A Conformation-Specific Monoclonal Antibody Reacting with Fusion-Active gp41 from the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein

The gp41 subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein plays a major role in the membrane fusion step of viral infection. The ectodomain of gp41 contains a six-helix structural domain that likely represents the core of the fusion-active conformation of the molecule. A monoclonal antibody (MAb), designated NC-1, was generated and cloned from a mouse immunized with the model polypeptide N36(L6)C34, which folds into a stable six-helix bundle. NC-1 binds specifically to both the α-helical core domain and the oligomeric forms of gp41. This conformation-dependent reactivity is dramatically reduced by point mutations within the N-terminal coiled-coil region of gp41 which impede formation of the gp41 core. NC-1 binds to the surfaces of HIV-1-infected cells only in the presence of soluble CD4. These results indicate that NC-1 is capable of reacting with fusion-active gp41 in a conformation-specific manner and can be used as a valuable biological reagent for studying the receptor-induced conformational changes in gp41 required for membrane fusion and HIV-1 infection.     

Soluble CD23 in Human Immunodeficiency Virus Type 1 Infected Patients

The level of sCD23 produced in the course of human immunodeficiency virus (HIV) infection was measured in patients grouped according to the Centers for Disease Control by using an immunoradiometric assay. Soluble CD23 was evaluated in supernatants of peripheral blood mononuclear cell (PBMC) (10⁶ cells/ml) stimulated by phytohemagglutinin (PHA). Compared with healthy controls (m±S.D. = 1.0 ±0.34 U/ml, n = 7), higher values were observed in some of the patients of group II (asymptomatic) (m±S.D. = 2±1.33, n = 9) and some of the patients of group IV (AIDS) (m±S.D. = 1.3 ±1.40, n = 8). Those results prompted us to compare the plasma levels of sCD23 in group II and group IV HIV-infected patients and in healthy individuals. Soluble CD23 plasma levels in healthy patients (n = 42) ranged from 0 to 1.5 U/ml (m±S.D. = 0.9±0.33), in group II patients (n = 17) from 0 to 3 U/ml (m±S.D. = 0.92±0.83) and in group IV patients (n =73) from 0 to 2.9 U/ml (m±S.D. = 1.15±0.71). The differences between the patients and the healthy individuals were not statistically significant but individual sCD23 values higher than 2 U/ml were obtained in 6% of the group II patients and 16.7% of the group IV patients. Increased values of sCD23 were obtained in plasma from patients with secondary infectious diseases (groups IV-C1 and IV-C2) and from patients without secondary infectious diseases (group II, group IV-A and group IV-B). Elevated values of sCD23 were detected even in patients with low counts of CD4⁺ T cells and CD8⁺ T cells in their peripheral blood. sCD23 has numerous activities including control of IgE synthesis and cytokine-like properties. Our results show a disarray of sCD23 in HIV-infected patients which could be involved in drug reactions, allergic manifestations and the IgE-level increase. Further investigations should attempt to define the role of sCD23 in clinical manifestations of HIV infection.     

Stabilization of Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Trimers by Disulfide Bonds Introduced into the gp41 Glycoprotein Ectodomain

Biochemical and structural studies of fragments of the ectodomain of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane envelope glycoprotein have demonstrated that the molecular contacts between alpha helices allow the formation of a trimeric coiled coil. By introducing cysteine residues into specific locations along these alpha helices, the normally labile HIV-1 gp160 envelope glycoprotein was converted into a stable disulfide-linked oligomer. Although proteolytic cleavage into gp120 and gp41 glycoproteins was largely blocked, the disulfide-linked oligomer was efficiently transported to the cell surface and was recognized by a series of conformationally dependent antibodies. The pattern of hetero-oligomer formation between this construct and an analogous construct lacking portions of the gp120 variable loops and of the gp41 cytoplasmic tail demonstrates that these oligomers are trimers. These results support the relevance of the proposed gp41 structure and intersubunit contacts to the native, complete HIV-1 envelope glycoprotein. Disulfide-mediated stabilization of the labile HIV-1 envelope glycoprotein oligomer, which has been suggested to possess advantages as an immunogen, may assist attempts to develop vaccines.     

Antagonism to and Intracellular Sequestration of Human Tetherin by the Human Immunodeficiency Virus Type 2 Envelope Glycoprotein

