid constructs that mimic receptor-coreceptor complexes can be used to dissect coreceptor function and its inhibition.

of HIV-1 binds to CD4 with high affinity. The critical residues in both molecules have been identified by mutagenesis and crystallography (10, 11, 32, 34, 40). The SU-binding site on CD4 is centered on the CDR2-like region of the N-terminal immunoglobulin (Ig)-like domain (D1), whereas residues in SU that make contact with CD4 are located in six distinct regions of the polypeptide (32). The binding of the envelope glycoprotein, Env, to CD4 induces conformational changes in the Env-CD4 complex (42, 43, 55) that appear to facilitate a subsequent interaction with the cognate chemokine receptor (32, 62, 65). The recruitment of a chemokine receptor could be a limiting step in the fusion process. It may promote fusion merely by placing the Env-CD4 complex in close proximity to the target cell membrane, or it may trigger a final fusogenic conformational change in the Env-CD4 complex. In order to explore the interactions between Env, CD4, and chemokine receptors in more detail we designed a series of CD4-chemokine receptor hybrids. When the orientation of CD4 to the rest of the hybrids is appropriate, such constructs might be predicted to enhance the functional affinity of SU for the chemokine receptor moieties by allowing two-point interactions on a single molecule. The hybrid constructs might also circumvent the potentially limiting step of coreceptor recruitment. The two most N-terminal Ig-like domains of CD4 (D1D2) were linked to the N termini of CXCR4, CCR5, CCR2b, and CXCR2. The latter two chemokine receptors are known to have only weak or no coreceptor function for HIV-1

The human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV) normally require the presence of both CD4 and a chemokine receptor at the cell surface for entry into a target cell. Different viral strains use distinct members of the chemokine receptor family as coreceptors (for reviews, see references 5 and 44). The chemokine receptors CCR5 and CXCR4, in particular, function prominently in HIV-1 infection. Viral strains are classified as R5, X4, or X4R5 according to whether they use CCR5, CXCR4, or both as coreceptors. While macrophage-tropic primary isolates preferentially use CCR5 and many T-cell-tropic isolates use both CXCR4 and CCR5, viruses adapted to growth in T-cell lines preferentially use CXCR4 (6, 66). Enveloped viruses enter cells by fusion with the plasma membrane or with the endosomal membrane after endocytosis (for a recent review, see reference 31). In HIV infection of model cell lines, the viral envelope fuses with the plasma membrane of the target cell (38, 46, 49, 61). Although the molecular mechanism of HIV fusion is not well understood, some of the interactions that precede it have been described in great detail. The outer envelope glycoprotein, gp120 or SU (surface unit), * Corresponding author. Mailing address: MRC Laboratory for Molecular Cell Biology, Dept. of Biochemistry & Molecular Biology, University College London, Gower St., London WC1E 6BT, United Kingdom. Phone: 44 171 419 3543. Fax: 44 171 380 7805. E-mail: [email protected]. 7453

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(5, 44), although they can function as coreceptors with CD4 in cell-to-cell fusion induced by an HIV-2 envelope glycoprotein (9). The hybrids between D1D2 of CD4 and CXCR4 were constructed with and without a Gly- and Asn-rich 39-residue spacer between the CD4 moiety and the N terminus of the chemokine receptor. In addition, the entire extracellular fourdomain fragment of CD4 was linked to the N terminus of CXCR4 (Fig. 1). The physiological ligand for CXCR4 is the CXC chemokine stromal cell-derived factor 1 (SDF-1) (7, 45), a peptide with a molecular mass of approximately 8 kDa. SDF-1 blocks X4 virus infection by two mechanisms: down-modulation of CXCR4 from the cell surface and competition with SU for binding to CXCR4 (2, 59). It could therefore be predicted that enhancement of SU-CXCR4 interactions will diminish the antiviral potency of SDF-1. A synthetic nonpeptide molecule, the bicyclam AMD3100, with a molecular mass of only 1 kDa, competes with both SDF-1 and the CXCR4-specific monoclonal antibody (MAb) 12G5 for binding to CXCR4. It also blocks X4 virus infection (15, 57, 58) by a mechanism that, as we demonstrate, does not involve down-modulation of CXCR4. In these and other regards we illustrate how hybrids between receptors and coreceptors can be useful tools in analyzing interactions with envelope glycoproteins and the antiviral mechanisms of coreceptor ligands. MATERIALS AND METHODS Cells. The cell line Mv-1-lu, derived from fetal mink lung epithelium, and Mv-1-lu cells stably expressing human CD4 (12), were obtained from the AIDS Reagent Project (ARP) of the United Kingdom Medical Research Council (Potters Bar, United Kingdom). The Mv-1-lu cells expressing wild-type CXCR4 and CD4 were previously described (59). All tissue culture media and reagents were from Gibco Ltd. (Paisley, United Kingdom). The Mv-1-lu cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, penicillin (10 IU/ml), and streptomycin (10 mg/ml). In addition, 1 mg of G418 per ml and 0.5 mg of hygromycin per ml were included for cell lines stably transfected with plasmids carrying the respective resistance markers. COS-7 cells were cultured in DMEM F12 containing 5% fetal calf serum, 2 mM glutamine, penicillin (10 IU/ml), and streptomycin (10 mg/ml). Virus. A stock of HIV-1LAI was prepared from chronically infected H9 cells provided by the ARP. The infectivity of this stock on Mv-1-lu cells expressing wild-type CXCR4 and CD4 was approximately 103 focus-forming units per ml. The X4R5 strain HIV-189.6 (17) and the R5 strain HIV-1SF162 (13), which had been cultured in peripheral blood mononuclear cells and yielded a 50% tissue culture infective dose on peripheral blood mononuclear cells of 106/ml, were donated by G. Simmons (The Institute of Cancer Research, London, United Kingdom). MAbs, recombinant proteins, and CXCR4 ligands. The MAb Q4120, which reacts with an epitope overlapping the SU-binding site in the N-terminal Ig-like domain, domain 1 (D1), of CD4 (24), was obtained from the ARP. The MAb 12G5 (20), directed to a discontinuous epitope, which involves the second extracellular loop of CXCR4 (8), was provided by J. Hoxie (University of Pennsylvania, Philadelphia, Pa.). The MAbs to HIV-1 Gag proteins 38:96K and EF7 (27) were obtained from the ARP. Q4120 and 12G5 were labeled with 125I as previously described (59). SDF-1␣ with an additional methionine at the N terminus (59) and the bicyclam AMD3100 (15, 57, 58) were donated by M. Luther (Glaxo-Wellcome Inc., Research Triangle Park, N.C.). Recombinant outer Env glycoprotein (SU) derived from the X4 strain HIV1LAI, ⬎90% pure, was obtained from the ARP. SU from the R5 strain HIV-1JRFL, ⬎95% pure, was generously donated by W. Olson and P. Maddon (Progenics Inc., Tarrytown, N.Y.). Both SU proteins were produced in Chinese hamster ovary (CHO) cells and had been characterized in CD4 and antibody binding assays. Construction of CD4-chemokine receptor hybrids. The signal sequence and the first two Ig-like domains (D1 and D2) of human CD4, from Met1 to Phe204 (54, 63), were fused to the N terminus of human CXCR4, yielding the hybrid designated CD4(2D)CXCR4. The corresponding linkages were made for CXCR2, CCR5, and CCR2b. CD4 fragments with overlaps to the N-terminal end of the respective chemokine receptors and the entire chemokine receptor genes with overlaps to the C-terminal end of the CD4 fragment were generated separately in initial PCRs with wild-type CD4 and the corresponding chemokine receptor cDNAs as templates. The primers were designed to include appropriate sites for restriction enzymes. The primer for the N-terminal end of CD4 was TAG AAG CTT ACC ATG AAC CGG GGA GTC CCT (sense). The primers

