Correlation between carbohydrate structures on the envelope glycoprotein gp120 of HIV-1 and HIV-2 and syncytium inhibition with lectins

The binding of 13 different lectins to gp120 partially purified from two HIV-1 isolates and one HIV-2 isolate was studied by in situ staining on electrophoretically separated and electroblotted HIV antigens. The lectins concanavalin A, wheat germ agglutinin, Lens culinaris agglutinin, Vicia faba agglutinin, Pisum sativum agglutinin and phytohaem(erythro)agglutinin bound to gp120 of all three isolates. The carbohydrate of gp120 recognized by lectins was thus arranged in at least four types of glycans: a high mannose type glycan, a bisected hybrid or complex type glycan, a biantennary fucosylated complex type glycan and a triantennary bisected complex type glycan. Only lectins which bound at least one of the four types of glycans were capable of inhibiting fusion of HIV-infected cells with CD4 cells by a carbohydrate-specific interaction with the HIV-infected cells. Thus, several different glycan structures may be implicated in CD4-gp120 binding.     

Oligosaccharide-mediated interactions of the envelope glycoprotein gp120 of HIV-1 that are independent of CD4 recognition

In this study carbohydrate-mediated interactions of the envelope glycoprotein, gp120, of HIV-1 were investigated. Oligosaccharide probes (neoglycolipids), prepared from the N-glycosidically-linked chains of the natural and recombinant forms of gp120, were used in conjunction with the intact glycoprotein to investigate reactivities with a soluble carbohydrate-binding protein (lectin) known as mannose-binding protein in human serum. Evidence is presented that the high-mannose-type oligosaccharides with seven, eight and nine mannose residues from both forms of gp120 are recognized by the serum lectin, and that these reactivities are unrelated to CD4 recognition. Reactivities of the two forms of envelope glycoprotein with macrophages derived from human blood monocytes and with the mannose-specific macrophage endocytosis receptor isolated from human placental membranes were also investigated. Evidence is presented that both forms of gp120 bind to the macrophage surface by multiple interactions in addition to CD4 binding, and that among these interactions is a carbohydrate-mediated binding to the endocytosis receptor. We propose that such carbohydrate-mediated interactions could form the basis of viral attachment to a variety of healthy and diseased tissues.     

Cell surface-associated Tat modulates HIV-1 infection and spreading through a specific interaction with gp120 viral envelope protein

Human immunodeficiency virus-1 (HIV-1) Tat, a nuclear transactivator of viral gene expression, has the unusual property of being released by infected cells. Recent studies suggest that extracellular Tat is partially sequestered by heparan sulfate proteoglycans. As a consequence, Tat is concentrated on the cell surface and protected from proteolytic degradation, thus remaining in a biologically active form. We show that Tat binds the surfaces of both HIV-1-infected and surrounding uninfected cells. We provide evidence for a specific interaction between Tat and the HIV-1 glycoprotein 120 (gp120) envelope protein, which enhances virus attachment and entry into cells. We map the interacting sites of both Tat and gp120 and show that synthetic peptides mimicking the gp120 site inhibit HIV-1 infection. Our data demonstrate that membrane-associated Tat is a novel modulator of virus entry and suggest that the Tat-gp120 interaction represents a critical step in HIV-1 spreading during the course of infection.     

Cooperative subunit interactions within the oligomeric envelope glycoprotein of HIV-1: functional complementation of specific defects in gp120 and gp41

The envelope glycoprotein (Env) of HIV-1 is displayed on the surface of the virion or infected cell as an oligomer of multiple gp120/gp41 complexes. We sought to unravel the relationships between this oligomeric structure and the requirements for sequential interactions with CD4 and coreceptor (CCR5 or CXCR4). We used a quantitative cell fusion assay to examine the effects of coexpressing pairs of Envs, each nonfunctional because of a specific defect in one of the essential properties. We observed efficient fusion activity upon coexpression of two Env variants, one containing a gp41 subunit with a mutated fusion peptide and the other containing a gp120 subunit with a mutated CD4 binding site or a mismatched coreceptor specificity. We also observed fusion upon coexpression of two Env variants with distinct gp120 defects, i.e., a CD4 binding site mutation and the incorrect coreceptor specificity determinants. Coimmunoprecipitation experiments verified the efficient formation of mixed oligomers, suggesting that the observed fusion reflected subunit complementation within the oligomeric complex. These results support a model in which cooperative subunit interactions within the Env oligomer result in concerted conformational changes upon receptor binding, resulting in activation for fusion. The implications of these findings for Env function and virus neutralization are discussed.     