Tetherin (CD317/BST-2), an interferon-induced membrane protein, restricts the release of nascent retroviral particles from infected cell surfaces. While human immunodeficiency virus type 1 (HIV-1) encodes the accessory gene vpu to overcome the action of tetherin, the lineage of primate lentiviruses that gave rise to HIV-2 does not. It has been previously reported that the HIV-2 envelope glycoprotein has a Vpu-like function in promoting virus release. Here we demonstrate that the HIV-2 Rod envelope glycoprotein (HIV-2 Rod Env) is a tetherin antagonist. Expression of HIV-2 Rod Env, but not that of HIV-1 or the closely related simian immunodeficiency virus (SIV) SIVmac1A11, counteracts tetherin-mediated restriction of Vpu-defective HIV-1 in a cell-type-specific manner. This correlates with the ability of the HIV-2 Rod Env to mediate cell surface downregulation of tetherin. Antagonism requires an endocytic motif conserved across HIV/SIV lineages in the gp41 cytoplasmic tail, but specificity for tetherin is governed by extracellular determinants in the mature Env protein. Coimmunoprecipitation studies suggest an interaction between HIV-2 Rod Env and tetherin, but unlike studies with Vpu, we found no evidence of tetherin degradation. In the presence of HIV-2 Rod Env, tetherin localization is restricted to the trans-Golgi network, suggesting Env-mediated effects on tetherin trafficking sequester it from virus assembly sites on the plasma membrane. Finally, we recapitulated these observations in HIV-2-infected CD4⁺ T-cell lines, demonstrating that tetherin antagonism and sequestration occur at physiological levels of Env expression during virus replication.Various stages of the replication cycle of primate lentiviruses can be targeted by host antiviral restriction factors (reviewed in reference 49). In addition to the well-characterized antiviral effects of members of the APOBEC3 family of cytidine deaminases, particularly APOBEC3G and -3F, and species-specific variants of tripartite motif family 5α, the release of nascent retroviral particles has recently been shown to be a target for a novel restriction factor, tetherin (CD317/bone marrow stromal cell antigen 2 [BST-2]) (31, 46). Tetherin is an interferon-inducible gene that was originally shown to impart a restriction on the release of mutants of human immunodeficiency virus type 1 (HIV-1) that lack a vpu gene (31, 46). In tetherin-positive cells, mature Vpu-defective HIV-1 particles are retained on the cell surface, linked to the plasma membrane (PM) and each other via protease-sensitive tethers, and can be subsequently endocytosed and accumulate in late endosomes (30, 31). Tetherin is not HIV specific and restricts the release of virus-like particles derived from all retroviruses tested (18), as well as those of filoviruses and arenaviruses (18, 19, 39).Tetherin is a small (181-amino-acid) type II membrane protein with an unusual topology that exists mainly as a disulfide-linked dimer (34). It consists of an N-terminal cytoplasmic tail, a transmembrane anchor, an extracellular domain that includes three cysteine residues important for dimerization, a putative coiled-coil, and finally a glycophosphatidyinosityl-linked lipid anchor (22) that is essential for restriction (31). Tetherin localizes to retroviral assembly sites on the PM (18, 31), and this unusual structure is highly suggestive that tetherin restricts virion release by incorporation into the viral membrane and cross-linking virions to cells. Such a mechanism would make tetherin a powerful antiviral effector that can target an obligate part of most, if not all, enveloped virus assembly strategies. Moreover, since tetherin restriction has no specific requirement for virus protein sequences, to avoid its action, mammalian viruses have evolved to encode several distinct countermeasures that specifically inhibit tetherin''s antiviral function.The Vpu accessory protein antagonizes tetherin-mediated restriction of HIV-1 (31, 46). In the presence of Vpu, tetherin is downregulated from the cell surface (2, 46) and is targeted for degradation (10, 13, 14), although whether these processes are required for antagonism of tetherin function is unclear (27). HIV-1 Vpu displays a distinct species specificity in that it is unable to target tetherin orthologues from rhesus macaques or African green monkeys (14, 25). This differential sensitivity maps to the tetherin transmembrane domain, particularly residues that are predicted to have been under high positive selection pressure during primate evolution (14, 16, 25). This suggests that tetherin evolution may have been driven in part by viral countermeasures like Vpu. Vpu, however, is only encoded by HIV-1 and its direct simian immunodeficiency virus (SIV) lineage precursors. The majority of SIVs, including the SIVsm, the progenitor of both HIV-2 and SIVmac, do not encode a Vpu protein (21). In some of these SIVs, tetherin antagonism was recently shown to map to the nef gene (16, 51). SIV Nef proteins, however, are generally ineffective against human tetherin because they target a (G/D)DIWK motif that was deleted from the human tetherin cytoplasmic tail sometime after the divergence of humans and chimpanzees (51). This raises the question of how HIV-2 is able to overcome human tetherin, as recent data show chronically HIV-2-infected CEM T cells have reduced tetherin levels on their surface (10).Interestingly, it has long been known that the envelope glycoprotein of certain HIV-2 isolates can stimulate the release of Vpu-defective HIV-1 virions from cells we now know to be tetherin positive (5, 6, 43). HIV and SIV Envs form trimeric spikes of dimers of the surface subunit (SU-gp105 in HIV-2/SIVmac and gp120 in HIV-1) that bind CD4 and the chemokine coreceptor and gp41 (the transmembrane [TM] subunit that facilitates fusion with and entry into the target cell). Envelope precursors (gp140 or gp160) are synthesized in the endoplasmic reticulum, where they become glycosylated and are exported to the surface via the secretory pathway (8). During transit through the Golgi apparatus and possibly in endosomal compartments, the immature precursors are cleaved by furin-like proteases to form mature spikes (15, 29). Multiple endocytosis motifs in the gp41 cytoplasmic tail lead to only minor quantities of Env being exposed at the cell surface at any given time (7, 40). Recent data demonstrated that the conserved GYxxθ motif, a binding site for the clathrin adaptor protein AP-2 (3), in the membrane-proximal region of HIV-2 gp41 is required to promote Vpu-defective HIV-1 release from HeLa cells (1, 32). Based on experiments with HIV-1/HIV-2 chimeric envelopes, an additional requirement in the extracellular component was suggested (1). In this study we set out to examine the Vpu-like activity of HIV-2 envelope in light of the discovery of tetherin. We demonstrate that the HIV-2 Env is a tetherin antagonist, and we provide mechanistic insight into the basis of this antagonism.     

Critical Amino Acids within the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein V4 N- and C-Terminals Contribute to Virus Entry

The importance of the fourth variable (V4) region of the human immunodeficiency virus 1 (HIV-1) envelope glycoprotein (Env) in virus infection has not been well clarified, though the polymorphism of this region has been found to be associated with disease progression to acquired immunodeficiency syndrome (AIDS). In the present work, we focused on the correlation between HIV-1 gp120 V4 region polymorphism and the function of the region on virus entry, and the possible mechanisms for how the V4 region contributes to virus infectivity. Therefore, we analyzed the differences in V4 sequences along with coreceptor usage preference from CCR5 to CXCR4 and examined the importance of the amino acids within the V4 region for CCR5- and CXCR4-tropic virus entry. In addition, we determined the influence of the V4 amino acids on Env expression and gp160 processing intracellularly, as well as the amount of Env on the pseudovirus surface. The results indicated that V4 tended to have a shorter length, fewer potential N-linked glycosylation sites (PNGS), greater evolutionary distance, and a lower negative net charge when HIV-1 isolates switched from a coreceptor usage preference for CCR5 to CXCR4. The N- and C-terminals of the HIV-1 V4 region are highly conserved and critical to maintain virus entry ability, but only the mutation at position 417 in the context of ADA (a R5-tropic HIV-1 strain) resulted in the ability to utilize CXCR4. In addition, 390L, 391F, 414I, and 416L are critical to maintain gp160 processing and maturation. It is likely that the hydrophobic properties and the electrostatic surface potential of gp120, rather than the conformational structure, greatly contribute to this V4 functionality. The findings provide information to aid in the understanding of the functions of V4 in HIV-1 entry and offer a potential target to aid in the development of entry inhibitors.     

