Synthesis of glycolipid analogues that disrupt binding of HIV-1 gp120 to galactosylceramide

HIV-1 has been shown to infect CD4 negative cells by the binding of HIV gp120 to the glycolipid galactosylceramide (1) (GalCer). Several analogues of 1 were prepared to investigate the specific orientation of 1 in the membrane bilayer that is involved in gp120 binding. Interestingly, N-stearyl-1-deoxynojirimycin (8) displayed potent and specific affinity for gp120 equal to that of 1, a finding that may shed light on the antiviral activity of N-butyl-1-deoxynojirimycin.     

Characterization of human immunodeficiency virus type 1 gp120 binding to liposomes containing galactosylceramide.

Human immunodeficiency virus type 1 (HIV-1) infects some cell types which lack CD4, demonstrating that one or more alternative viral receptors exist. One such receptor is galactosylceramide (GalCer), a glycosphingolipid distributed widely in the nervous system and in colonic epithelial cells. Using a liposome flotation assay, we found that the HIV-1 surface glycoprotein, gp120, quantitatively bound to liposomes containing GalCer but not to liposomes containing phospholipids and cholesterol alone. Binding was saturable and was inhibited by preincubating liposomes with anti-GalCer antibodies. We observed less efficient binding of gp120 to liposomes containing lactosylceramide, glucosylceramide, and galactosylsulfate, whereas no binding to liposomes containing mixed gangliosides, psychosine, or sphingomyelin was detected. Binding to GalCer was rapid, largely independent of temperature and pH, and stable to conditions which remove most peripheral membrane proteins. By contrast, gp120 bound to lactosylceramide could be removed by 2 M potassium chloride or 3 M potassium thiocyanate, demonstrating a less stable interaction. Removal of N-linked oligosaccharides on gp120 did not affect binding efficiency. However, as previously observed for CD4 binding, heat denaturation of gp120 prevented binding to GalCer. Finally, binding was critically dependent on the concentration of GalCer in the target membrane, suggesting that binding to glycolipid-rich domains occurs and that GalCer conformation may be important for gp120 recognition.     

Synthetic multimeric peptides derived from the principal neutralization domain (V3 loop) of human immunodeficiency virus type 1 (HIV-1) gp120 bind to galactosylceramide and block HIV-1 infection in a human CD4-negative mucosal epithelial cell line.

The glycosphingolipid galactosylceramide (GalCer), which binds gp120 with high affinity and specificity, is a potential alternative receptor for human immunodeficiency virus type 1 (HIV-1) in some CD4-negative neural and epithelial human cells, including the human colonic epithelial cell line HT-29. In the present study, we demonstrate that synthetic multibranched peptides derived from the consensus sequence of the HIV-1 V3 loop block HIV-1 infection in HT-29 cells. The most active peptide was an eight-branched multimer of the motif Gly-Pro-Gly-Arg-Ala-Phe which at a concentration of 1.8 microM induced a 50% inhibition of HIV-1 infection in competition experiments. This peptide was not toxic to HT-29 cells, and preincubation with HIV-1 did not affect viral infectivity, indicating that the antiviral activity was not due to a nonspecific virucidal effect. Using a high-performance thin-layer chromatography binding assay, we found that multibranched V3 peptides recognized GalCer and inhibited binding of recombinant gp120 to the glycosphingolipid. In addition, these peptides abolished the binding of an anti-GalCer monoclonal antibody to GalCer on the surface of live HT-29 cells. These data provide additional evidence that the V3 loop is involved in the binding of gp120 to the GalCer receptor and show that multibranched V3 peptides are potent inhibitors of the GalCer-dependent pathway of HIV-1 infection in CD4-negative mucosal epithelial cells.     