J. VIROL. for the C-terminal ends of the chemokine receptors were as follows: for CXCR4, ACC GAA TTC TTA GCT GGA GTG AAA ACT TGA (antisense); for CXCR2, CAC GAA TTC CTA TTA GAG AGT AGT GGA AGT (antisense); for CCR5, CAC GGA TCC TCA CAA GCC CAC AGA TAT TTC (antisense); and for CCR2b, CAC GGA TCC CTA TTA TAA ACC AGC CGA GAC TTC (antisense). The primers used to create the overlaps for fusing the CD4 and chemokine receptor moieties were as follows: for CD4(2D)CXCR4, GTG CTA GCT TTC ATG GAG GGG ATC AGT ATA TAC (sense) and GTA TAT ACT GAT CCC CTC CAT GAA AGC TAG CAC CAC GAT GTC (antisense); for CD4(2D)CXCR2, GTG CTA GCT TTC ATG GAA GAT TTT AAC ATG (sense) and CAT GTT AAA ATC TTC CAT GAA AGC TAG CAC CAC GAT GTC (antisense); for CD4(2D)CCR5, GTG CTA GCT TTC ATG GAT TAT CAA GTG TCA AGT (sense) and TCA TGA CAC TTG ATA ATC CAT GAA AGC TAG CAC CAC GAT GTC (antisense); for CD4(2D)CCR2b, GTG CTA GCT TTC ATG CTG TCC ACA TCT CGT TCT CGG (sense) and AGA TGT GGA CAG CAT GAA AGC TAG CAC CAC GAT GTC (antisense). The CD4 and chemokine receptor fragments were joined in a PCR ligation. The fusion constructs were then inserted into the HindIII-EcoRI-BamHI sites of the pTEJ8 vector (28), and the DNA sequence was confirmed. In addition, the CD4-D1D2 fragment was fused to CXCR4 via a spacer sequence of 39 residues, thus yielding the hybrid CD4(2D)-Sp-CXCR4. This spacer sequence was derived from the hinge B region of a cellulase from the fungus Humicola insolens (GGGSNNGGGN NNGGGNNNGG GGNNNGG GNN NGGGNTGGG) (courtesy of Helle Woldike, Novo Nordisk, Bagsvaerd, Denmark). The primers used for making CD4(2D)-Sp-CXCR4 were as follows: spacer-CXCR4, AAC ACC GGT GGC GGG ATG GAG GGG ATC AGT ATA TAC (sense); CXCR4-spacer, GTA TAT ACT GAT CCC CTC CAT CCC GCC ACC GGT GTT ACC (antisense); CD4-spacer, ATC GTG GTG CTA GCT TTC GGC GGT GGA AGC AAC AAT GGT (sense); and spacer-CD4, GTT GCT TCC ACC GCC GAA AGC TAG CAC CAC GAT GTC (antisense). Furthermore, the extracellular part of CD4 comprising all four Ig-like domains (D1 to D4), residues Met1 to Pro396, was directly fused to the N terminus of CXCR4. The primers used in making CD4(4D)CXCR4 were ACA TGT AGC CCC ATT ATG GAG GGG ATC AGT ATA TAC (sense) and GTA TAT ACT GAT CCC CTC CAT TGG CTG CAC CGG GGT GGA CCA TG (antisense). Fig. 1 illustrates the CD4-CXCR4 hybrids schematically. Transfection of CD4-chemokine receptor hybrid genes into Mv-1-lu cells. Mv-1-lu cells (2 ⫻ 106) were washed in HEBS buffer (20 mM HEPES, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, and 6 mM D-glucose [adjusted to pH 7.0]) and transferred to a 0.4-cm-path-length electroporation cuvette containing 10 ␮g of DNA in 250 ␮l of HEBS buffer. The cells were electroporated with a single pulse in a Gene Pulser (Bio-Rad, Hercules, Calif.) set to 250 ␮F, 400 V, and infinite resistance. Clones expressing the hybrids were selected by limiting dilution in medium containing 1 mg of G418 per ml. Flow cytometric analysis of antibody and SU binding. Cells grown to confluence were detached in 10 mM EDTA in phosphate-buffered saline (PBS) and transferred to U-bottomed 96-well plates (Costar, Cambridge, Mass.) at approximately 106 cells per well. All subsequent steps were performed at 4°C. Cells were spun and resuspended in 200 ␮l of fluorescence-activated cell sorter (FACS) wash buffer (FWB; 2% FCS and 0.02% NaN3 in PBS). The cells were incubated with primary antibody at various concentrations in 100 ␮l of FWB for 2 h on a shaker. Subsequently the cells were washed twice and then incubated for 45 min with fluorescein isothiocyanate-conjugated goat anti-mouse antibodies (Pierce and Warriner UK Ltd., Chester, United Kingdom) diluted 1/200 in FWB. After three more washes, the cell-bound fluorescence was detected with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif.). The relative MAb binding was calculated as the mean fluorescence intensity (mfi) for each concentration of MAb (MAb[XnM] in the formula below) divided by the mfi at the plateau of binding [MAb(plateau)], after subtraction from both of the background mfi for antigen-negative cells (bd), i.e., [mfiMAb[XnM] ⫺ mfibd]/[mfiMAb(plateau) ⫺ mfibd]. Binding of recombinant SU to Mv-1-lu cells was detected as previously described (41). Briefly, we measured the ability of recombinant R5 (HIV-1JR-FL) and X4 (HIV-1LAI) virus SU to compete with the MAbs Q4120 and 12G5 for the binding to the hybrids and wild-type CD4 or CXCR4. Cells (2 ⫻ 105) in 90 ␮l of FWB were preincubated with SU at a range of concentrations in 96-well plates at 4°C with shaking for 2 h. MAb in 10 ␮l was added to the cell suspension giving final concentrations of 15 nM for 12G5 and of 2.0 nM for Q4120. The incubation was then continued for 1 h. After two washes, antibody binding was detected by flow cytometry as described above. Electron microscopy. The ultrastructural localization of hybrid receptors was determined by the use of colloidal gold labeling of cryosections essentially as described previously (35). Two days after passage, Mv-1-lu cells stably expressing CD4(2D)CXCR4 or CD4(4D)CXCR4 were fixed for 100 min in 4% paraformaldehyde, washed, embedded in 10% gelatin, infiltrated with 2.3 M sucrose, and frozen in liquid nitrogen. Cryosections (approximately 60 nm thick) were labeled with the MAb Q4120 at 5 ␮g/ml and a goat anti-mouse antibody conjugated to 10-nm-diameter gold particles (British Biocell International, Cardiff, United Kingdom), or with the latter alone as a control, and then examined with a transmission electron microscope (EM 420; Phillips, Eindhoven, The Netherlands).

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FIG. 1. Schematic representations of CD4-CXCR4 hybrids. Wild-type CD4, including its four Ig-like domains, single transmembrane segment, and C-terminal cytoplasmic tail, is shown in schematic outline next to CXCR4, which has seven transmembrane segments. The CD4-CXCR4 hybrids are illustrated below. The two N-terminal Ig-like domains of CD4 were linked directly to the N terminus of CXCR4 [CD4(2D)CXCR4]. Similar CD4(2D) hybrids were also constructed with CCR2b, CCR5, or CXCR2 as the chemokine receptor moiety. As shown, the two N-terminal Ig-like domains of CD4 were also linked via a spacer to CXCR4 [CD4(2D)-Sp-CXCR4]. In addition, all four extracellular CD4 domains were linked to CXCR4 [CD4(4D)CXCR4].

Western blotting. Mv-1-lu cells stably expressing hybrids or wild-type CD4 and CXCR4 were lysed in 20 mM Tris-HCl buffer, pH 8, containing 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 ␮g each of chymostatin, leupeptin, antipain, and pepstatin per ml. The nuclei were spun down at 3,000 rpm for 10 min in a benchtop centrifuge. Approximately 20 ␮g of protein was loaded per lane in 10% sodium dodecyl sulfate-polyacrylamide gels. Electrophoresis and blotting under nonreducing conditions without preheating were carried out as previously described (48). The MAb Q4120 was used at a concentration of 7 ␮g/ml for detecting CD4 and hybrids, and antimouse IgG–peroxidase conjugate (Pierce and Warriner) was diluted 1/2,000. The blots were developed with enhanced chemiluminescence (Super Signal; Pierce and Warriner). Quantitative infectivity assay. Mv-1-lu cells stably expressing CD4, CXCR4, or hybrids were seeded in 96-well plates at 8 ⫻ 103 cells/well 24 h before challenge. The cells were incubated with 50 ␮l of a 1/5 dilution of HIV-1 for 3 h at 37°C. The inoculum was then aspirated and replaced with medium. After two more days of culture, the cells were fixed in cold methanol/acetone (1/1) and the viral Gag protein was detected (9) with the MAbs 38:96K and EF7 (27) followed by ␤-galactosidase-conjugated sheep anti-mouse antibody (Genosys, The Woodlands, Tex.). Then the substrate chlorophenol red ␤-D-galactopyranoside (Boehringer) at a concentration of 2 mM was incubated with shaking at 37°C for 3 h, and the absorbance was measured in a spectrophotometer at 562 nm. With this modification of the readout, the experiments to measure SDF-1␣ inhibition of infection were carried out as previously described (59). Similar experiments were performed to measure the inhibition of infection by the bicyclam AMD3100 (58). SDF-1␣ binding to hybrids and wild-type CXCR4 transiently expressed in COS-7 cells. SDF-1␣ was labeled with 125I by the oxidative iodination procedure