Reevaluation of the Requirement for TIP47 in Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Incorporation

Incorporation of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins into assembling particles is crucial for virion infectivity. Genetic and biochemical data indicate that the matrix (MA) domain of Gag and the cytoplasmic tail of the transmembrane glycoprotein gp41 play an important role in coordinating Env incorporation; however, the molecular mechanism and possible role of host factors in this process remain to be defined. Recent studies suggested that Env incorporation is mediated by interactions between matrix and tail-interacting protein of 47 kDa (TIP47; also known as perilipin-3 and mannose-6-phosphate receptor-binding protein 1), a member of the perilipin, adipophilin, TIP47 (PAT) family of proteins implicated in protein sorting and lipid droplet biogenesis. We have confirmed by nuclear magnetic resonance spectroscopy titration experiments and surface plasmon resonance that MA binds TIP47. We also reevaluated the role of TIP47 in HIV-1 Env incorporation in HeLa cells and in the Jurkat T-cell line. In HeLa cells, TIP47 overexpression or RNA interference (RNAi)-mediated depletion had no significant effect on HIV-1 Env incorporation, virus release, or particle infectivity. Similarly, depletion of TIP47 in Jurkat cells did not impair HIV-1 Env incorporation, virus release, infectivity, or replication. Our results thus do not support a role for TIP47 in HIV-1 Env incorporation or virion infectivity.     

The Selection of Low Envelope Glycoprotein Reactivity to Soluble CD4 and Cold during Simian-Human Immunodeficiency Virus Infection of Rhesus Macaques

Envelope glycoprotein (Env) reactivity (ER) describes the propensity of human immunodeficiency virus type 1 (HIV-1) Env to change conformation from the metastable unliganded state in response to the binding of ligands (antibodies and soluble CD4 [sCD4]) or incubation in the cold. To investigate Env properties that favor in vivo persistence, we inoculated rhesus macaques with three closely related CCR5-tropic simian-human immunodeficiency viruses (SHIVs) that differ in ER to cold (ER_cold) and ER to sCD4 (ER_sCD4); these SHIVs were neutralized by antibodies equivalently and thus were similar in ER_antibody. All three SHIVs achieved high levels of acute viremia in the monkeys without alteration of their Env sequences, indicating that neither ER_cold nor ER_sCD4 significantly influences the establishment of infection. Between 14 and 100 days following infection, viruses with high ER_cold and ER_sCD4 were counterselected. Remarkably, the virus variant with low ER_cold and low ER_sCD4 did not elicit a neutralizing antibody response against the infecting virus, despite the generation of high levels of anti-Env antibodies in the infected monkeys. All viruses that achieved persistent viremia escaped from any autologous neutralizing antibodies and exhibited low ER_cold and low ER_sCD4. One set of gp120 changes determined the decrease in ER_cold and ER_sCD4, and a different set of gp120 changes determined resistance to autologous neutralizing antibodies. Each set of changes contributed to a reduction in Env-mediated entry. During infection of monkeys, any Env replication fitness costs associated with decreases in ER_cold and ER_sCD4 may be offset by minimizing the elicitation of autologous neutralizing antibodies.     

A V3 Loop-Dependent gp120 Element Disrupted by CD4 Binding Stabilizes the Human Immunodeficiency Virus Envelope Glycoprotein Trimer