Human immunodeficiency virus type 1 challenge of chimpanzees immunized with recombinant envelope glycoprotein gp120.

The major envelope glycoprotein, gp120, of human immunodeficiency virus type 1 (HIV-1) was purified from a Chinese hamster ovary cell line transfected with a truncated form of the HIV-1 env gene. The recombinant glycoprotein (rgp120) was formulated with aluminum hydroxide adjuvant and was used to immunize chimpanzees. The recombinant preparation was effective in eliciting cellular and humoral immunity as well as immunologic memory. Anti-rgp 120 antibodies reacted with authentic viral gp120 in immunological blot assays and were able to neutralize HIV-1 infectivity in vitro. Sera from the rgp120-immunized animals were able to neutralize HIV-1 pseudotypes of vesicular stomatitis virus prepared from the IIIB isolate, from which the gene encoding rgp120 was derived, as well as two heterologous isolates, ARV-2 and RF. The immune response elicited against the rgp120 was not effective in preventing viral infection after intravenous challenge with HIV-1. The implications of these results on HIV-1 vaccine development are discussed.     

HIV-1 gp120 and chemokines activate ion channels in primary macrophages through CCR5 and CXCR4 stimulation

HIV type 1 (HIV-1) uses the chemokine receptors CCR5 and CXCR4 as coreceptors for entry into target cells. Here we show that the HIV-1 envelope gp120 (Env) activates multiple ionic signaling responses in primary human macrophages, which are important targets for HIV-1 in vivo. Env from both CCR5-dependent JRFL (R5) and CXCR4-dependent IIIB (X4) HIV-1 opened calcium-activated potassium (K(Ca)), chloride, and calcium-permeant nonselective cation channels in macrophages. These signals were mediated by CCR5 and CXCR4 because macrophages lacking CCR5 failed to respond to JRFL and an inhibitor of CXCR4 blocked ion current activation by IIIB. MIP-1beta and SDF-1alpha, chemokine ligands for CCR5 and CXCR4, respectively, also activated K(Ca) and Cl(-) currents in macrophages, but nonselective cation channel activation was unique to gp120. Intracellular Ca(2+) levels were also elevated by gp120. The patterns of activation mediated by CCR5 and CXCR4 were qualitatively similar but quantitatively distinct, as R5 Env activated the K(Ca) current more frequently, elicited Cl(-) currents that were approximately 2-fold greater in amplitude, and elevated intracellular Ca(+2) to higher peak and steady-state levels. Env from R5 and X4 primary isolates evoked similar current responses as the corresponding prototype strains. Thus, the interaction of HIV-1 gp120 with CCR5 or CXCR4 evokes complex and distinct signaling responses in primary macrophages, and gp120-evoked signals differ from those activated by the coreceptors' chemokine ligands. Intracellular signaling responses of macrophages to HIV-1 may modulate postentry steps of infection and cell functions apart from infection.     

HIV-1 envelope trimer elicits more potent neutralizing antibody responses than monomeric gp120

HIV-1 envelope glycoprotein is the primary target for HIV-1-specific antibodies. The native HIV-1 envelope spike on the virion surface is a trimer, but trimeric gp140 and monomeric gp120 currently are believed to induce comparable immune responses. Indeed, most studies on the immunogenicity of HIV-1 envelope oligomers have revealed only marginal improvement over monomers. We report here that suitably prepared envelope trimers have nearly all the antigenic properties expected for native viral spikes. These stable, rigorously homogenous trimers have antigenic properties markedly different from those of monomeric gp120s derived from the same sequences, and they induce potent neutralizing antibody responses for a cross-clade set of tier 1 and tier 2 viruses with titers substantially higher than those elicited by the corresponding gp120 monomers. These results, which demonstrate that there are relevant immunologic differences between monomers and high-quality envelope trimers, have important implications for HIV-1 vaccine development.     