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and purified by high-performance liquid chromatography (52). Binding of 125Ilabeled SDF-1␣ to COS-7 cells transiently transfected by the calcium phosphate method (23) with CD4-CXCR4 hybrids and wild-type CXCR4 was measured as previously described (52, 60). Briefly, the transfected COS-7 cells were transferred to 24-well culture plates 1 day after transfection, i.e., 1 day before the binding experiments. The numbers of cells per well were adjusted to give 5 to 10% binding of the added radiolabeled ligand. Binding was performed for 3 h at 4°C in binding buffer (50 mM HEPES, 5 mM MgCl2, 1 mM CaCl2, and 0.5% bovine serum albumin) with increasing concentrations of cold ligand. The reactions were terminated by washing the wells four times in binding buffer supplemented with 0.5 M NaCl. All determinations were performed in duplicate, and the nonspecific binding was determined in the presence of 1 ␮M SDF-1␣. The data were analyzed with Inplot (GraphPad Software, San Diego, Calif.). Internalization of CD4-CXCR4 hybrids. Endocytosis assays on adherent cells were performed essentially as described previously (59). Briefly, cells were seeded in 16-mm-diameter wells in 24-well plates and grown for 2 days to a final density of 1 ⫻ 105 to 2 ⫻ 105 cells per well. The cells were cooled on ice, washed with binding medium (BM; see reference 59), and incubated for 2 h at 4°C with 250 ␮l of 0.3 nM 125I-labeled Q4120 antibody in BM. Subsequently, the cells were washed in BM to remove free antibody and then warmed by addition of 250 ␮l of BM (37°C) with or without SDF-1␣ (125 nM). At the indicated times, the cells were returned to 4°C and washed with cold BM. For each time point six wells were used. From half of the wells, the cells were collected directly in 400 ␮l of NaOH (0.2 M) and transferred to tubes for ␥-counting (as a measure of total cell-associated activity). To determine the intracellular activity, the remaining wells were rinsed twice with 0.5 ml of BM at 4°C and adjusted to pH 2.0 and then incubated twice for 3 min each time with 1 ml of the same medium to remove cell surface-bound antibody. The cells were harvested in NaOH as described above. The proportion of internalized antibody at each time point was determined by dividing the acid-resistant activity by the total cell-associated activity. We also monitored the down-modulation of CD4-CXCR4 hybrids by measuring their disappearance from the cell surface after incubation with or without chemokine. This allowed comparison with wild-type CXCR4, for which SDF-1induced endocytosis could not be measured directly since the prebinding of the MAb 12G5 interferes with SDF-1 binding (59). Cells plated in 16-mm-diameter wells were incubated at 37°C in BM with or without SDF-1␣, as indicated. After treatment, the cells were placed on ice, cooled by addition of 1 ml of ice-cold BM, and washed four times with ice-cold BM. Receptors expressed at the cell surface were then detected with iodinated 12G5 or Q4120 antibodies as previously described (59, 60). For detection with 12G5, the cells were washed for 5 min in cold BM adjusted to pH 3.0 and subsequently returned to pH 7.4 in cold BM, before incubation in 250 ␮l of 125I-labeled 12G5 at 1 nM for 2 h at 4°C. For detection with Q4120, the cells were directly labeled for 2 h at 4°C in 250 ␮l of BM containing 0.3 nM of 125I-labeled Q4120. Unlabeled Q4120 was included when an excess of the antibody over the antigen concentration was required. Subsequently, the cells were washed again in cold BM and harvested in 400 ␮l of 0.2 M NaOH, and the radioactivity was determined as described above.

RESULTS Expression of hybrids of CD4 and chemokine receptors in Mv-1-lu cells. Mv-1-lu cells were used for these studies because they are resistant to infection by HIV-1 when CD4 (12) or CXCR4 is expressed alone but show strong susceptibility when these two molecules are expressed together (59). Furthermore, the rate and extent of SDF-1␣- and phorbol ester-induced endocytosis of CXCR4 in Mv-1-lu cells are similar to those seen in T cell lines (59). Using the MAb Q4120 in immunofluorescence studies on nonpermeabilized cells, we found that CD4-CXCR4 hybrids were localized at the cell surface two days after transfection (data not shown). We established stable cell lines by G418 selection and confirmed flow cytometrically with Q4120 that the three CD4-chemokine receptor hybrids CD4(2D)CXCR4, CD4(2D)-Sp-CXCR4, and CD4(4D)CXCR4 (Fig. 1) were expressed. Examples of FACS profiles are shown in Fig. 2A. The anti-CXCR4 MAb 12G5 also bound to the stably expressed hybrids, and the ratios of the Q4120 to the 12G5 binding plateaus were similar for the three hybrids (data not shown). Since both Q4120 and 12G5 bind to epitopes that are sensitive to denaturation, we conclude that the CD4 and CXCR4 moieties of the hybrids folded correctly. Western blotting indicated that the relative molecular mobilities of the receptor hybrids and wild-type CD4 expressed by the Mv-1-lu lines were as expected (Fig. 2B). Q4120 was used

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FIG. 2. CD4-CXCR4 hybrid expression in Mv-1-lu cells. (A) Flow cytometric detection of stably expressed CD4 and CD4-CXCR4 hybrids on intact Mv-1-lu cells. The fluorescence intensity is depicted on the x axis, and the cell counts are on the y axis. The binding of the anti-CD4-D1 MAb Q4120 at a saturating concentration of 10 nM is illustrated. The labels 1 through 5 above the peaks indicate the following: 1, parental Mv-1-lu cells (mfi ⫽ 12.6); 2, wild-type CD4 (mfi ⫽ 882); 3, Mv-1-lu stably expressing CD4(2D)-Sp-CXCR4 (mfi ⫽ 159); 4, CD4(2D)CXCR4 (clone B; mfi ⫽ 580); and 5, CD4(4D)CXCR4 (mfi ⫽ 1340). (B) Western blot of lysates of Mv-1-lu cells expressing wild-type and hybrid receptors. Two CD4(2D)CXCR4-positive clones (clones B and H) were included. The CD4 moiety was detected with the MAb Q4120. Clone H had a similar level of cell-surface expression in flow cytometry to that of the CD4(2D)-Sp-CXCR4-expressing cells. Molecular standards (the masses are given in kilodaltons) migrated as indicated in the right-hand margin. The mass of the CXCR4 moiety predicted from the amino acid sequence is approximately 40 kDa; for two and four domains of CD4 the masses are 20 and 40 kDa, respectively.

to detect the antigens, since 12G5 does not recognize CXCR4 on Western blots. Under the nonreducing conditions used, the electrophoretic mobilities may not precisely reflect the molecular masses. Nevertheless, wild-type CD4 showed a relative mobility corresponding to approximately 55 kDa and CD4(2D) CXCR4 yielded a band at approximately 60 kDa. CD4(4D) CXCR4 gave a band corresponding to approximately 80 to 90 kDa, as expected for a CXCR4 hybrid containing the whole extracellular portion of CD4. However, the CD4(2D)Sp-CXCR4 showed no reactivity, even though the level of expression of this hybrid, as revealed by FACS analysis, was similar to that of the CD4(2D)CXCR4 Mv-1-lu clone H. This lack of reactivity may be due to epitope masking by the spacer on blotted antigen. To examine the cellular localization of the hybrids further, cells expressing CD4(2D)CXCR4 and CD4(4D)CXCR4 were processed for cryosectioning and electron microscopy. Ultrathin sections were labeled with the anti-CD4 MAb Q4120, followed by colloidal gold-conjugated anti-mouse antibodies. As shown in Fig. 3, the CD4(2D)CXCR4 and CD4(4D)CXCR4 hybrids were localized mainly on the plasma membrane, including on microvilli. Occasionally gold particles were seen in coated pits, small vesicles, and membrane-containing vesicles resembling multivesicular bodies. Staining was specific, because gold particles were rarely seen over the cytoplasm, nuclei, or mitochondria. No gold particles were observed on sec-

tions labeled with gold-conjugated secondary antibody in the absence of the primary MAb (Fig. 3C). We conclude that most CD4(2D)CXCR4 and CD4(4D)CXCR4 molecules were located on the plasma membrane, although some were associated with endocytic organelles. There was no indication of significant quantities of hybrids sequestered in intracellular compartments, although we cannot exclude the possibility of undetected misfolded molecules. To determine whether the Q4120 and 12G5 epitopes on the hybrids were presented similarly to those on wild-type molecules, we estimated the affinity of these MAbs for their respective CD4 and CXCR4 epitopes on the hybrids, using flow cytometry. Figure 4 shows the relative MAb binding as a function of Q4120 and 12G5 concentrations. The Mv-1-lu cells expressing wild-type CD4 and CXCR4, alone or together, or the hybrid CD4(2D)CXCR4, CD4(2D)-Sp-CXCR4, or CD4(4D) CXCR4 showed no significant differences in the concentrations yielding half-maximal binding of either Q4120 (0.4 to 1 nM) or 12G5 (2 to 4 nM [this agrees with the affinity given in reference 59]). These half-maximal binding concentrations approximate the equilibrium dissociation constant, Kd, of the binding reaction (for a derivation, see reference 30). The half-maximal MAb binding concentrations for clones expressing distinct levels of the same hybrid did not differ significantly (data not shown), which validates the approximation of the total MAb concentrations to the free MAb concentrations. Thus, as judged by these analyses, the