Human immunodeficiency virus (HIV-1) entry into cells is mediated by a trimeric complex consisting of noncovalently associated gp120 (exterior) and gp41 (transmembrane) envelope glycoproteins. The binding of gp120 to receptors on the target cell alters the gp120-gp41 relationship and activates the membrane-fusing capacity of gp41. Interaction of gp120 with the primary receptor, CD4, results in the exposure of the gp120 third variable (V3) loop, which contributes to binding the CCR5 or CXCR4 chemokine receptors. We show here that insertions in the V3 stem or polar substitutions in a conserved hydrophobic patch near the V3 tip result in decreased gp120-gp41 association (in the unliganded state) and decreased chemokine receptor binding (in the CD4-bound state). Subunit association and syncytium-forming ability of the envelope glycoproteins from primary HIV-1 isolates were disrupted more by V3 changes than those of laboratory-adapted HIV-1 envelope glycoproteins. Changes in the gp120 β2, β19, β20, and β21 strands, which evidence suggests are proximal to the V3 loop in unliganded gp120, also resulted in decreased gp120-gp41 association. Thus, a gp120 element composed of the V3 loop and adjacent beta strands contributes to quaternary interactions that stabilize the unliganded trimer. CD4 binding dismantles this element, altering the gp120-gp41 relationship and rendering the hydrophobic patch in the V3 tip available for chemokine receptor binding.The entry of human immunodeficiency virus type 1 (HIV-1) is mediated by the viral envelope glycoproteins (9, 79). The HIV-1 envelope glycoproteins are synthesized as an ∼850-amino acid precursor, which trimerizes and is posttranslationally modified by carbohydrates to create a 160-kDa glycoprotein (gp160). The gp160 envelope glycoprotein precursor is proteolytically processed in the Golgi apparatus, resulting in a gp120 exterior envelope glycoprotein and a gp41 transmembrane envelope glycoprotein (16, 17, 66, 76). In the mature HIV-1 envelope glycoprotein trimer, the three gp120 subunits are noncovalently bound to three membrane-anchored gp41 subunits (32).HIV-1 entry involves the binding of gp120 in a sequential fashion to CD4 and one of the chemokine receptors, CCR5 or CXCR4 (1, 8, 15, 18, 25, 36). CD4 binding triggers the formation of an activated intermediate that is competent for binding to CCR5 or CXCR4 (29, 69, 73, 78). These chemokine receptors are G protein-coupled, 7-transmembrane segment receptors with relatively short N termini. The choice of chemokine receptors is dictated primarily by the sequence of a gp120 region, the third variable (V3) loop, that exhibits variability among HIV-1 strains and becomes exposed upon CD4 binding (4, 8, 10, 33, 37, 38, 49, 59, 75). X-ray crystal structures of CD4-bound HIV-1 gp120 have revealed that the gp120 “core” consists of a gp41-interactive inner domain, a surface-exposed and heavily glycosylated outer domain, and a conformationally flexible bridging sheet (38, 43, 79). In the CD4-bound state, the V3 loop projects 30 Å from the gp120 core, toward the chemokine receptor (38). The V3 loop in these structures consists of three elements: (i) conserved antiparallel β strands that contain a disulfide bond at the base of the loop; (ii) a conformationally flexible stem; and (iii) a conserved tip (37, 38). During the virus entry process, the base of the gp120 V3 loop and elements of the bridging sheet interact with the CCR5 N terminus, which is acidic and contains sulfotyrosine residues (12-14, 23, 24). Sulfotyrosine 14 of CCR5 is thought to insert into a highly conserved pocket near the V3 base, driving further conformational rearrangements that result in the rigidification of the V3 stem (37). The conserved β-turn at the tip of the V3 loop, along with some residues in the V3 stem, is believed to bind the “body” of CCR5, i.e., the extracellular loops and membrane-spanning helices. CCR5 binding is thought to induce further conformational changes in the HIV-1 envelope glycoproteins, leading to the fusion of the viral and target cell membranes by the gp41 transmembrane envelope glycoproteins.CCR5 binding involves two points of contact with the gp120 V3 loop: (i) the CCR5 N terminus with the V3 base and (ii) the CCR5 body with the V3 tip and distal stem (12-14, 23, 24, 37, 38). The intervening V3 stem can tolerate greater conformational and sequence variation, features that might decrease HIV-1 susceptibility to host antibodies (30). Despite amino acid variation, the length of the V3 loop is well conserved among naturally occurring group M (major group) HIV-1 strains (30, 42). This conserved length may be important for aligning the two CCR5-binding elements of the V3 loop. In addition to allowing optimal CCR5 binding, the conserved V3 length and orientation may be important for CCR5 binding to exert effects on the conformation of the HIV-1 envelope glycoproteins. We examine here the consequences of introducing extra amino acid residues into the V3 stem. The residues were introduced either into both strands of the V3 loop, attempting to preserve the symmetry of the structure, or into one of the strands, thereby kinking the loop. The effects of these changes on assembly, stability, receptor binding, and the membrane-fusing capacity of the HIV-1 envelope glycoproteins were assessed. In addition to effects on chemokine receptor binding, unexpected disruption of gp120-gp41 association was observed. Further investigation revealed a conserved patch in the tip of the V3 loop that is important for the association of gp120 with the trimeric envelope glycoprotein complex, as well as for chemokine receptor binding. Apparently, the V3 loop and adjacent gp120 structures contribute to the stability of the trimer in the unliganded HIV-1 envelope glycoproteins. These structures are known to undergo rearrangement upon CD4 binding, suggesting their involvement in receptor-induced changes in the virus entry process.     

Characterization of Simian-Human Immunodeficiency Virus Envelope Glycoprotein Epitopes Recognized by Neutralizing Antibodies from Infected Monkeys

We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein epitopes recognized by neutralizing antibodies from monkeys recently infected by molecularly cloned simian-human immunodeficiency virus (SHIV) variants. The early neutralizing antibody response in each infected animal was directed mainly against a single epitope. This primary neutralizing epitope, however, differed among individual monkeys infected by identical viruses. Two such neutralization epitopes were determined by sequences in the V2 and V3 loops of the gp120 envelope glycoprotein, while a third neutralization epitope, apparently discontinuous, was determined by both V2 and V3 sequences. These results indicate that the early neutralizing antibody response in SHIV-infected monkeys is monospecific and directed against epitopes composed of the gp120 V2 and V3 variable loops.     

Antibody-Dependent Cellular Cytotoxicity Directed against Cells Expressing Human Immunodeficiency Virus Type 1 Envelope of Primary or Laboratory-Adapted Strains by Human and Chimpanzee Monoclonal Antibodies of Different Epitope Specificities