CD4-induced epitopes in the HIV envelope glycoprotein, gp120

The HIV surface glycoprotein, gp120, contains conserved and functional domains that exist within a viral envelope spike that is otherwise highly variable with respect to conformation, sequence and structure. Termed CD4-induced epitopes, these domains are stabilized on transition state gp120 structures as a consequence of CD4 receptor engagement. These nuggets of conservation naturally attract the attention of those seeking to develop vaccine and therapeutic strategies to fight infection by diverse HIV strains. However, an appreciation for the immunological relevance and practical value of CD4-induced epitopes is still evolving. This review covers the current findings related to the accessibility, antigenicity and immunogenicity of CD4-induced epitopes on gp120.     

gp120 envelope glycoproteins of human immunodeficiency viruses competitively antagonize signaling by coreceptors CXCR4 and CCR5

Signal transductions by the dual-function CXCR4 and CCR5 chemokine receptors/HIV type 1 (HIV-1) coreceptors were electrophysiologically monitored in Xenopus laevis oocytes that also coexpressed the viral receptor CD4 and a G protein-coupled inward-rectifying K⁺ channel (Kir 3.1). Large Kir 3.1-dependent currents generated in response to the corresponding chemokines (SDF-1α for CXCR4 and MIP-1α; MIP-1β and RANTES for CCR5) were blocked by pertussis toxin, suggesting involvement of inhibitory guanine nucleotide-binding proteins. Prolonged exposures to chemokines caused substantial but incomplete desensitization of responses with time constants of 5–7 min and recovery time constants of 12–19 min. CXCR4 and CCR5 exhibited heterologous desensitization in this oocyte system, suggesting possible inhibition of a common downstream step in their signaling pathways. In contrast to chemokines, perfusion with monomeric or oligomeric preparations of the glycoprotein of M_r 120,000 (gp120) derived from several isolates of HIV-1 did not activate signaling by CXCR4 or CCR5 regardless of CD4 coexpression. However, adsorption of the gp120 from a T-cell-tropic virus resulted in CD4-dependent antagonism of CXCR4 response to SDF-1α, whereas gp120 from macrophage-tropic viruses caused CD4-dependent antagonism of CCR5 response to MIP-1α. These antagonisms could be partially overcome by high concentrations of chemokines and were specific for coreceptors of the corresponding HIV-1 isolates, suggesting that they resulted from direct interactions of gp120–CD4 complexes with coreceptors and that they did not involve the desensitization pathway. These results indicate that monomeric or oligomeric gp120s specifically antagonize CXCR4 and CCR5 signaling in response to chemokines, but they do not exclude the possibility that gp120s might also function as weak agonists in some cells. The gp120-mediated disruption of CXCR4 and CCR5 signaling may contribute to AIDS pathogenesis.     

CCR5/CD4/CXCR4 oligomerization prevents HIV-1 gp120IIIB binding to the cell surface

CCR5 and CXCR4, the respective cell surface coreceptors of R5 and X4 HIV-1 strains, both form heterodimers with CD4, the principal HIV-1 receptor. Using several resonance energy transfer techniques, we determined that CD4, CXCR4, and CCR5 formed heterotrimers, and that CCR5 coexpression altered the conformation of both CXCR4/CXCR4 homodimers and CD4/CXCR4 heterodimers. As a result, binding of the HIV-1 envelope protein gp120_IIIB to the CD4/CXCR4/CCR5 heterooligomer was negligible, and the gp120-induced cytoskeletal rearrangements necessary for HIV-1 entry were prevented. CCR5 reduced HIV-1 envelope-induced CD4/CXCR4-mediated cell-cell fusion. In nucleofected Jurkat CD4 cells and primary human CD4⁺ T cells, CCR5 expression led to a reduction in X4 HIV-1 infectivity. These findings can help to understand why X4 HIV-1 strains infection affect T-cell types differently during AIDS development and indicate that receptor oligomerization might be a target for previously unidentified therapeutic approaches for AIDS intervention.For HIV-1 to enter a target cell, the viral envelope glycoprotein gp120 must interact with a set of cell surface molecules that include the primary receptor, CD4 (1), and a chemokine receptor (CCR5 or CXCR4) that acts as a coreceptor (2, 3). These molecules form CD4/chemokine receptor complexes, as deduced from coprecipitation data for CXCR4 or CCR5 with CD4 (4–8).Most HIV-1 variants isolated from newly infected individuals use CCR5 and CD4 to enter host cells; these M-tropic R5 strains are predominant in acute and asymptomatic phases of HIV infection. CD4⁺ T helper type 1 (Th1) cells, which express high CCR5 levels (9, 10), are implicated in maintaining asymptomatic status (11, 12). The “viral shift” from R5 to T-tropic X4 HIV-1 strains correlates with AIDS progression (13, 14). X4 strains infect mainly CD4⁺ Th2 cells, which express little CCR5 and whose CXCR4 levels resemble those of Th1 cells (15, 16), which suggests that cell susceptibility to HIV-1 infection depends on the CD4/coreceptor ratio and on receptor levels during cell activation and/or differentiation (17). CXCR4 and CCR5 are present as homodimers and heterodimers at the plasma membrane (18–20). In addition, gp120-mediated CD4/CXCR4 and CD4/CCR5 association and clustering is reported (21–23). Nonetheless, little is known of how CCR5 expression influences the CD4/CXCR4 interaction, or of the molecular basis that underlies the differences in X4 strains infection relative to CCR5 levels at the cell surface.Here, we identify CD4/CXCR4/CCR5 oligomers at the cell membrane, even in the absence of ligands. CCR5 expression in these complexes modifies the heterodimeric CD4/CXCR4 conformation and blocks gp120_IIIB binding, without altering binding of the CXCR4 ligand CXCL12 and its subsequent signaling. gp120_IIIB-triggered LIMK1 activation, cofilin dephosphorylation, and the actin cytoskeleton rearrangement necessary for cell-cell fusion were impeded in CD4/CXCR4/CCR5-expressing cells. The data obtained using recombinant gp120_IIIB glycoprotein were confirmed by experiments showing that X4 HIV-1 infection of Jurkat and primary T cells is regulated by CCR5 expression.     