Polyvalent interactions of HIV-gp120 protein and nanostructures of carbohydrate ligands

This paper presents the initial effort in anti-HIV infection using glycosphingolipid-based nanostructures. HIV infection of CD4 negative cells is initiated by the binding of the viral envelope glycoprotein gp120 to galactosylceramide (GalCer), a glycosphingolipid that serves as the cellular receptor for viral recognition. A series of nanostructures of GalCer are designed and produced using an AFM-based lithography method known as nanografting. The geometry dependence of recombinant gp120 binding to these nanostructures is monitored using high-resolution AFM imaging. Gp120 molecules are found to favor binding sites that allow for polyvalent interactions. Increased adsorption at the intersection of two lines, or between two parallel lines with matching separation for trimeric binding, strongly suggests that trivalent interactions are dominant in gp120-GalCer nanostructure interactions. Systematic distance-dependence studies, using parallel nanolines with various separations, reveal a separation of 4.8 nm, matching the separation of V3 loops in gp120 trimers. This investigation demonstrates that nanotechnology provides a powerful tool for investigating and guiding polyvalent interactions among biological systems.     

The virotoxin model of HIV-1 enteropathy: Involvement of GPR15/Bob and galactosylceramide in the cytopathic effects induced by HIV-1 gp120 in the HT-29-D4 intestinal cell line

BACKGROUND: Malabsorption and diarrhea are common, serious problems in AIDS patients, and are in part due to the incompletely understood entity HIV enteropathy. Our prior in vitro work has shown that increased transepithelial permeability and glucose malabsorption, similar to HIV enteropathy, are caused by HIV surface protein gp120, although the mechanism remains unclear. RESULTS: We studied the effects of HIV surface protein gp120 on the differentiated intestinal cell line HT-29-D4, specifically the effects on microtubules, transepithelial resistance, and sodium glucose cotransport. gp120 induced extensive microtubule depolymerization, an 80% decrease in transepithelial resistance, and a 70% decrease in sodium-dependent glucose transport, changes closely paralleling those of HIV enteropathy. The effects on transepithelial resistance were used to study potential inhibitors. Neutralizing antibodies to GPR15/Bob but not to CXCR4 (the coreceptor allowing infection with these HIV strains) inhibited these effects. Antibodies to galactosylceramide (GalCer) and a synthetic analog of GalCer also inhibited the gp120-induced changes, suggesting the involvement of GalCer-enriched lipid rafts in gp120 binding to intestinal epithelial cells. CONCLUSION: We conclude that direct HIV infection and gp120-induced cytopathic effects are distinct phenomena. While in vivo confirmation is needed to prove this, gp120 could be a virotoxin significantly contributing to HIV enteropathy.     

The binding of HIV-1 gp41 membrane proximal domain to its mucosal receptor, galactosyl ceramide, is structure-dependent

The peptide of HIV-1 envelope gp41 (a.a 628-683), referred to herein as P5, contains P1, a conserved galactose-specific lectin domain for binding the mucosal HIV-1-receptor, galactosyl ceramide (GalCer), as shown earlier, and a potential calcium-binding site (a.a 628-648). P1 contains contiguous epitopes recognized by the broadly neutralizing antibodies 2F5, 4E10, Z13. However, similar neutralizing antibodies could not be raised in animal model using immunogens based on these epitopes. We now show that the structure of both P5 and P1 peptides, as measured by circular dichroism, differs according to their environment: aqueous or lipidic, and as a function of calcium concentration. P5, but not P1, binds to calcium with a low binding affinity constant in the order of 2.5x10(4). Calcium binding results in a conformational change of P5, leading in turn to a decrease in affinity for GalCer. Hence, the affinity of the gp41-lectin site for the galactose harbored by the mucosal HIV-1 receptor GalCer is modulated by the peptide secondary and tertiary structure and the local environment. Therefore, definition of the conformation of this novel extended gp41 membrane proximal region, containing the conserved peptide P1 and the Ca(2+) binding site, could help designing an immunogen efficient at inducing neutralizing anti-HIV-1 antibodies.     