The characteristics of antibody-dependent cellular cytotoxicity (ADCC) directed by a panel of human and chimpanzee antienvelope (anti-Env) monoclonal antibodies (MAbs) of different epitope specificities were studied; this was accomplished by using target cells expressing human immunodeficiency virus type 1 (HIV-1) Envs of either primary or laboratory-adapted strains. Human MAbs of similar apparent affinities (1 × 10⁹ to 2 × 10⁹ liters/mol) against either a “cluster II”-overlapping epitope of gp41 or against the CD4 binding site, V3 loop, or C5 domain of gp120 directed substantial and comparable levels of specific lysis against targets infected with laboratory-adapted strains of HIV-1. As expected, those MAbs specific for relatively conserved regions of Env generally exhibited ADCC activity against a broader range of HIV-1 strains than those directed against variable epitopes. Significant ADCC activities of selected MAbs against primary isolate Env-expressing cells were demonstrated. In addition, a new ADCC epitope in the V2 domain of gp120 was defined. CD56⁺ cells were demonstrated to be the effector cells in these studies by fluorescence-activated cell sorting followed by ADCC assays. Notably, all anti-Env MAbs tested in this study, including MAbs directed against each of the known neutralization epitope clusters in gp120, directed significant levels of ADCC against targets expressing Env of one or more HIV-1 strains. These results imply that many, if not most, HIV-1-neutralizing human Abs of high affinity (≥3 × 10⁸ liters/mol in these studies) and of the immunoglobulin G1 (IgG1) subclass (i.e., the predominate IgG subclass) are capable of directing ADCC. Since neutralizing Abs have been associated with long-term survival following HIV-1 infection, this suggests that ADCC activity may be beneficial in vivo.The in vivo role(s) of antibodies (Abs) that can direct antibody-dependent cellular cytotoxicity (ADCC) against human immunodeficiency virus type 1 (HIV-1) Env-expressing cells in vitro remains unclear. In ADCC, anti-Env Abs direct effector cells to kill target cells bearing HIV-1 envelope on their surfaces; this is accomplished via specific binding of the Abs’ antigen-binding sites to Envs and their Fc regions to Fc receptors on the effector cells. Broadly strain reactive, ADCC-directing Abs arise early in the immune response to HIV-1 infection in vivo (14) and may be partially responsible for the initial clearance of viremia.Earlier in the HIV-1 epidemic, concerns were raised that shed soluble gp120 in HIV-1-infected individuals might bind to CD4⁺ cells, including uninfected ones, and could target these cells for “innocent bystander” killing by ADCC (6). However, effector cells armed with serum Abs able to direct ADCC in vitro against either innocent bystanders or HIV-1-infected cells were found at highest frequency in asymptomatic, seropositive individuals; patients with AIDS-related complex and AIDS showed progressively diminished reactivities (20). Furthermore, in a recent study (1), the ability of monoclonal Abs (MAbs) against three distinct gp120 epitopes to direct ADCC against uninfected CD4⁺ cells to which rgp120_SF2 had been adsorbed (i.e., innocent bystanders) was demonstrated to be less efficient by at least an order of magnitude than their ability to direct ADCC against HIV-1-infected cells.The existing data from in vivo studies (reviewed in reference 1) supports the efficacy, rather than the pathogenicity, of ADCC-directing Abs against HIV-1. Consistent with this data is our recent characterization of two MAbs, 42F and 43F, isolated from a long-term survivor of HIV-1 infection (1); these MAbs directed significant levels of ADCC and defined a new, conserved ADCC epitope in the C5 domain of HIV-1 gp120. Preliminary evidence indicated that concentrations of 42F- and 43F-like Abs in the serum of the donor were in the range required to direct high levels of ADCC, and these MAbs were shown to bind both oligomeric primary-isolate and laboratory-adapted Env efficiently (1).Because of the potential importance of ADCC-directing Abs against HIV-1, in this study we have evaluated ADCC directed against cells expressing HIV-1 Envs of primary or laboratory-adapted strains by a panel of human and chimpanzee anti-Env MAbs of different epitope specificities. Significant ADCC activities of selected MAbs against primary-isolate Env-expressing cells were demonstrated, and a new ADCC epitope in the V2 domain of gp120 was defined. Finally, a MAb’s ability to direct ADCC against a specific target cell type was shown to be dependent on additional factors beyond its ability to efficiently bind antigen on the target cell and its possession of an Fc region of the appropriate isotype to engage FcγR on effector cells.     

Vaccine-Induced Linear Epitope-Specific Antibodies to Simian Immunodeficiency Virus SIVmac239 Envelope Are Distinct from Those Induced to the Human Immunodeficiency Virus Type 1 Envelope in Nonhuman Primates

To evaluate antibody specificities induced by simian immunodeficiency virus (SIV) versus human immunodeficiency virus type 1 (HIV-1) envelope antigens in nonhuman primate (NHP), we profiled binding antibody responses to linear epitopes in NHP studies with HIV-1 or SIV immunogens. We found that, overall, HIV-1 Env IgG responses were dominated by V3, with the notable exception of the responses to the vaccine strain A244 Env that were dominated by V2, whereas the anti-SIVmac239 Env responses were dominated by V2 regardless of the vaccine regimen.     

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

Chemokine receptor CXCR4 (also known as LESTR and fusin) has been shown to function as a coreceptor for T-cell-tropic strains of human immunodeficiency virus type 1 (HIV-1). We have developed a binding assay to show that HIV envelope (Env) can interact with CXCR4 independently of CD4 but that this binding is markedly enhanced by the previous interaction of Env with soluble CD4. We also show that nonglycosylated HIV-1_SF-2 gp120 or sodium metaperiodate-treated oligomeric gp160 from HIV-1₄₅₁ bound much more readily to CXCR4 than their counterparts with intact carbohydrate residues did.In the recent past, several members of the family of chemokine receptors have been identified as cofactors for human immunodeficiency virus type 1 (HIV-1) entry (1, 6, 8, 10). Specifically, CCR5 (as well as CCR3 and CCR2b in some instances) has been shown to mediate entry of viruses characterized as macrophage tropic or dual tropic (1, 5–8), while CXCR4 has been shown to mediate entry of T-cell-tropic or dual-tropic strains (7, 10). While several ligands have been found for CCR5, CXC chemokine stromal derivative factor (SDF1) remains the only known ligand for CXCR4 (4, 24). Coimmunoprecipitation studies have shown that HIV-1 Env from T-cell-tropic strains forms a complex with CD4 and CXCR4 (18), but the nature of the binding events leading to the formation of this complex and the possibility of a direct interaction between HIV Env and CXCR4 remained speculative. Data from Hesselgesser et al. (15) have more recently shown that gp120 from the T-cell-tropic strains IIIB or BRU was able to compete with SDF1 for binding to CXCR4 in hNT cells (a neuronal CD4-negative cell line), indicating the possibility of a direct interaction between CXCR4 and gp120, but no information was presented on the relevance of the interaction with CD4. Other data have shown that gp120 from macrophage-tropic strains of HIV might be able to bind directly to CCR5 and that the affinity for binding between the two molecules can be increased significantly by the presence of soluble CD4 (sCD4) (34), although this effect could not be reproduced by a different group (32).We have performed the following studies to determine if HIV Env binds to CXCR4 independently of CD4 and, if so, what would be the effect of previous binding of HIV Env to sCD4.

CD4-independent binding of HIV Env to CXCR4.