Positive in vivo selection of the HIV-1 envelope protein gp120 occurs at surface-exposed regions

The rapid evolution of human immunodeficiency virus (HIV) envelope represents a major challenge to vaccine and drug development, particularly because the underlying mechanisms are not completely understood. To explore whether distinct patterns of positive selection within the envelope glycoprotein (gp) 120 exist and are associated with functionally relevant domains, we conducted a long-term survey of sequence evolution in 20 HIV-1-infected persons who interrupted antiretroviral therapy. In total, 1753 clonal sequences encompassing the C2-V3-C3 region of gp120 were derived. Strikingly, positively selected amino acids mapped almost exclusively (P=.0003) to externally accessible residues on the gp120 crystal structure. The current understanding of envelope structure and function associates the main determinants of viral entry and the targets for neutralizing antibodies with these exterior regions of gp120, strongly suggesting that the observed adaptive evolution of these sites occurs in response to respective selective forces.     

HIV type 1 mother-to-child transmission facilitated by distinctive glycosylation sites in the gp120 envelope glycoprotein

The human immunodeficiency virus type 1 (HIV-1) characteristics associated with mother-to-child transmission (MTCT) are still poorly understood. We studied a cohort of 30 mothers from Rwanda infected with HIV-1 subtype A or C viruses of whom seven infected their children either during gestation or soon after birth. CD4 counts and viral load did not significantly differ between nontransmitting mother (NTM) versus transmitting mother (TM) groups. In contrast to earlier studies we not only analyzed and compared the genotypic characteristics of the V1-V5 region of the gp120 envelope of viruses found in TM and their infected children, but also included data from the NTM. No differences were found with respect to length and number of potential N-glycosylation sites (PNGS) in the V1-V2 and the V1-V5 region. We identified that viruses with a PNGS on positions AA234 and AA339 were preferably transmitted and that viruses with PNGS-N295 showed a disadvantage in transmission. We also showed that the frequency of PNGS-N339 in the viruses of TM and infected children was significantly higher than the frequency in NTM in our cohort and in viruses undergoing sexual transmission while the frequency of PNGS-N295 in children was significantly lower than the frequency in TM and acute horizontal infections. Collectively, our results provide evidence that the presence of the PNGS-N339 site and absence of the PNGS-N295 site in the gp120 envelope confers an advantage to HIV-1 when considering MTCT.     