Non-natural glycosphingolipids and structurally simpler analogues bind HIV-1 recombinant Gp120

Interactions of recombinant gp120 (rgp120) with non-natural glycosphingolipids (GSLs) and structurally simpler analogues have been studied using a competitive adhesion assay. Conjugates of cellobiosyl ceramide and melibiosyl ceramide were synthetically prepared as water-soluble GSL analogues. These ligands were screened against a panel of biologically relevant analogues, and the results show that their interactions with rgp120 are comparable to natural cellular receptors. Glycolipid interactions with rgp120 were probed further by the synthesis and testing of structurally simpler analogues that were obtained by reductive amination of lactose, cellobiose, and melibiose with a biotinylated amino ethylene glycol moiety. RGp120 did not recognize conjugates lacking a lipid component. However, palmitoylation of the secondary amino alditols yielded compounds with comparable rgp120 affinity to the natural cellular receptor, galactosyl ceramide (GalCer). Taken together, the SAR showed that both a hydrophobic and a hydrophilic component are required for rgp120 recognition. Moreover, structural variability in the carbohydrate headgroup did not significantly alter rgp120 recognition indicating that this interaction is not highly specific.     

Synthesis of novel, multivalent glycodendrimers as ligands for HIV-1 gp120

Multivalent neoglycoconjugates are valuable tools for studying carbohydrate-protein interactions. To study the interaction of HIV-1 gp120 with its reported alternate glycolipid receptors, galactosyl ceramide (GalCer) and sulfatide, galactose- and sulfated galactose-derivatized dendrimers were synthesized, analyzed as ligands for rgp120 by surface plasmon resonance, and tested for their ability to inhibit HIV-1 infection of CXCR4- and CCR5-expressing indicator cells. Four different series of glycodendrimers were made by amine coupling spacer-arm derivatized galactose residues, either sulfated or nonsulfated, to poly(propylenimine) dendrimers, generations 1-5. One series of glycodendrimers was prepared from the ceramide saccharide derivative of purified natural GalCer, and another was from chemically synthesized 3-(beta-D-galactopyranosylthio)propionic acid. Synthesis of 3-sulfogalactopyranosyl-derivatized dendrimers was accomplished using the novel compound, 3-(beta-D-3-sulfogalactopyranosylthio)propionic acid. The fourth series was made by random sulfation of the 3-(beta-D-galactopyranosylthio)propionic acid functionalized dendrimers. Structures of the carbohydrate moieties were confirmed by NMR, and the average molecular weights and polydispersities of the different glycodendrimers were determined using MALDI-TOF MS. Surface plasmon resonance studies found that rgp120 IIIB bound to the derivatized dendrimers tested with nanomolar affinity, and to dextran sulfate with picomolar affinity. In vitro studies of the effectiveness of these compounds at inhibiting infection of U373-MAGI-CCR5 cells by HIV-1 Ba-L indicated that the sulfated glycodendrimers were better inhibitors than the nonsulfated glycodendrimers, but not as effective as dextran sulfate.     

Synthesis, gp120 binding and anti-HIV activity of fatty acid esters of 1,1-linked disaccharides

Inspired by the anti-human immunodeficiency virus (HIV) activity of analogues of β-galactosylceramide (GalCer), a set of mono- and di-saccharide fatty acid esters were designed as GalCer mimetics and their binding to the V3 loop peptide of HIV-1 and anti-HIV activity evaluated. 1,1-linked Gal-Man and Glu-Man disaccharides with an ester on the Man subunit bound the V3 loop peptide and inhibited HIV infectivity in single round infection assays with the TZM-bl cell line. IC(50)'s were in the 50 μM range with no toxicity to the cells at concentrations up to 200 μM. These compounds appear to inhibit virus entry at early steps in viral infection since they were inactive if added post viral entry. Although these compounds were found to bind to the V3 loop peptide of gp120, it is not clear that this interaction is responsible for their anti-HIV activity because the relative binding affinity of closely related analogues did not correlate with their antiviral behavior. The low cytotoxicity of these 1,1-linked disaccharide fatty acid esters, combined with the easy accessibility to structurally diverse analogues make these molecules attractive leads for new topical anti-viral agents.     

Synthesis of novel glycolipids that bind HIV-1 Gp120.

As part of a research effort to design and prepare high affinity ligands for the galactosyl ceramide (GalCer) binding site on the HIV cell surface glycoprotein, gp120, several GalCer analogues have been prepared and characterized. The molecular design of analogues permits independent variations of the carbohydrate, the length of a hydrophilic spacer between the ligand and the lipid, and the composition of the hydrophobic lipid chains. Five different galactosyl analogues were synthesized having hydrophilic spacers of tri-, tetra-, and penta-ethylene glycol separating the carbohydrate from the lipid region which has either oleoyl or stearoyl lipid chains. The synthetic design allows for a convergent synthesis of the three components of the glycolipid conjugate. The structural characterization includes the proton and carbon chemical shifts, which were assigned after analysis of 1D and 2D NMR spectra.     