The phenotypes of the T-cell lines CEM-SS and Jurkat 25 (J25) were evaluated with respect to surface expression of both CD4 and CXCR4. J25 clone 22F6 cells (3, 21) were grown in complete medium (RPMI 1640, 2% penicillin-streptomycin, 2% l-glutamine; BioWhittaker, Walkersville, Md.) containing heat-inactivated 10% fetal calf serum at 37°C in a 5% CO₂ atmosphere. CEM-SS is a T-cell line that was obtained from the AIDS Research and Reference Reagent Program and maintained in complete medium. CEM-SS cells were derived from a human lymphoblastoid tumor (22, 23). Commercial monoclonal antibody (MAb) to CD4 (mouse immunoglobulin G2a [IgG2a], clone S3.5), fluorescein isothiocyanate (FITC) labeled, and the necessary isotypic controls were obtained from Caltag Laboratories (San Francisco, Calif.). Mouse MAb 12G5 against CXCR4 was raised in BALB/c mice and has been described previously (9). Goat anti-mouse IgG–FITC was purchased from Becton Dickinson (San Jose, Calif.). Flow cytometric analysis was performed on a Becton Dickinson FACScan cytometer equipped with a 15-mW argon laser emitting at 488 nm. Dead cells were detected on the basis of their scatter and eliminated from the analysis. Live cells (10,000) were analyzed for each marker. CXCR4 surface expression was determined by washing the cells taken in logarithmic growth phase with phosphate-buffered saline (PBS) containing 1% horse serum and incubating them with 10 μl of 12G5 antibody/100 μl (0.16 mg/ml) at 4°C for 30 min. The cells were then washed again in PBS, and a secondary goat anti-mouse IgG–FITC (Becton Dickinson) was incubated with the cells for another 30 min at 4°C. Finally, the cells were washed with PBS and fixed with 2% paraformaldehyde. As a control, equal amounts of mouse IgG2a (the same isotype as 12G5) were used. Both cell lines expressed significant levels of CXCR4 on their surfaces (Fig. ​(Fig.1),1), but only CEM-SS had measurable levels of surface CD4. This characteristic of the phenotype of J25 cells, with respect to CD4 expression, has been reported before (3). To assess binding of HIV Env to CXCR4, the following binding assay was developed. Oligomeric gp160 (ogp160) was purified from cell cultures (obtained from T. C. Van Cott (Henry M. Jackson Foundation, Rockville, Md.) infected with HIV₄₅₁ (17). The cells were washed once with PBS and then incubated with ogp160 for 1 h at 37°C in RPMI medium. The cells were washed again in PBS and incubated with 10 μg of human MAb 1331A [IgG3(λ)]/ml, which is specific for the C terminus of gp120 (i.e., amino acids 510 to 516 of HIV_LAI), or with a human MAb against p24 (MAb 71-31) as a control (12) for 30 min at 4°C. The secondary antibody was a goat anti-human IgG phycoerythrin labeled (Caltag). The cells were fixed in 2% paraformaldehyde, and the fluorescence intensity was determined by flow cytometry. Background was obtained by adding MAb 1331 and goat anti-human IgG, phycoerythrin labeled, to the cells in the absence of ogp160. The results of the binding assay with ogp160 from HIV₄₅₁ and both cell lines are shown in Fig. ​Fig.2A.2A. By using the high-affinity human MAb 1331A against the C-terminal region of gp120, our assay was able to detect significant binding of the ogp160 molecule to the surfaces of both cell lines even at concentrations of only 88 nM. The very high relative affinity of MAb 1331A for the gp120 molecule appears to be critical to demonstrate this interaction, as other antibodies with lower relative affinities for gp120 were incapable of detecting this low-level binding (data not shown). The binding of ogp160 to the CD4-expressing CEM-SS cells was several orders of magnitude higher than that to the J25 cells. To prove the specificity of the binding assay for CXCR4, a synthetic form of SDF1 was produced and tested for its ability to block infection by the HIV-1 strain NL_4-3 in HeLa CD4-positive long terminal repeat (LTR)-LacZ cells. These data have been published elsewhere (2). SDF1 synthesis and composition have been described previously (24). Exposure of J25 cells to SDF1 was shown to produce a dose-dependent blockage of the binding of ogp160 to the surfaces of the J25 cells (Fig. ​(Fig.2B),2B), indicating the specific nature of the assay. Open in a separate windowFIG. 1Phenotype analysis of CEM-SS and J25 cell lines. Thin solid line, background; thick solid line, CD4; dashed line, CXCR4.Open in a separate windowFIG. 2(A) Binding of ogp160 from HIV₄₅₁ to the surfaces of CEM-SS or J25 cells. Fluorescence intensity is expressed on a logarithmic scale on the x axis, with each line representing one-half log. Concentrations of ogp160 are shown at the right of each graph. The experiments were done in duplicate to ensure consistency of results. (B) Effect of RANTES (250 nM) or increasing amounts of SDF1 (up to 250 nM) on binding of ogp160 (355 nM) to J25 cells. The results are expressed as mean channel fluorescence. Experiments were repeated twice to ensure consistency of results.To further test the fact that HIV Env binding to CXCR4 could occur independently of CD4, and to evaluate the effect of prior binding of Env to sCD4, the following experiments were performed. We preexposed CEM-SS as well as J25 cells to either the anti-CD4 antibody Leu3a (Becton Dickinson), which blocks the CD4 binding domain of HIV Env, or OKT4 (Ortho Diagnostics, Costa Mesa, Calif.), which does not block binding of HIV Env to CD4. The cells were then tested for their ability to bind ogp160 to their surfaces. As shown in Fig. ​Fig.3,3, OKT4 had no significant effect on the binding of ogp160 to either CEM-SS or J25 cells while Leu3a readily inhibited binding of ogp160 to CEM-SS cells but had no such effect on J25 cells. Furthermore, when ogp160 was allowed to react in advance with recombinant sCD4 produced in CHO cells (Intracel, Issaquah, Wash.) for 30 min at 4°C at a concentration of 1 μg/ml, we were able to show a clear decrease in the surface binding of ogp160 to CEM-SS cells while the opposite, an obvious enhancement in surface binding, was demonstrated for J25 cells (Fig. ​(Fig.3).3). Open in a separate windowFIG. 3Binding of ogp160 to CEM-SS or J25 cells after exposure of the cells to the anti-CD4 antibodies Leu3a (thin solid lines), OKT4 (dotted lines), or a combination of ogp160 with sCD4 (dashed lines). The shaded areas represent background. The thick solid lines represent binding in the absence of antibodies or sCD4. The experiments were performed in quadruplicate with similar results. Mean channel fluorescence is represented on the x axis.Taken together, these data indicate that HIV Env can bind to CXCR4 independently of CD4. On the other hand, prior interaction of HIV Env with CD4 results in a clear increase in the binding of HIV Env to CXCR4.