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FIG. 3. Ultrastructural localization of CD4-CXCR4 hybrids. The MAb Q4120, bound to its epitope on the first Ig-like domain (D1) of CD4 in sections of Mv-1-lu cells stably expressing CD4(2D)CXCR4 (A) or CD4(4D)CXCR4 (B), was detected with anti-mouse antibody conjugated to 10-nm-diameter gold particles. (C) Lack of nonspecific binding of gold-conjugated antibody to Mv-1-lu cells expressing CD4(4D)CXCR4. The section was incubated without primary antibody and only with the gold-conjugated second antibody. The scale bar represents 100 nm.

Q4120 epitope on CD4 is not compromised by being expressed in the context of the hybrids and does not obscure or conformationally perturb the 12G5 epitope. CD4-CXCR4 hybrids mediate HIV-1LAI infection. In order to determine whether the CD4-chemokine receptor hybrids can function as virus receptors, we challenged the hybrid-expressing Mv-1-lu cells with different strains of HIV-1. Infection

was determined by immunodetection of newly synthesized viral Gag protein and quantitated spectrophotometrically (see Materials and Methods). In each experiment the mean optical density (OD) of at least three wells was determined for each cellular clone and virus combination; in some experiments Mv-1-lu clones expressing different levels of the hybrid receptors were included, as indicated.

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FIG. 4. MAb binding to CD4 and CXCR4 epitopes on receptor hybrids. The relative MAb binding (see Materials and Methods) determined from flow cytometric analyses is plotted on the y axis as a function of MAb concentration, which is plotted on the x axis. The value of x corresponding to y ⫽ 0.5 is an approximation of the Kd for MAb binding. (A) Titration curves for the binding of the CD4 MAb Q4120 to Mv-1-lu cells expressing wild-type CD4 (䊐), wild-type CD4 together with wild-type CXCR4 (■), or the hybrid CD4(2D)CXCR4 (F), CD4(2D)-Sp-CXCR4 (Œ), or CD4(4D)CXCR4 (}). (B) Titration curves for the binding of anti-CXCR4 MAb 12G5 to Mv-1-lu cells expressing wild-type CXCR4 (䊐) or other receptors (symbols are as described for panel A).

When the CD4(2D)-chemokine receptor hybrids, CD4(2D) CXCR2, CD4(2D)CXCR4, CD4(2D)CCR2b, and CD4(2D) CCR5, were transiently expressed, only the cells expressing CD4(2D)CXCR4 were susceptible to infection by HIV-1LAI (data not shown). Likewise, after incubation of stably expressing lines with HIV-1LAI, HIV-189.6, and HIV-1SF162, the only hybrid conferring strong susceptibility to infection was CD4 (2D) CXCR4. The X4R5 virus HIV-189.6, which gave strong infections of CD4⫹ CXCR4⫹ and CD4⫹ CCR5⫹ Mv-1-lu cells, showed significant infection with the CXCR4 hybrid but not with the CCR5 hybrid. HIV-1SF162, although it infected CD4⫹ CCR5⫹ cells, failed to infect cells expressing any of the hybrid receptors, including CD4(2D)CCR5 (Fig. 5A). To determine whether infection was related to the levels of hybrid expression, we compared Q4120 binding on the hybrid-expressing Mv-1-lu cells by FACS. The mfi values at the saturating concentration of 10 nM of Q4120 differed for the clones: for CD4 (2D)CCR5, mfi was 100 to 140; for CD4(2D)CCR2b, it was 70; for CD4(2D)CXCR2, it was 40; and for CD4(2D)CXCR4, it was 140 to 200 for clone H and 400 to 500 for clone B. Since the two CD4(2D)CXCR4 clones showed little difference in their susceptibilities to infection, despite different expression levels, and since one of them (clone H) was close in expression level to the CD4(2D)CCR5 clone, the lack of coreceptor function for the CCR5 hybrid is unlikely to be due to its lower expression level. It is possible that some susceptibility to infection via CD4(2D)CCR2b and CD4(2D)CXCR2 might be detected in more sensitive infectivity assays or if clones with higher levels of expression were available. However, these constructs still did not give any susceptibility to infection when expressed transiently or in experiments with cell populations showing heterogeneous expression levels while undergoing G418 selection (data not shown). The HIV-1LAI infectivities on Mv-1-lu lines stably expressing CD4(2D)CXCR4, CD4(2D)-Sp-CXCR4, and CD4 (4D) CXCR4 were compared (Fig. 5B). In addition to the clones with the highest level of expression (illustrated in Fig. 2), others with lower expression levels were also included. The mean OD of different clones is plotted for each hybrid receptor: CD4(2D)CXCR4 (four clones), CD4(2D)-Sp-CXCR4 (two clones), and CD4(4D)CXCR4 (eight clones). The results indicate that the CD4(2D)CXCR4 hybrid functioned more efficiently for infection than the 4D hybrid. Four different CD4

(2D)CXCR4 and five CD4(4D)CXCR4 clones with a 10-fold expression range were retested for infection. The CD4(4D) CXCR4 hybrid gave weak infection that correlated with expression level (r ⫽ 0.7), whereas the CD4(2D)CXCR4 conferred stronger susceptibility that did not correlate with expression level (r ⫽ 0.002) (data not shown). We therefore selected clones of all three hybrids with Q4120 binding plateaus in FACS analysis ranging from 100 to 200 mfi. The susceptibilities of these clones to HIV-1LAI infection were compared. As shown in Fig. 5C, where the susceptibilities are expressed relative to that of the wild-type CD4⫹ CXCR4⫹ Mv-1-lu cells, the CD4(2D)CXCR4 hybrid functioned more efficiently than CD4 (2D)-Sp-CXCR4, which in turn was more efficient than CD4 (4D)CXCR4. Together these data indicate that the envelope protein on HIV-1LAI virions can interact with the CD4(2D) CXCR4 hybrid efficiently and that this interaction allows the conformational changes required for fusion. SU competition with anti-CD4 and -CXCR4 MAbs for binding to CD4-CXCR4 hybrids. As the CD4-CXCR4 hybrids exhibited different abilities to mediate infection, we examined their interactions with recombinant SU. Initially, we detected bound SU with an antiserum to its C-terminal region. The concentration of SULAI that yielded half-maximal binding to cells expressing CD4, both CD4 and CXCR4, and CD4 (2D) CXCR4 or CD4(2D)-Sp-CXCR4 was in the range from 1 to 3 nM (data not shown), demonstrating that the recombinant SU had the requisite conformation for high-affinity binding at least to CD4. However, since the C-terminal part of SU may influence the interaction with chemokine receptors (41), we used a competition assay in which the ability of X4 or R5 SU to impede the subsequent binding of MAbs to either the CD4 or CXCR4 epitopes was detected. In Fig. 6A, SULAI (X4) and SUJR-FL (R5) are shown to compete potently for binding to the Q4120 epitope on wild-type CD4 expressed together with CXCR4 as well as on the CD4-CXCR4 hybrid constructs. Similar curves were obtained for CD4 expressed alone or together with CCR5 (data not shown). Whereas SULAI and SUJR-FL proteins showed comparable binding to wild-type CD4, SULAI was marginally more potent than SUJR-FL in competing with Q4120 for binding to the hybrid receptors. The half-maximal inhibitory concentration, IC50, of SULAI competition with Q4120 for binding to wild-type CD4 was 8 nM; for the 2D hybrid constructs CD4(2D)CXCR4 and CD4(2D)-Sp-CXCR4