Synthesis of single- and double-chain fluorocarbon and hydrocarbon galactosyl amphiphiles and their anti-HIV-1 activity

Galactosylceramide (GalCer) is an alternative receptor allowing HIV-1 entry into CD4(-)/GalCer(+) cells. This glycosphingolipid recognizes the V3 loop of HIV gp120, which plays a key role in the fusion of the HIV envelope and cellular membrane. To inhibit HIV uptake and infection, we designed and synthesized analogs of GalCer. These amphiphiles and bolaamphiphiles consist of single and double hydrocarbon and/or fluorocarbon chain beta-linked to galactose and galactosamine. They derive from serine (GalSer), cysteine (GalCys), and ethanolamine (GalAE). The anti-HIV activity and cytotoxicity of these galactolipids were evaluated in vitro on CEM-SS (a CD4(+) cell line), HT-29, a CD4(-) cell line expressing high levels of GalCer receptor, and/or HT29 genetically modified to express CD4. GalSer and GalAE derivatives, tested in aqueous medium or as part of liposome preparation, showed moderate anti-HIV-1 activities (IC50 in the 20-220 microM range), whereas none of the GalCys derivatives was found to be active. Moreover, only some of these anti-HIV active analogs inhibited the binding of [3H]suramin (a polysulfonyl compound which displays a high affinity for the V3 loop) to SPC3, a synthetic peptide which contains the conserved GPGRAF region of the V3 loop. Our results most likely indicate that the neutralization of the virion through masking of this conserved V3 loop region is not the only mechanism involved in the HIV-1 antiviral activity of our GalCer analogs.     

HIV-1 gp41 envelope residues 650-685 exposed on native virus act as a lectin to bind epithelial cell galactosyl ceramide

The initial step in the interaction between human immunodeficiency virus (HIV-1) and epithelial cells is the binding of HIV-1 envelope glycoproteins to the epithelial cell galactosyl ceramide (GalCer). Here we show that HIV-1 envelope gp41 residues 650-685 bind GalCer in a galactose-specific manner. The gp41 residues that display this lectin activity are highly conserved among HIV-1 isolates and constitute three regions: residues 650-661, which encompass a charged helix; residues 662-667, referred to as the conserved epitope ELDKWA, the epitope recognized by antibodies that neutralize HIV-1 entry in epithelial and CD4(+)-mononucleated cells; and residues 668-685, a hydrophobic Trp-rich sequence that stabilizes the structure of the galactose binding site. Similar to other galactose-specific lectins, the gp41 lectin site is active only as an oligomer. Finally the orientation of the galactose toward the gp41 lectin site appears to be controlled by the lipid microenvironment of the epithelial membrane. From the experimental data we construct a theoretical model of the interaction between gp41 and GalCer based on thermodynamic considerations. This model integrates the dynamics and the spatial organization of the viral envelope glycoproteins, GalCer organized in raft microdomains in the apical region of the epithelial cell membrane and the interfacial water. Characterization of the minimal sequence and structure of gp41 in direct interaction with GalCer may help unravel the still unknown immunogenic determinant able to elicit antibodies against ELDKWA and target of one of the rare neutralizing antibodies against gp41.     

Human immunodeficiency virus type 1 infection of SK-N-MC cells: domains of gp120 involved in entry into a CD4-negative, galactosyl ceramide/3' sulfo-galactosyl ceramide-positive cell line.