Relevance of the glycosylation state of HIV Env in binding to CXCR4.

The binding of HIV Env to CD4 is dependent on the appropriate conformation of the Env molecule (27), which can be significantly altered by changes in its carbohydrate content. We next tested the hypothesis that alterations in the carbohydrate moieties of Env would affect its binding to CXCR4. To do so, we used the gp120 molecule from HIV_SF2, produced in CHO cells, and its counterpart, nonglycosylated HIV_SF2 Env 2-3, produced in yeast strain 2150, and tested both in the binding assay with CEM-SS or J25 cells. HIV_SF-2 gp120 and its nonglycosylated counterpart, Env 2-3, were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, from Kathelyn Steimer, Chiron Corp. (13, 14, 19, 26, 29–31). The results are shown in Fig. ​Fig.4.4. As expected, nonglycosylated HIV_SF2 Env 2-3 bound to the surfaces of the CEM-SS cells to a lesser extent than did HIV_SF2 gp120. On the other hand, and unexpectedly, nonglycosylated HIV_SF2 Env 2-3 bound much more readily to the surfaces of the J25 cells than its glycosylated counterpart, HIV_SF-2 gp120, even when used at equal molar concentrations. To determine whether these findings could be generalized to other Env molecules that lacked intact carbohydrate molecules, we treated ogp160 with sodium metaperiodate. ogp160 from HIV₄₅₁ at 1.25 μg/ml was treated with sodium metaperiodate (Sigma, St. Louis, Mo.) in acetate buffer for 2 h at 4°C in the dark (33). The cells to be tested had been treated previously with 1% glycine (Sigma) for 30 min at 37°C. Such treatment results in the oxidation and cleavage of the carbohydrate hydroxyl groups without affecting the structure of the polypeptide chains (33). Nonspecific binding by the resulting aldehyde groups was prevented by blocking the target cells beforehand with 1% glycine. The results are shown in Fig. ​Fig.4.4. Sodium metaperiodate treatment of ogp160 resulted in a marked inhibition of the binding of ogp160 to the surfaces of the CEM-SS cells. In contrast, sodium metaperiodate treatment of ogp160 resulted in a very clear increase in the binding of HIV Env to the surfaces of the J25 cells. The preexposure of CEM-SS cells to SDF1 did not significantly affect the binding of ogp160 or sodium metaperiodate-treated ogp160. On the other hand, preexposure of J25 cells to 250 nM SDF1 resulted in a marked decrease in binding of both ogp160 and sodium metaperiodate-treated ogp160. These data indicate the specificity of the interaction of the deglycosylated form of ogp160 with CXCR4. The results of these experiments suggest that the alteration in the carbohydrate content of the HIV Env molecules resulted in a better exposure of the epitopes involved in gp120 binding to CXCR4. Open in a separate windowFIG. 4Binding of HIV_SF-2 gp120 or the nonglycosylated form, HIV_SF-2 Env 2-3 (Non-glyc SF-2 gp120), to CEM-SS or J25 cells. The concentration was 355 nM for both. The binding of ogp160 and sodium metaperiodate-treated ogp160 (De-glyc ogp160), each at a concentration of 355 nM, to CEM-SS or J25 cells is also shown. The two right-hand bars in each graph show results for cells preexposed to SDF1 at 150 nM. The results are expressed as mean channel fluorescence. The experiments were performed in duplicate with similar results.The understanding of the underlying mechanisms by which HIV Env, CD4, and the newly discovered HIV coreceptors interact to mediate viral entry remains a very significant issue. The way that HIV Env and CD4 interact is well established (28), and some information exists about the interaction between HIV Env, CCR5, and CD4 (34). In this paper we have shown that HIV Env is able to interact in a CD4-independent manner with CXCR4. Still, the extent of such interaction was clearly lower than that of the sCD4-HIV Env complex and CXCR4. This effect of sCD4 seems to be consistent with the observation that the complexing of this molecule with HIV Env from the strains JRFL or BAL resulted in a significant increase in the affinity of HIV Env for CCR5 (34). We speculate that this interaction between sCD4 and HIV Env results in a conformational change that exposes the binding epitopes in HIV Env relevant for binding to CXCR4, as it does with other gp120 epitopes (16). A different scenario would involve a change in both molecules, resulting in a newly formed common binding epitope. This second alternative seems less likely given our data showing CD4-independent binding of HIV Env to CXCR4, as well as previous data showing the existence of HIV strains capable of CD4-independent entry into target cells (9, 15).The gp120 molecule from HIV contains 20 potential N-linked glycosylation sites, with N-linked glycans representing at least 50% of the molecular mass. Their role in CD4 binding has been studied extensively, although some of the results remain somewhat controversial. Most of the available data seem to indicate that complete lack of glycosylation completely (20), or at least partially (25), inhibits HIV Env binding to CD4. Also, enzymatic manipulation of the carbohydrate residues results in a significant decrease but not in complete abrogation of the binding of HIV Env to CD4 (11, 20, 25). It was therefore somewhat unexpected to find that the nonglycosylated form, as well as the sodium metaperiodate-treated form, of HIV Env was able to bind in such an enhanced way to CXCR4. This would appear to reinforce the concept of the existence of a binding epitope for CXCR4 within HIV Env which is different from the one for CD4. It also suggests that the changes occurring as a consequence of the manipulation of the carbohydrate residues likely result in a better exposure of the CXCR4 binding epitope(s) within the HIV Env molecule.In summary, we have shown that HIV Env can interact with CXCR4 in a CD4-independent manner. We have also shown how the interaction of CD4 with HIV Env results in a significant increase in the binding of the latter to CXCR4 and how the alterations in the carbohydrate composition of the HIV Env molecule affect its binding to CXCR4. The complete definition of these interactions may result in novel approaches to protect against cell infection by HIV.     