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FIG. 5. HIV infection of Mv-1-lu cells expressing CD4-chemokine receptor hybrids. (A) On the horizontal line the hybrids or receptors stably expressed by clones of Mv-1-lu cells are indicated. Cells were incubated with HIV-1LAI ( ), HIV-1SF162 ( ), or HIV-189.6 (■) for 3 h at 37°C. Bars represent averages ⫾ standard errors of means of OD at 562 nm (OD562) in the immunostaining assay from two experiments with at least three replicates in each. (B) The bars represent susceptibility to infection by HIV-1LAI. The hybrids and receptors, CD4 and CXCR4 (one clone), CD4(2D)CXCR4 (four clones), CD4(2D)-Sp-CXCR4 (two clones), CD4(4D)CXCR4 (eight clones), and CXCR4 (one clone), expressed in Mv-1-lu cells, are indicated under each bar. The bars show the means ⫾ standard errors of means from six to eight experiments with at least three replicates for each clone in each. (C) As in panel B, the susceptibility of Mv-1-lu cells to infection by HIV-1LAI is represented by blocks above the labels on the horizontal line. Only clones with similar expression levels have been selected (mfi ⫽ 100 to 200); three CD4(2D)CXCR4, two CD4(2D)-Sp-CXCR4, and four CD4(4D) CXCR4 clones were included. The relative OD shown on the y axis is the OD for the respective CD4-CXCR4 hybrid divided by the OD for CD4⫹ CXCR4⫹ after subtraction from both of the background OD for CXCR4. The bars represent the means ⫾ standard errors of means from four to five experiments.

,

the IC50s were 20 and 15 nM, respectively; for binding to CD4 (4D)CXCR4 it was 4 nM. Thus the affinity of monomeric recombinant SULAI for binding to the CD4 moiety was slightly increased for the 4D hybrid and somewhat reduced for the 2D constructs compared with wild-type CD4. Since HIV-1 infection was mediated more efficiently by the 2D than the 4D hybrid constructs (Fig. 5C), the SU affinity for CD4 as determined here does not appear to be a limiting factor in that process. To monitor interactions of SU with CXCR4, we measured SU competition with the CXCR4-specific MAb, 12G5. No significant SU competition with 12G5 was seen on the CXCR4expressing CD4⫹ cells (Fig. 6B). Previously, higher concentrations of SULAI gave ⬍25% block of 12G5 binding to T cell lines in a similar assay, in which the competition correlated

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with cell surface expression of CD4 (41). Lower density of cell-surface CD4 and lower CD4/CXCR4 ratios than on some T cell lines may explain the lack of 12G5 competition on the CD4⫹ CXCR4⫹ Mv-1-lu cells. SULAI and SUJR-FL competed with high affinity but variable, limited efficacy for binding to the 12G5 epitope on the CXCR4 moiety of the hybrid constructs (Fig. 6B). In all cases SULAI blocked the binding of 12G5 more efficiently than SUJR-FL, but this difference was negligible for the 4D hybrid. The inhibition of 12G5 binding by SULAI was more efficient (up to 60%) on the CD4(2D)CXCR4 hybrid than on the CD4(2D)-Sp-CXCR4 and CD4(4D)CXCR4 constructs (40 and 30%, respectively). The IC50 of SULAI in the competition with 12G5 for binding to the CD4(2D)CXCR4 construct was 20 nM, i.e., similar to that in the Q4120 competition for binding to CD4. Prebinding of Q4120 (50 nM) to the CD4 part of the CD4 (2D)CXCR4 hybrid did not significantly impair the binding of 125 I-labeled 12G5. SULAI at 100 nM gave 66% ⫾ 6% of the binding in the absence of competitor, SUJR-FL gave 86% ⫾ 1.3% binding, and 50 nM Q4120 gave 95% ⫾ 5% binding (averages from two experiments with triplicate samples in each). This indicates that the X4 SU competition is largely attributable to specific SU-CXCR4 interactions. Furthermore, it suggests that the ability of the R5 SUJR-FL to block the binding of 12G5 to CXCR4 was not solely due to steric hindrance resulting from its binding to the CD4 part of CD4 (2D)CXCR4. Inhibition of infection of hybrid-expressing cells by SDF-1␣ and AMD3100. Both the chemokine SDF-1␣ and the nonpeptide compound AMD3100 are known to inhibit HIV infection mediated by CXCR4. However, low concentrations of SDF-1␣, which blocked HIV-1LAI infection of CD4⫹ CXCR4⫹ Mv-1lu cells, slightly enhanced the infection of cells expressing the 2D hybrids (Fig. 7A). At higher concentrations, SDF-1␣ blocked the infection mediated by the 2D hybrids but less efficiently than for wild-type receptors. In contrast, the effects of the nonpeptide AMD3100 were very similar on the CD4⫹ CXCR4⫹ cells and those expressing the 2D hybrids, whereas it was markedly more potent in blocking infection mediated by the 4D construct (Fig. 7B). In conclusion, the clear differences in the ability of SDF-1␣ and AMD3100 in blocking HIV-1LAI infection mediated by wild-type and hybrid receptors indicate that these two compounds block by distinct mechanisms. However, these results raise the questions of whether SDF-1␣ binds with the same affinity to hybrids as to wild-type CXCR4 and whether the hybrids are internalized as efficiently in response to SDF-1␣ binding as wild-type CXCR4. SDF-1␣ binding to CXCR4 and hybrid receptors. Using 125 I-labeled SDF-1␣ as a tracer, we found that wild-type CXCR4 as well as the CD4-CXCR4 hybrid receptors bound SDF-1␣ with dissociation constants in the nanomolar range (Fig. 8A), similar to (14, 60) or somewhat lower than (25) what has previously been reported for CXCR4. The CD4 (4D) CXCR4 hybrid and wild-type CXCR4 bound SDF-1␣ very similarly in a monocomponent fashion. In contrast, both the CD4(2D)CXCR4 and the CD4(2D)-Sp-CXCR4 hybrid constructs gave competition curves with Hill coefficients of magnitude less than unity, indicating more than one binding mode for the ligand (Table 1). Two-component analysis showed that SDF-1␣ bound to the 2D hybrids with affinities corresponding to that observed for the wild-type receptor, i.e., with IC50s of 1.1 and 2.6 nM (compared with 1.3 nM for the wild-type receptor), as well as with higher affinities with IC50s of 0.10 and 0.11 nM (Table 1). The 12G5 antibody displaced approximately 60% of the radioactive SDF-1␣ from wild-type CXCR4 in a monocompo-

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FIG. 6. SU competition with the MAbs Q4120 and 12G5 for binding to CD4, CXCR4, and CD4-CXCR4 hybrids. After preincubation of cells with the various concentrations of SU, the binding of the MAb Q4120 to CD4 (A) or 12G5 to CXCR4 (B) was measured by FACS. The mfi in the flow cytometric analyses is indicated as a proportion of binding relative to that at the plateau of binding after subtraction of a background for the parental Mv-1-lu cells. The relative MAb binding expressed as a percent is plotted on the y axis, as a function of the SU concentration (shown on the x axis). F, X4 SULAI; 䊐, R5 SUJR-FL. The receptors and hybrids are indicated above the diagrams.

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FIG. 7. Inhibition of HIV-1 infection of Mv-1-lu cells expressing CD4 and CXCR4 or CD4-CXCR4 hybrids by SDF-1␣ and AMD3100. SDF-1␣ and AMD3100 inhibition of HIV-1LAI infection was assayed as described in Materials and Methods. The OD562 determined in the immunostaining assay is expressed as a proportion of that seen for cells infected in the absence of inhibitor after subtraction of the background obtained with nonpermissive cells (y axis). The bars are plotted above the concentrations of SDF-1␣ (A) and AMD3100 (B) and represent data for Mv-1-lu cells expressing CD4 and CXCR4, CD4(2D)CXCR4, CD4(2D)-Sp-CXCR4, and CD4 (4D)CXCR4. The means ⫾ standard errors of means from two to four experiments are shown.