Specific inhibition of HIV-1 coreceptor activity by synthetic peptides corresponding to the predicted extracellular loops of CCR5

We used synthetic peptides to the extracellular loops (ECLs) of CCR5 to examine inhibitory effects on HIV infection/fusion with primary leukocytes and cells expressing recombinant CCR5. We show for the first time that peptides derived from the first, second, or third ECL caused dose-dependent inhibition of fusion and infection, although with varying potencies and specificities for envelope glycoproteins (Envs) from different strains. The first and third ECL peptides inhibited Envs from the R5 Ba-L strain and the R5X4 89.6 strain, whereas the second ECL peptide inhibited Ba-L but not 89.6 Env. None of the peptides affected fusion mediated by Env from the X4 LAV strain. Fusion mediated by Envs from several primary HIV-1 isolates was also inhibited by the peptides. These findings suggest that various HIV-1 strains use CCR5 domains in different ways. Experiments involving peptide pretreatment and washing, modulation of the expression levels of Env and CCR5, analysis of CCR5 peptide effects against different coreceptors, and inhibition of radiolabeled glycoprotein (gp) 120 binding to CCR5 suggested that the peptide-blocking activities reflect their interactions with gp120. The CCR5-derived ECL peptides thus provide a useful approach to analyze structure-function relationships involved in HIV-1 Env-coreceptor interactions and may have implications for the design of drugs that inhibit HIV infection.     

Modeling of CCR5 Recognition by HIV-1 gp120: How the Viral Protein Exploits the Conformational Plasticity of the Coreceptor

The chemokine receptor CCR5 is a key player in HIV-1 infection. The cryo-EM 3D structure of HIV-1 envelope glycoprotein (Env) subunit gp120 in complex with CD4 and CCR5 has provided important structural insights into HIV-1/host cell interaction, yet it has not explained the signaling properties of Env nor the fact that CCR5 exists in distinct forms that show distinct Env binding properties. We used classical molecular dynamics and site-directed mutagenesis to characterize the CCR5 conformations stabilized by four gp120s, from laboratory-adapted and primary HIV-1 strains, and which were previously shown to bind differentially to distinct CCR5 forms and to exhibit distinct cellular tropisms. The comparative analysis of the simulated structures reveals that the different gp120s do indeed stabilize CCR5 in different conformational ensembles. They differentially reorient extracellular loops 2 and 3 of CCR5 and thus accessibility to the transmembrane binding cavity. They also reshape this cavity differently and give rise to different positions of intracellular ends of transmembrane helices 5, 6 and 7 of the receptor and of its third intracellular loop, which may in turn influence the G protein binding region differently. These results suggest that the binding of gp120s to CCR5 may have different functional outcomes, which could result in different properties for viruses.     

Stable,uncleaved HIV-1 envelope glycoprotein gp140 forms a tightly folded trimer with a native-like structure