CD4-Induced Conformational Changes in the Human Immunodeficiency Virus Type 1 gp120 Glycoprotein: Consequences for Virus Entry and Neutralization

Human immunodeficiency virus type 1 (HIV-1) entry into target cells involves sequential binding of the gp120 exterior envelope glycoprotein to CD4 and to specific chemokine receptors. Soluble CD4 (sCD4) is thought to mimic membrane-anchored CD4, and its binding alters the conformation of the HIV-1 envelope glycoproteins. Two cross-competing monoclonal antibodies, 17b and CG10, that recognize CD4-inducible gp120 epitopes and that block gp120-chemokine receptor binding were used to investigate the nature and functional significance of gp120 conformational changes initiated by CD4 binding. Envelope glycoproteins derived from both T-cell line-adapted and primary HIV-1 isolates exhibited increased binding of the 17b antibody in the presence of sCD4. CD4-induced exposure of the 17b epitope on the oligomeric envelope glycoprotein complex occurred over a wide range of temperatures and involved movement of the gp120 V1/V2 variable loops. Amino acid changes that reduced the efficiency of 17b epitope exposure following CD4 binding invariably compromised the ability of the HIV-1 envelope glycoproteins to form syncytia or to support virus entry. Comparison of the CD4 dependence and neutralization efficiencies of the 17b and CG10 antibodies suggested that the epitopes for these antibodies are minimally accessible following attachment of gp120 to cell surface CD4. These results underscore the functional importance of these CD4-induced changes in gp120 conformation and illustrate viral strategies for sequestering chemokine receptor-binding regions from the humoral immune response.     

CD4 Glycoprotein Degradation Induced by Human Immunodeficiency Virus Type 1 Vpu Protein Requires the Function of Proteasomes and the Ubiquitin-Conjugating Pathway

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic β subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E₁ or (ii) following expression of a mutant form of ubiquitin (Lys₄₈ mutated to Arg₄₈) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.

The human immunodeficiency virus type 1 (HIV-1)-specific accessory protein Vpu performs two distinct functions in the viral life cycle (11, 12, 29, 34, 46, 47, 50–52; reviewed in references 31 and 55): enhancement of virus particle release from the cell surface, and the selective induction of proteolysis of newly synthesized membrane proteins. Known targets for Vpu include the primary virus receptor CD4 (63, 64) and major histocompatibility complex (MHC) class I molecules (28). Vpu is an oligomeric class I integral membrane phosphoprotein (35, 48, 49) with a structurally and functionally defined domain architecture: an N-terminal transmembrane anchor and C-terminal cytoplasmic tail (20, 34, 45, 47, 50, 65). Vpu-induced degradation of endoplasmic reticulum (ER) membrane proteins involves the phosphorylated cytoplasmic tail of the protein (50), whereas the virion release function is mediated by a cation-selective ion channel activity associated with the membrane anchor (19, 31, 45, 47).CD4 is a 55-kDa class I integral membrane glycoprotein that serves as the primary coreceptor for HIV entry into cells. CD4 consists of a large lumenal domain, a transmembrane peptide, and a 38-residue cytoplasmic tail. It is expressed on the surface of a subset of T lymphocytes that recognize MHC class II-associated peptides, and it plays a pivotal role in the development and maintenance of the immune system (reviewed in reference 30). Down regulation of CD4 in HIV-1-infected cells is mediated through several independent mechanisms (reviewed in references 5 and 55): intracellular complex formation of CD4 with the HIV envelope protein gp160 (8, 14), endocytosis of cell surface CD4 induced by the HIV-1 nef gene product (1, 2), and ER degradation induced by the HIV-1 vpu gene product (63, 64).Vpu-induced degradation of CD4 is an example of ER-associated protein degradation (ERAD). ERAD is a common outcome when proteins in the secretory pathway are unable to acquire their native structure (4). Although it was thought that ERAD occurs exclusively inside membrane vesicles of the ER or other related secretory compartments, this has gained little direct experimental support. Indeed, there are several recent reports that ERAD may actually represent export of the target protein to the cytosol, where it is degraded by cytosolic proteases. It was found that in yeast, a secreted protein, prepro-α-factor (pαF), is exported from microsomes and degraded in the cytosol in a proteasome-dependent manner (36). This process was dependent on the presence of calnexin, an ER-resident molecular chaperone that interacts with N-linked oligosaccharides containing terminal glucose residues (3). In mammalian cells, two human cytomegalovirus (HCMV) proteins, US2 and US11, were found to cause the retranslocation of MHC class I molecules from the ER to the cytosol, where they are destroyed by proteasomes (61, 62). In the case of US2, class I molecules were found to associate with a protein (Sec61) present in the channel normally used to translocate newly synthesized proteins into the ER (termed the translocon), leading to the suggestion that the ERAD substrates are delivered to the cytosol by retrograde transport through the Sec61-containing pore (61). Fujita et al. (24) reported that, similar to these findings, the proteasome-specific inhibitor lactacystin (LC) partially blocked CD4 degradation in transfected HeLa cells coexpressing CD4, Vpu, and HIV-1 Env glycoproteins. In the present study, we show that Vpu-induced CD4 degradation can be completely blocked by proteasome inhibitors, does not require the ER chaperone calnexin, but requires the function of the cytosolic polyubiquitination machinery which apparently targets potential ubiquitination sites within the CD4 cytoplasmic tail. Our findings point to differences between the mechanism of Vpu-mediated CD4 degradation and ERAD processes induced by the HCMV proteins US2 and US11 (61, 62).     

CD4-Chemokine Receptor Hybrids in Human Immunodeficiency Virus Type 1 Infection

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-1(LAI) to nearly the same extent as the wild-type molecules, whereas CD4(4D)CXCR4 was less efficient. Recombinant SU(LAI) 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-1(LAI) 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-1(LAI) and the X4R5 virus HIV-1(89. 6), unlike the R5 strain HIV-1(SF162), 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.