nent fashion with an IC50 of 3.7 nM (Fig. 8B and Table 1). The 12G5 IC50 for the CD4(4D)CXCR4 hybrid was similar, i.e., 2.2 nM, but the maximal displacement of SDF-1␣ was greater, i.e., approximately 90% (Fig. 8B). Furthermore, the 12G5 displacement curves for the CD4(2D)CXCR4 and CD4(2D)-SpCXCR4 hybrid receptors were more shallow. Two-component analysis indicated an approximately equal distribution between a higher-affinity site and a site with the same affinity as on the wild-type receptor (Table 1). Thus, the radioligand binding studies showed that both SDF-1␣ and 12G5 bound with at least as high affinity to the CD4-CXCR4 hybrid constructs as to the wild-type CXCR4 receptor. Surprisingly, the two-component analysis of the shallow binding curves for the CD4(2D)CXCR4 and the CD4 (2D)-Sp-CXCR4 hybrid receptors indicated that for these constructs approximately half of the binding sites occur in a form showing 10-fold higher affinity than wild-type CXCR4 for SDF-1␣. SDF-1␣-induced down-modulation of CD4-CXCR4 hybrids. Although the affinity of SDF-1␣ for the hybrids was at least as high as that for the wild type, it remained a possibility that they

differed from the wild type in their capacity to undergo SDF1␣-induced down-modulation. Fig. 9A shows the efficient and rapid down-modulation from the cell surface of the CD4(2D) CXCR4, CD4(2D)-Sp-CXCR4, and CD4(4D)CXCR4 hybrids as a result of incubation with SDF-1␣ in a range of concentrations similar to those used for SDF-1␣ inhibition of infection. In this experiment the MAb 12G5 was used in order to allow comparison with wild-type CXCR4. Since this antibody and SDF1␣ partially compete for binding to CXCR4, SDF-1␣ was first allowed to bind and receptor internalization proceeded at 37°C. Then the amount of CXCR4 remaining at the cell surface was determined after acid stripping of the cell surfacebound SDF-1␣ (59). This method showed a similar downmodulation of hybrid and wild-type CXCR4 both as a function of SDF-1␣ concentration and kinetically (Fig. 9A and B). The presence of the Q4120 epitope on the CD4 moiety of the hybrids offered an alternative means of measuring the internalization of CXCR4 in response to SDF-1␣: Q4120 can be prebound to hybrid-expressing cells which are subsequently incubated with SDF-1␣, because the binding of 125I-labeled Q4120 does not interfere with the SDF-1␣ binding. Thus en-

TABLE 1. Binding constants for SDF-1␣ and the anti-CXCR4 MAb 12G5 determined by radioligand competition on COS-7 cells expressing CXCR4 and CD4-CXCR4 hybrid receptorsa SDF-1␣

12G5

Hybrid

Average IC50 (nM)

Hill coefficient

IC50 for high-affinity site, nMb

IC50 for low-affinity site, nMc

Bmax, fmol/105 cells (n)

Average IC50 (nM)

Hill coefficient

IC50 for high-affinity site, nMb

IC50 for low-affinity site, nMc

CXCR4 CD4(2D)CXCR4 CD4(2D)-Sp-CXCR4 CD4(4D)CXCR4

1.3 0.44 0.27 0.78

⫺1.1 ⫺0.79 ⫺0.65 ⫺1.0

0.10 (43) 0.11 (65)

1.1 (57) 2.6 (35)

66 ⫾ 24 (8) 18 ⫾ 5 (4) 17 ⫾ 3 (4) 34 ⫾ 7 (8)

3.7 0.70 0.55 2.2

⫺1.0 ⫺0.50 ⫺0.64 ⫺0.85

0.08 (54) 0.12 (51)

4.6 (46) 2.3 (49)

a Receptor constructs were transiently expressed in COS-7 cells, and competition binding experiments were performed by using 125I-labeled SDF-1␣ on whole cells as described in the text. IC50s and Hill coefficients were calculated from the sum curves. For competition curves having Hill coefficients with magnitudes of less than 0.8, a two-component analysis was performed and the IC50s of both the high- and low-affinity (corresponding to wild-type) sites are indicated. Bmax values are means ⫾ standard errors of the means. n, number of experiments. b The percent high-affinity sites to total sites is shown in parentheses. c The percent low-affinity sites to total sites is shown in parentheses.

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receptor hybrids and the only marginally lower affinity for wild-type CXCR4 do not explain the strong sensitivity to inhibition of infection of the CD4(4D)CXCR4-expressing cells. Since AMD3100 blocks 12G5 binding equally well after having bound at temperatures that will or will not allow endocytosis, it has been suggested that it does not induce downmodulation of CXCR4 (58). By use of the single-molecule CD4-CXCR4 hybrid receptors, we directly addressed this suggestion. The masking of the 12G5 epitope by AMD3100 (Fig. 10A) in an acid-resistant fashion (data not shown) precluded the use of 12G5 for measuring effects of AMD3100 on the cell surface expression of CXCR4. However, the MAb Q4120 could be used instead, since AMD3100 does not affect MAb binding to the CD4 moiety of the hybrids. As shown in Fig. 10B, we found no effect of AMD3100 on the cell surface expression of the hybrids. DISCUSSION

FIG. 8. SDF-1␣ binding to wild-type CXCR4 and CD4-CXCR4 hybrid receptors. 125I-SDF-1␣ binding displaced by SDF-1␣ (A) or the MAb 12G5 (B) from CXCR4 wild-type receptor (F), CD4(2D)CXCR4 (■), CD4(2D)-SpCXCR4 (䊐), and CD4(4D)CXCR4 (E) expressed transiently in COS-7 cells. For CD4(2D)CXCR4 and CD4(2D)-Sp-CXCR4 the curves are calculated from twosite competition binding analysis.

docytosis rates can be measured directly as percentages of hybrid molecules at the cell surface that become internalized over time (47). This approach also showed rapid induction of endocytosis of the three CD4-CXCR4 hybrids by SDF-1␣ (Fig. 9D), which reached steady state when 80 to 90% of the molecules were internal. The rates of endocytosis from two experiments with 250 nM SDF-1␣ were 7.0 to 7.2%/min for CD4 (2D)CXCR4, 7.2 to 8.2%/min for CD4(2D)-Sp-CXCR4, and 3.8 to 4.7%/min for CD4(4D)CXCR4. All results shown in Fig. 8 were obtained with the same clones as used for the experiment presented in Fig. 2A. Hence, the somewhat slower internalization of CD4(4D)CXCR4 than of the other two hybrids could be related to its higher expression level and may not translate into smaller numbers of molecules internalized per minute. Previously we showed that in T cells CD4 is not internalized with CXCR4 after SDF-1␣ binding to the latter (59). In contrast, when the hybrids were internalized, the CD4 moiety by necessity disappeared from the cell surface (Fig. 9C). However, it is noteworthy that this concomitant loss of both receptor and coreceptor nevertheless resulted in weaker inhibition of infection than down-modulation of CXCR4 alone (Fig. 7A). AMD3100 binding to hybrid receptors and effect on their cell surface expression. We assessed the AMD3100 binding to Mv-1-lu cell-expressed CXCR4 and hybrid receptors in a 125Ilabeled 12G5 competition assay. AMD3100 was titrated from 10⫺2 to 102 nM and allowed to bind to Mv-1-lu cells expressing wild-type CXCR4, CD4(2D)CXCR4, or CD4(4D)CXCR4 for 1 h at 4°C before the addition of 125I-labeled 12G5. The AMD3100 concentration yielding half-maximal 12G5 competition was approximately 5 nM for wild-type CXCR4 and 2 nM for CD4(2D)CXCR4 and CD4(4D)CXCR4 (Fig. 10A). Hence, the similar apparent AMD3100 affinities for the latter two

Evidence is accumulating that direct physical interactions between the viral outer Env protein, SU, CD4, and specific chemokine receptors are required in the fusion and entry of most HIVs (4, 25, 41, 62, 65). CD4 binding is known to induce conformational changes in the Env oligomeric complex (55). Furthermore, an epitope on SU that is induced by CD4 binding is implicated in interactions with the chemokine receptor (32, 62, 65). This suggests a chronology of events in which CD4 binding precedes interactions with the chemokine receptor. The recognition of the chemokine receptor by SU may serve as a trigger for further conformational changes in Env that are conducive to fusion. Alternatively, the chemokine receptor may be an additional point of contact for SU that brings it closer to the membrane. The distinct properties that we identified for three different CD4-CXCR4 hybrids shed light on several functions of the receptor-coreceptor complexes in HIV-1 entry. We found that hybrids between CD4 and CXCR4 could be expressed efficiently on the cell surface and that both the CD4 and chemokine receptor components of these hybrid molecules adopted conformations similar to those of their parent proteins. These hybrids were able to mediate infection by X4 HIV-1; moreover, their capacities to do so correlated with the efficiency of SU interactions with their CXCR4 moieties. The fusion activity of these hybrids may occur through an ability to act in trans, i.e., by an intermolecular mechanism in which one hybrid molecule provides the CD4 function and another provides the CXCR4 function. Complementation of CXCR4 with CD4(2D)CCR5 gave some susceptibility to X4 virus infection, indicating that a CD4 moiety on a noncognate hybrid can function together with a cognate nonhybrid chemokine receptor (data not shown). Although it is thus plausible that the hybrids can function in trans, several lines of evidence argue against this as a dominant mechanism. First, in an intermolecular mechanism, the CD4(4D)CXCR4 and CD4(2D)-SpCXCR4 hybrids might be expected to function as efficiently as the CD4(2D)CXCR4 hybrid, whereas our results demonstrated differential efficacy. Second, the level of hybrid expression at the cell surface correlated with infection mediated by CD4(4D)CXCR4 but not with CD4(2D)CXCR4-mediated infection. This suggests that a dominant intermolecular mechanism may only occur for the 4D hybrid. Third, the competition by X4 SULAI with the MAb 12G5 for binding to the hybrids suggests that one SU molecule can interact with both the CD4 and the CXCR4 moieties on one and the same 2D hybrid molecule. Fourth, the less potent antiviral effect of SDF-1␣ on the 2D hybrids than on wild-type CXCR4 may be suggestive of