The HIV-1 envelope spike [trimeric (gp160)₃, cleaved to (gp120/gp41)₃] is the mediator of viral entry and the principal target of humoral immune response to the virus. Production of a recombinant preparation that represents the functional spike poses a challenge for vaccine development, because the (gp120/gp41)₃ complex is prone to dissociation. We have reported previously that stable HIV-1 gp140 trimers, the uncleaved ectodomains of (gp160)₃, have nearly all of the antigenic properties expected for native viral spikes. Because of recent claims that uncleaved gp140 proteins may adopt a nonnative structure with three gp120 moieties “dangling” from a trimeric gp41 ectodomain in its postfusion conformation, we have inserted a long, flexible linker between gp120 and gp41 in our stable gp140 trimers to assess their stability and to analyze their conformation in solution. The modified trimer has biochemical and antigenic properties virtually identical to those of its unmodified counterpart. Both forms bind a single CD4 per trimer, suggesting that the trimeric conformation occludes two of the three CD4 sites even when a flexible linker has relieved the covalent constraint between gp120 and gp41. In contrast, an artificial trimer containing three gp120s flexibly tethered to a trimerization tag binds three CD4s and has antigenicity nearly identical to that of monomeric gp120. Moreover, the gp41 part of both modified and unmodified gp140 trimers has a structure very different from that of postfusion gp41. These results show that uncleaved gp140 trimers from suitable isolates have compact, native-like structures and support their use as candidate vaccine immunogens.The HIV-1 envelope glycoprotein mediates initial steps of virus infection by engaging cellular receptors and facilitating fusion of viral and target-cell membranes (1). Biosynthesis of the virus-encoded envelope glycoprotein yields a precursor, gp160, which following trimerization undergoes cleavage by a furin-like protease into two noncovalently associated fragments: the receptor-binding fragment, gp120, and the fusion fragment, gp41 (1). Three copies each of gp120 and gp41 form the mature envelope spikes (gp120/gp41)₃, the major viral surface antigen. Binding, through a site on gp120, to the host primary receptor, CD4, and then, through a second site, to a coreceptor (e.g., CCR5 or CXCR4) triggers large conformational changes that include reduced interaction between gp120 and gp41 (probably leading to dissociation of the former) and a cascade of ensuing gp41 refolding events (2, 3). Within the precursor gp160, gp41, with its C-terminal transmembrane (TM) segment anchored in the viral membrane, folds into a prefusion conformation. Cleavage of gp160 makes this prefusion conformation metastable with respect to a rearranged, postfusion conformation. Thus, the loss of constraint on gp41 that accompanies coreceptor binding to gp120 triggers a transition in gp41 to an extended, membrane-bridging conformation (sometimes called a “prehairpin” conformation) (4) with a hydrophobic “fusion peptide” at its N terminus inserted into the target-cell membrane and the TM segment in the viral membrane. This relatively long-lived, transient conformation is the target of fusion inhibitors, such as enfuvirtide (5), and of several broadly neutralizing antibodies (bnAbs) (6–8). Folding back of each chain into an α-helical hairpin creates a stable, six-helix bundle—the “postfusion conformation”—placing the fusion peptide and TM segment at the same end of the molecule. This irreversible refolding of gp41 brings the two membranes together, leading to bilayer fusion and viral entry. Thus, during the fusion process, there are at least three distinct conformational states of the envelope protein: the prefusion conformation of (gp120/gp41)₃, the extended intermediate of gp41, and the postfusion conformation of gp41 (with release of free gp120). Moreover, conformational changes of the prefusion form occur upon CD4 and perhaps also coreceptor binding.The envelope glycoprotein is also the primary target of humoral responses in HIV-1–infected individuals. Studies of human monoclonal antibodies (mAbs) have identified a subset members that neutralize a wide range of HIV isolates (see Table S1 for a partial catalog and original references) (9, 10). These bnAbs are of particular interest, because they may guide a search for immunogens to elicit them in vaccinees. Epitopes on gp120 recognized by human bnAbs include the CD4-binding site, a trimer-specific epitope in the relatively invariant parts of the V2 and V3 loops, and a site near the base of the V3 loop involving an N-linked glycan at position 332. A glycan-dependent epitope spans both gp120 and gp41. The membrane-proximal external region (MPER) of gp41 binds a set of bnAbs that were among the earliest broad neutralizers discovered. The domain-swapped, dimeric antibody, 2G12, recognizes only glycans at defined positions, and its reactivity therefore depends on specific glycosylation patterns but not on many other aspects of the gp120 amino acid sequence.Interesting groups of nonneutralizing antibodies, or with a very narrow range of isolates neutralized, include those that bind the so-called CD4-induced (CD4i) epitope, which overlaps the coreceptor site, on the bridging sheet of gp120, when the epitope becomes exposed by the conformational changes that accompany CD4 binding (Table S1). Nonneutralizing antibodies that interact with gp41 fall into two “clusters”: those in cluster I, which recognize the “immunodominant” C-C loop of gp41, and those in cluster II, which bind strongly with a segment just preceding the MPER in the gp41 polypeptide chain, but only when gp41 is in the postfusion conformation. Most of the antibodies listed in Table S1 recognize conformation-dependent epitopes and thus are excellent molecular probes for defining the conformational state of the envelope trimer.A form of gp140, stabilized by a disulfide crosslink between gp120 and gp41 (perhaps related to the disulfide between surface and TM subunits in many oncoretroviruses) was introduced over a decade ago (11) and subsequently modified by introducing an Ile-to-Pro mutation in gp41, to retard formation of the six-helix bundle (12). The product, known as SOSIP (SOS to designate the double cysteine mutations and IP to denote the isoleucine to proline change), is possible only with certain isolates, and the most widely studied has been BG505 SOSIP.664 (13–16). This modified gp140 trimer, which can be cleaved with furin without compromising stability, has greatly facilitated structural analysis, probably by eliminating large-scale conformational fluctuations (13, 14). In this variant, the MPER has been deleted, and the furin site has been replaced with a string of six arginines. Its structure, determined by both electron cryomicroscopy and X-ray crystallography, shows that (as expected from other fusion proteins) the conformation of gp41 in the prefusion state is distinct from the postfusion six-helix bundle (3, 17). The SOSIP.664 structure, which is an extremely important contribution to our understanding of envelope trimer molecular architecture, has some puzzling features. Docking CD4 onto the model suggests that it can bind all three gp120 sites with no clashes. This observation is at odds with a large body of evidence, including a recent biophysical study of the same trimer, showing that CD4 binding induces a substantial structural rearrangement (18–22). Moreover, uncleaved BG505 gp140 without the SOSIP modifications is unstable and heterogeneous. Can one really conclude, as suggested (14), that all HIV-1 uncleaved gp140 trimers are misfolded products resembling three gp120 moieties flexibly linked to a trimer gp41 in the postfusion, six-helix bundle conformation? The answer to this question is of considerable consequence, as it relates directly to design and production of candidate HIV-1 vaccine immunogens.In the work reported here, we have inserted a flexible, 20-residue linker between gp120 and gp41 in the context of a previously characterized, stable gp140. The linker releases the tight covalent constraint between gp120 and gp41, and it should therefore mimic to some extent the furin cleavage (which is effectively a linker of infinite length). If an uncleaved gp140 trimer truly resembles “three balls on a string,” as asserted (14), addition of a linker that can extend as much as 70 Å should exaggerate this property, causing the trimer with the inserted linker to have antigenic properties resembling three monomeric gp120s and a very large hydrodynamic radius. We find, to the contrary, that gp140 with the flexible linker, 20-residue insert (gp140–FL20) has antigenic properties essentially identical to those of the unmodified trimer and very different from those of free gp120 or of a construct with three gp120s held together by a heterologous trimerization tag. Moreover, the linker barely affects the hydrodynamic radius of the trimer, which is even slightly smaller than that of SOSIP. These results show that the stable, uncleaved gp140 trimers we have characterized previously are compact and native-like, and they support our suggestion that they are promising, envelope-based immunogens for clinical testing in vaccine development.     