The primary receptor for human immunodeficiency virus (HIV) is the CD4 molecule; however, in vitro evidence suggests that a neutral glycolipid, galactosyl ceramide (GalCer) or a derivative molecule, 3' sulfogalactosyl ceramide (GalS), may serve as an alternative receptor for HIV type 1 (HIV-1) in cells of neural and colonic origin. Biochemical studies have demonstrated that recombinant gp120 envelope protein binds to GalCer/GalS in both solid-phase enzyme-linked immunosorbent assay and high-performance thin-layer chromatography overlays. We have used the SK-N-MC cell line, a CD4-negative, GalCer/GalS-positive cell line previously characterized as susceptible to HIV-1 infection, to identify virus isolates with either a positive infection phenotype, HIVHxB2, or a negative infection phenotype, HIV-1(89.6). Using a solid-phase virus binding assay, we determined the level of restriction in HIV-1(89.6) infection to be at the level of virus-glycolipid binding. Furthermore, using HIV-1HxB2-HIV-1(89.6) chimeras, we have identified a 193-amino-acid fragment from the envelope region of HIV-1HxB2 containing the V3, V4, and V5 regions which confers a positive infection phenotype on the HIV-1(89.6) background. Recombinant viruses which separate this 193-amino-acid fragment into two distinct chimeras are each able to confer a positive infection phenotype on the background of HIV89.6, suggesting that a stable GalCer/GalS-envelope interaction is dependent on the conformation of the envelope protein in the context of the viral membrane. Alternatively, the GalCer/GalS-gp120 bond may involve multiple sites on the oligomeric envelope protein.     

Synthesis of biotinylated glycoconjugates and their use in a novel ELISA for direct comparison of HIV-1 Gp120 recognition of GalCer and related carbohydrate analogues

As part of our program directed toward the design and synthesis of high-affinity ligands for the GalCer-binding site on the HIV cell surface glycoprotein, gp120, we required a reliable method for qualitatively assessing relative binding affinities for related analogues. Due to the hydrophilic nature of these synthetic conjugates, difficulties were encountered with typical ELISA methods, which rely upon hydrophobic interactions to anchor the ligand to a microtiter plate. Other types of assays were also problematic due to nonspecific binding of gp120. Therefore, we developed a general method for plating water-soluble ligands on microtiter plates using biotin/NeutrAvidin recognition for adhesion. A water-soluble GalCer analogue was prepared by conjugating psychosine to biotin using a novel tetraethylene glycol linker. In a similar manner, LacCer and GlcCer analogues were prepared and these conjugates were plated into microtiter wells containing NeutrAvidin. Unoccupied sites were blocked using biotin functionalized as a primary amide. Gp120 binding to galactosyl sphingosine, GalSph (19), GlcSph (22), and LacSph (23) conjugates was assessed through incubation with recombinant HRP-gp120. It was determined that LacSph has the strongest interaction with gp120. The binding affinities of GalSph and GlcSph were similar to each other and less strong than LacSph. These data contradict earlier studies where HPTLC showed that LacCer and GlcCer do not significantly bind gp120. They also contradict liposome-based assays that reported psychosine is not recognized by gp120. The extent of plating for each biotinylated molecule was quantified using HRP-biotin, allowing direct comparison of ligand plating efficiencies for the first time. Several other synthetic biotin conjugates were prepared and tested, demonstrating the feasibility of performing ELISA on water-soluble ligands.     

Determinants of human immunodeficiency virus type 1 entry in the CDR2 loop of the CD4 glycoprotein.

Various roles for the viral receptor, CD4, have been proposed in facilitating human immunodeficiency virus type 1 (HIV-1) entry, including virion binding to the target cell and the induction of conformational changes in the viral envelope glycoproteins required for the membrane fusion reaction. Here, we compare the structural requirements in the CDR2-like loop of CD4 domain 1, the major contact site of the gp120 envelope glycoprotein, for gp120 binding and virus entry. For every CD4 mutant examined, the level of cell surface expression and the gp120 binding affinity were sufficient to explain the relative ability to function as a viral receptor. The decrease in relative infectibility associated with decreased gp120 binding affinity was more pronounced at lower cell surface CD4 concentrations. These results imply that both receptor density and affinity determine the efficiency of HIV-1 entry and that specific structures in the CD4 residues examined are probably not required for HIV-1 entry functions other than gp120 binding.     