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FIG. 9. SDF-1␣-induced internalization of CD4-CXCR4 hybrids. Mv-1-lu cells expressing wild-type CXCR4 (䊐) or the hybrids CD4(2D)CXCR4 (F), CD4 (2D)-Sp-CXCR4 (Œ), and CD4(4D)CXCR4 (}) were treated with increasing concentrations of SDF-1␣ for 30 min (A) or with 125 nM SDF-1␣ for up to 60 min (B through D). The means of triplicate values from one experiment are shown. The error bars, each representing 1 standard deviation, are sometimes hidden within the symbols. (A and B) Comparisons of the SDF-1␣-induced down-modulation of CXCR4 and hybrids from the cell surface. Panel A shows the dependence of down-modulation on SDF-1␣ concentration, and panel B shows down-modulation as a function of time. After incubation in BM with SDF-1␣ at 37°C, the cells were cooled down on ice, acid washed, and then labeled with 125I-12G5 at 4°C. The graphs show the cell-associated 12G5 binding for SDF-1␣-treated cells as a proportion of 12G5 binding on untreated cells for the indicated concentrations and time points. (C) SDF-1␣-induced down-modulation of CD4-CXCR4 hybrids detected with the anti-CD4 MAb Q4120. The cells were labeled with 125I-Q4120 at 4°C. The results are expressed as described for panels A and B. (D) Direct measurement of the endocytosis of CD4-CXCR4 hybrids detected with Q4120. Cells were incubated first on ice with 125I-Q4120 for 2 h and then in BM with SDF-1␣ at 37°C for the indicated period. Each time point indicates the acid-resistant (internalized) radioactivity as a proportion of the total cell-associated activity (y axis).

a mechanism in which both components of a single hybrid molecule are used (see below). Fifth, the R5 and X4R5 viruses did not infect CD4(2D)CCR5-expressing cells, indicating that an intermolecular mechanism was not active in those cases. However, the CXCR4 hybrids that functioned in HIV-1 entry did not enhance susceptibility to infection. Why these molecules were not more efficient than the wild-type receptors is unclear. One possibility is that the recruitment of CXCR4 into fusion complexes is not a limiting step. Alternatively, even the most permissive hybrids may be geometrically disadvantaged for interactions with the Env protein. The relative susceptibilities to infection suggest that such disadvantages would be less pronounced for the 2D hybrids, in particular, CD4(2D) CXCR4, than for CD4(4D)CXCR4. However, the two- to fourfold less potent SU competition with the anti-CD4 MAb on the 2D hybrids than on the wild-type CD4 and CD4(4D) CXCR4 (Fig. 6A) may indicate that conditions favoring simultaneous CD4 and CXCR4 interactions on the 2D hybrids are suboptimal. The CD4 hybrids with chemokine receptors other than CXCR4 did not provide receptor-coreceptor function for the X4, X4R5, or R5 strains tested (Fig. 5A): expressing CD4 in juxtaposition to a chemokine receptor did not overcome the specific chemokine receptor requirements for infection. We did observe some R5 SUJR-FL competition with 12G5 for binding to CD4(2D)CXCR4, which although it was weaker than that of X4 SULAI, suggests physical contact of the R5 SU with CXCR4. However, this R5-SU association with CXCR4 was not sufficient to allow infection by the R5 strain HIV-1SF162.

The chemokine receptor binding site on SU is likely to contain conserved elements from the interdomain bridging sheet (32, 51), which may be the basis for low-affinity, nonspecific chemokine receptor interactions. Other more variable adjacent segments, e.g., the V3 region (41, 62, 65), may determine receptor preference; however, the exposure of these sites on monomeric, recombinant SU may differ from that on virionassociated Env oligomers. The segments of CXCR4 and CCR5 that are necessary for their respective coreceptor functions are being mapped (3, 8, 16, 18, 19, 22, 36, 53). Whereas the second extracellular loop is most strongly implicated in the coreceptor function of CXCR4 in HIV-1LAI infection, other strains have additional requirements, such as for the N-terminal segment. This may explain why the CXCR4 hybrids functioned more efficiently for HIV1LAI than for HIV-189.6 in relation to the infection mediated by the wild-type molecules. Similarly, the lack of infection via the CCR5 hybrid even for the R5 virus HIV-1SF162 and the X4R5 virus HIV-189.6 may be attributed to differences in the importance of the N-terminal segment of the coreceptor. Since the capacity of HIV-189.6 to use CCR2b as a coreceptor is weak (17), we cannot answer the question of whether CD4(2D) CCR2b may be used with similar efficiency. It has recently been reported that a hybrid of all four domains of CD4 linked to CCR5 can function in infection of virus pseudotyped with Env of HIV-1JR-FL, HIV-2, and SIV (26), albeit at a 10-fold lower efficiency than wild-type molecules. Although this cannot be directly compared with the lack of detectable function of the CD4(2D)CCR5 hybrid for HIV-1SF-162, the possibility remains

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FIG. 10. AMD3100 binding to CXCR4 and CD4-CXCR4 hybrids. (A) Binding of AMD3100 to Mv-1-lu cells expressing CXCR4 (䊐), CD4(2D)CXCR4 (F), and CD4(4D)CXCR4 (}) detected by competition with 125I-12G5 binding. The 12G5 binding, expressed as a percentage of binding in the absence of AMD3100 (y axis), is shown as a function of the AMD3100 concentration (x axis). (B) The effect of AMD3100 binding on the cell surface expression of CD4-CXCR4 hybrids. Mv-1-lu cells expressing CD4(2D)CXCR4 (F and E) or CD4(4D) CXCR4 (} and {) were incubated with 100 nM AMD3100 in BM for up to 1 h. The cells were then cooled on ice, washed, and labeled on ice with 125I-12G5 (F and }) or 125I-Q4120 (E and {). The y axis represents the amount of cellassociated iodinated antibody for AMD3100-treated cells expressed as a proportion of antibody bound to untreated cells at the indicated time points. The data in panels A and B are from one of two similar experiments and represent the means ⫾ standard deviations of three replicates.

that a CD4(2D)CCR5 hybrid might give relatively efficient infection with specific R5 viruses. We and others have previously postulated two mechanisms for chemokine inhibition of HIV-1 infection: direct competition with SU for binding and down-modulation of the chemokine receptor from the cell surface (1, 2, 37, 59, 64). Here we found that SDF-1␣ inhibition of HIV-1LAI infection via the CD4-CXCR4 hybrids was weak compared with that via wildtype CD4 and CXCR4. The average affinity of SDF-1␣ for the 2D hybrids, which showed the least sensitivity to SDF-1␣, was two- to threefold higher than those for wild-type CXCR4 and CD4(4D)CXCR4. This affinity difference was in the opposite direction to a change that would most readily explain the relative insensitivity of the 2D hybrids to SDF-1␣, although it cannot be ruled out that the higher-affinity SDF-1␣ binding to these hybrids may have qualitatively different effects; for example, it might explain the weak enhancement of infection via CD4(2D)CXCR4 by SDF-1␣ at a low concentration (Fig. 7A). In contrast to the differences in inhibition of infection, the