The Src kinase Lyn is required for CCR5 signaling in response to MIP-1beta and R5 HIV-1 gp120 in human macrophages

CCR5 is a receptor for several beta chemokines and the entry coreceptor used by macrophage-tropic (R5) strains of HIV-1. In addition to supporting viral entry, CCR5 ligation by the HIV-1 envelope glycoprotein 120 (gp120) can activate intracellular signals in macrophages and trigger inflammatory mediator release. Using a combination of in vitro kinase assay, Western blotting for phospho-specific proteins, pharmacologic inhibition, CCR5 knockout (CCR5Delta32) cells, and kinase-specific blocking peptide, we show for the first time that signaling through CCR5 in primary human macrophages is linked to the Src kinase Lyn. Stimulation of human monocyte-derived macrophages with either HIV-1 gp120 or MIP-1beta results in the CCR5-mediated activation of Lyn and the concomitant Lyn-dependent activation of the mitogen-activated protein (MAP) kinase ERK-1/2. Furthermore, activation of the CCR5/Lyn/ERK-1/2 pathway is responsible for gp120-triggered production of TNF-alpha by macrophages, which is believed to contribute to HIV-1 pathogenesis. Thus, Lyn kinase may play an important role both in normal CCR5 function in macrophages and in AIDS pathogenesis in syndromes such as AIDS dementia where HIV-1 gp120 contributes to inappropriate macrophage activation, mediator production, and secondary injury.     

Proliferative responses to human immunodeficiency virus type 1 (HIV-1) gp120 peptides in HIV-1-infected individuals immunized with HIV-1 rgp120 or rgp160 compared with nonimmunized and uninfected controls

The proliferative responses to a series of peptides constituting the human immunodeficiency virus type 1 (HIV-1) gp120 sequence were evaluated in 19 HIV-1-infected rgp160 vaccine recipients, 17 HIV-1-infected rgp120 vaccine recipients, 15 HIV-1-infected placebo recipients, and 18 HIV-1-uninfected controls. Many regions of the gp120 molecule were found to contribute proliferative epitopes, although there were clearly regions of relative dominance and silence. Vaccine recipients tended to have broader, more robust, and more frequent peptide recognition than the placebo recipients. Despite the considerable variability in the pattern of peptide recognition among individuals, there was a striking similarity between the rgp160 and rgp120 vaccinee groups as a whole. Low-risk HIV-1-uninfected individuals may react to a few peptides within the gp120 sequence as well, despite a lack of significant response to the whole gp120 protein.