Molecular mechanism of HIV-1 gp120 mutations that reduce CD4 binding affinity

The interaction of the HIV-1 fusion protein gp120 with its cellular receptor CD4 represents a crucial step of the viral infection process, thus rendering gp120 a promising target for the intervention with anti-HIV drugs. Naturally occurring mutations of gp120, however, can decrease its affinity for anti-infective ligands like therapeutic antibodies or soluble CD4. To understand this phenomenon on a structural level, we performed molecular dynamics simulations of two gp120 variants (termed gp120_3-2 and gp120_2-1), which exhibit a significantly decreased binding of soluble CD4. In both variants, the exchange of a nonpolar residue byglutamate was identified as an important determinant for reduced binding. However, those glutamates are located at different sequence positions and affect different steps of the recognition process: E471 in gp120_3-2 predominantly affects the CD4-bound conformation, whereas E372 in gp120_2-1 mainly modulates the conformational sampling of free gp120. Despite these differences, there exists an interesting similarity between the two variants: both glutamates exert their function by modulating the conformation and interactions of glycine-rich motifs (G366–G367, G471–G473) resulting in an accumulation of binding incompetent gp120 conformations or a loss of intermolecular gp120–CD4 hydrogen bonds. Thus, the present data suggests that interference with the structure and dynamics of glycine-rich stretches might represent a more widespread mechanism, by which gp120 mutations reduce binding affinity. This knowledge should be helpful to predict the resistance of novel gp120 mutations or to design gp120–ligands with improved binding properties.

An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:41     

Structural and functional characterization of an epitope in the conserved C-terminal region of HIV-1 gp120.

Through an integrated study of the reactivity of a monoclonal antibody, 803-15.6, with synthetic peptides and native recombinant HIV-1 envelope glycoprotein gp120, we have obtained structure-functional information on a region of rgp120 not yet elucidated by X-ray crystallography. mAb 803-15.6 binds with high affinity and broad cross-clade specificity to the conserved C-terminal region (amino acids 502-516) of HIV-1 rgp120. Phage display selection from a random peptide library identified the core binding motif as AXXKXRH, homologous to residues 502-508. Using quantitative binding analyses, the affinity of mAb 803-15.6 for native, monomeric recombinant gp120HXB2 (rgp120) was found to be similar to that for the synthetic gp120 peptide (502-516). Circular dichroism studies indicate that the synthetic peptide largely has a random coil conformation in solution. The results therefore suggest that the 803-15.6 epitope is fully accessible on rgp120 and that this region of rgp120 is as flexible as the synthetic peptide. Residues 502-504 are on the edge of a putative gp41 binding site that has been postulated to change conformation on CD4 binding. However, the affinity of mAb 803-15.6 for rgp120 is not affected by binding of CD4 and vice-versa. These results suggest either that the 502-504 region does not change conformation upon CD4 binding, or that recombinant gp120 does not undergo the same changes as occur in the native viral gp120-gp41 oligomer. The detailed characterization of the 803-15.6 epitope may be useful for further study of the role of the C5 region of gp120 in the viral attachment and fusion process.     

A region in domain 1 of CD4 distinct from the primary gp120 binding site is involved in HIV infection and virus-mediated fusion.

The high affinity binding site for human immunodeficiency virus (HIV) envelope glycoprotein gp120 resides within the amino-terminal domain (D1) of CD4. Mutational and antibody epitope analyses have implicated the region encompassing residues 40-60 in D1 as the primary binding site for gp120. Outside of this region, a single residue substitution at position 87 abrogates syncytium formation without affecting gp120 binding. We describe two groups of CD4 monoclonal antibodies (mAbs) which recognize distinct epitopes associated with these regions in D1. These mAbs distinguish between the gp120 binding event and virus infection and virus-induced cell fusion. One cluster of mAbs, which bind at or near the high affinity gp120 binding site, blocked gp120 binding to CD4 and, as expected, also blocked HIV infection of CD4+ cells and virus-induced syncytium formation. A second cluster of mAbs, which recognize the CDR-3 like loop, did not block gp120 binding as demonstrated by their ability to form ternary complexes with CD4 and gp120. Yet, these mAbs strongly inhibited HIV infection of CD4+ cells and HIV-envelope/CD4-mediated syncytium formation. The structure of D1 has recently been solved at atomic resolution and in its general features resembles IgVk regions as predicted from sequence homology and mAb epitopes. In the D1 structure, the regions recognized by these two groups of antibodies correspond to the C'C" (Ig CDR2) and FG (Ig CDR3) hairpin loops, respectively, which are solvent-exposed beta turns protruding in two different directions on a face of D1 distal to the D2 domain. This face is straddled by the longer BC (Ig CDR1) loop which bisects the plain formed by C'C' and FG. This structure is consistent with C'C' and FG forming two distinct epitope clusters within D1. We conclude that the initial interaction between gp120 and CD4 is not sufficient for HIV infection and syncytium formation and that CD4 plays a critical role in the subsequent virus-cell and cell-cell membrane fusion events. We propose that the initial binding of CD4 to gp120 induces conformational changes in gp120 leading to subsequent interactions of the FG loop with other regions in gp120 or with the fusogenic gp41 potion of the envelope gp160 glycoprotein.     