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2D hybrids were similar to wild-type CXCR4 and CD4(4D) CXCR4 in their susceptibilities to SDF-1␣-induced endocytosis (Fig. 9). As the hybrids contain both the CD4 and coreceptor moieties within a single molecule, the possibility of downmodulating both the receptor and coreceptor from the surface of hybrid-expressing cells might have been expected to augment the inhibition of infection. On the contrary, we found weaker SDF-1␣ inhibition of infection via the hybrids than wild-type receptors; infection via CD4(2D)CXCR4, which was enhanced at the lowest SDF-1␣ concentration, was the most insensitive. However, we also found that infection did not correlate with the level of CD4(2D)CXCR4 cell surface expression, whereas it did for CD4(4D)CXCR4. Thus differences in the amounts of receptor at the cell surface that are required for infection may partly explain the insensitivity to SDF-1␣. The direct block of SU binding to CXCR4 may also differ between hybrids and wild-type CXCR4. SDF-1␣ may compete less efficiently the greater the capacity of SU to establish twopoint binding. In fact, the 12G5 competition experiments suggest that SU-CXCR4 binding ranks similarly to the relative SDF-1␣ insensitivity for wild-type CXCR4 and the three CD4CXCR4 hybrids. An additional possibility is that, for wild-type molecules, SDF-1␣ may block the formation of CD4-CXCR4 complexes. Such a mechanism may be hampered when the CD4 moiety is covalently linked to CXCR4. Unlike the larger molecule SDF-1, AMD3100 might not interfere with CD4-CXCR4 interactions. Indeed, AMD3100 gave similar inhibition of infection via wild-type receptor, CD4 (2D)CXCR4, and CD4(2D)-Sp-CXCR4. However, infection via CD4(4D)CXCR4 showed significantly greater sensitivity to AMD3100, which could not be attributed to any detectable differences in the binding of the bicyclam to this hybrid. One possible explanation is that AMD3100 blocks CXCR4 function irreversibly. It has been suggested that AMD3100 interacts with residues in transmembrane segment 4 of CXCR4 that might contribute to irreversible binding (33); furthermore, we found that AMD3100 binding to CXCR4 at the surface of Mv-1-lu cells was resistant to acid washes, suggesting irreversibility. CD4(4D)CXCR4 might be more sensitive to an irreversible block, because efficient infection via this hybrid required higher levels of cell-surface expression than for the other hybrids. The stronger AMD3100 inhibition of infection via CD4(4D)CXCR4 than via wild-type molecules or the 2D hybrids is also in keeping with these hybrids’ SU interactions. For, unlike the SDF-1␣ effect on infection, the antiviral potency of an irreversible blocker added to cells before exposure to virus is not likely to depend on the strength of the SUreceptor interactions. In conclusion, the distinct sensitivities of the wild-type and hybrid receptors to SDF-1␣ and AMD3100 inhibition of infection indicate that the modes of action of these two CXCR4 ligands differ in more than just their capacities to down-modulate CXCR4. From a practical point of view, the most efficiently functioning CD4-CXCR4 hybrids may prove useful in the construction of receptor-coreceptor-bearing virus-like particles in order to target HIV-1 Env-expressing cells, as has been achieved for CD4 and chemokine receptors (21, 39, 56). By varying the CD4 linkage it may be possible to overcome the nonfunctionality of the hybrids for R5 virus (26). However, the hybrids also offer experimental advantages: the covalent linkage of receptor to coreceptor allows measurement of SU-chemokine receptor interactions that are otherwise barely detectable. Furthermore, the coexpression of receptor and coreceptor as one polypeptide provides a means of varying the expression level of the two moieties at a constant stoichiometric ratio, which may prove valuable in the study of the dependence of infectivity on cell

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surface concentrations of the two receptor components (29, 50). Another possible use of the hybrids is in facilitating the cocrystallization of Env, CD4, and chemokine receptor for structural studies. We conclude that a simulation of complexes between receptors and coreceptors is feasible. The study of these molecules covalently linked in different manners may help elucidate their interactions with viral envelope proteins. ACKNOWLEDGMENTS We are very grateful to Michael Luther, Glaxo-Wellcome, Inc., Research Triangle Park, N.C., for SDF-1␣ and AMD3100, to W. Olson and P. Maddon, Progenics Inc., Tarrytown, N.Y., for providing recombinant JR-FL SU, to R. Tedder and P. Balfe, University College Medical School, London, for use of their P3 laboratory, and to Q. Sattentau, J. Hoxie, and W. Olson for critical comments on the manuscript. This work was supported by grants from the UK MRC and GlaxoWellcome, Inc., to M.M. and from the Danish MRC and the Danish AIDS Foundation to T.W.S. P.J.K. is an MRC Research Fellow. N.S. is supported by a European Union TMR Marie Curie Research Training grant (ERBFMBICT961751). REFERENCES 1. Alkhatib, G., M. Locati, P. E. Kennedy, P. M. Murphy, and E. A. Berger. 1997. HIV-1 coreceptor activity of CCR5 and its inhibition by chemokines: independence from G protein signaling and importance of coreceptor downmodulation. Virology 234:340–348. 2. Amara, A., S. L. Gall, O. Schwartz, J. Salamero, M. Montes, P. Loetscher, M. Baggiolini, J. L. Virelizier, and F. Arenzana-Seisdedos. 1997. HIV coreceptor downregulation as antiviral principle: SDF-1␣-dependent internalization of the chemokine receptor CXCR4 contributes to inhibition of HIV replication. J. Exp. Med. 186:139–146. 3. Atchison, R. E., J. Gosling, F. S. Monteclaro, C. Franci, L. Digilio, I. F. Charo, and M. A. Goldsmith. 1996. Multiple extracellular elements of CCR5 and HIV-1 entry: dissociation from response to chemokines. Science 274: 1924–1926. 4. Bandres, J. C., Q. F. Wang, J. O’Leary, F. Baleaux, A. Amara, J. A. Hoxie, S. Zolla-Pazner, and M. K. Gorny. 1998. Human immunodeficiency virus (HIV) envelope binds to CXCR4 independently of CD4, and binding can be enhanced by interaction with soluble CD4 or by HIV envelope deglycosylation. J. Virol. 72:2500–2504. 5. Berger, E. A. 1997. HIV entry and tropism: the chemokine receptor connection. AIDS 11:S3–S16. 6. Berger, E. A., R. W. Doms, E. M. Fenyo, B. T. Korber, D. R. Littman, J. P. Moore, Q. J. Sattentau, H. Schuitemaker, J. Sodroski, and R. A. Weiss. 1998. A new classification for HIV-1. Nature 391:240. 7. Bleul, C. C., M. Farzan, H. Choe, C. Parolin, I. Clark-Lewis, J. Sodroski, and T. A. Springer. 1996. The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry. Nature 382:829–833. 8. Brelot, A., N. Heveker, O. Pleskoff, N. Sol, and M. Alizon. 1997. Role of the first and third extracellular domains of CXCR-4 in human immunodeficiency virus coreceptor activity. J. Virol. 71:4744–4751. 9. Bron, R., P. J. Klasse, D. Wilkinson, P. R. Clapham, A. Pelchen-Matthews, C. Power, T. N. Wells, J. Kim, S. C. Peiper, J. A. Hoxie, and M. Marsh. 1997. Promiscuous use of CC and CXC chemokine receptors in cell-to-cell fusion mediated by a human immunodeficiency virus type 2 envelope protein. J. Virol. 71:8405–8415. 10. Choe, H., K. A. Martin, M. Farzan, J. P. Sodroski, N. P. Gerard, and C. Gerard. 1998. Structural interactions between chemokine receptors, gp120 Env and CD4. Semin. Immunol. 10:249–257. 11. Choe, H. R., and J. Sodroski. 1992. Contribution of charged amino acids in the CDR2 region of CD4 to HIV-1 gp120 binding. J. Acquir. Immune Defic. Syndr. 5:204–210. 12. Clapham, P. R., D. Blanc, and R. A. Weiss. 1991. Specific cell surface requirements for the infection of CD4-positive cells by human immunodeficiency virus types 1 and 2 and by simian immunodeficiency virus. Virology 181:703–715. 13. Collman, R., N. F. Hassan, R. Walker, B. Godfrey, J. Cutilli, J. C. Hastings, H. Friedman, S. D. Douglas, and N. Nathanson. 1989. Infection of monocyte-derived macrophages with human immunodeficiency virus type 1 (HIV1). Monocyte-tropic and lymphocyte-tropic strains of HIV-1 show distinctive patterns of replication in a panel of cell types. J. Exp. Med. 170:1149–1163. 14. Crump, M. P., J. H. Gong, P. Loetscher, K. Rajarathnam, A. Amara, F. Arenzana-Seisdedos, J. L. Virelizier, M. Baggiolini, B. D. Sykes, and I. Clark-Lewis. 1997. Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding

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