Designed cyclic permutants of HIV-1 gp120: implications for envelope trimer structure and immunogen design

Most HIV-1 broadly neutralizing antibodies are directed against the gp120 subunit of the env surface protein. Native env consists of a trimer of gp120-gp41 heterodimers, and in contrast to monomeric gp120, preferentially binds CD4 binding site (CD4bs)-directed neutralizing antibodies over non-neutralizing ones. Some cryo-electron tomography studies have suggested that the V1V2 loop regions of gp120 are located close to the trimer interface. We have therefore designed cyclically permuted variants of gp120 with and without the h-CMP and SUMO2a trimerization domains inserted into the V1V2 loop. h-CMP-V1cyc is one such variant in which residues 153 and 142 are the N- and C-terminal residues, respectively, of cyclically permuted gp120 and h-CMP is fused to the N-terminus. This molecule forms a trimer under native conditions and binds CD4 and the neutralizing CD4bs antibodies b12 with significantly higher affinity than wild-type gp120. It binds non-neutralizing CD4bs antibody F105 with lower affinity than gp120. A similar derivative, h-CMP-V1cyc1, bound the V1V2 loop-directed broadly neutralizing antibodies PG9 and PG16 with ～20-fold higher affinity than wild-type JRCSF gp120. These cyclic permutants of gp120 are properly folded and are potential immunogens. The data also support env models in which the V1V2 loops are proximal to the trimer interface.     

Galactosylceramide domain microstructure: impact of cholesterol and nucleation/growth conditions

Galactosylceramide (GalCer), a glycosphingolipid, is believed to exist in the extracellular leaflet of cell membranes in nanometer-sized domains or rafts. The local clustering of GalCer within rafts is thought to facilitate the initial adhesion of certain viruses, including HIV-1, and bacteria to cells through multivalent interactions between receptor proteins (gp120 for HIV-1) and GalCer. Here we use atomic force microscopy (AFM) to study the effects of cholesterol on solid-phase GalCer domain microstructure and miscibility with a fluid lipid 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) in supported lipid bilayers. Using "slow-cooled vesicle fusion" to prepare the supported lipid bilayers, we were able to overcome the nonequilibrium effects of the substrate (verified by comparison to results for giant unilamellar vesicles) and accurately quantify the dramatic effect of cholesterol on the GalCer domain surface area/perimeter ratio (A(D)/P) and DLPC-GalCer miscibility. We compare these results to a supported lipid bilayer system in which the bilayer is rapidly cooled (nonequilibrium conditions), "quenched vesicle fusion", and find that the microstructures are remarkably similar above a cholesterol mol fraction of approximately 0.06. We determined that GalCer domains were contained in one leaflet distal to the mica substrate through qualitative binding experiments with Trichosanthes kirilowii agglutinin (TKA), a galactose-specific lectin, and AFM of Langmuir-Blodgett deposited GalCer/DLPC supported lipid bilayers. In addition, GalCer domains in bilayers containing cholesterol rearranged upon tip-sample contact. Our results further serve to clarify why discrepancies exist between different model membrane systems and between model membranes and cell membranes. In addition, these results offer new insight into the effect of cholesterol and surrounding lipid on domain microstructure and behavior. Finally, our observations may be pertinent to cell membrane structure, dynamics, and HIV infection.