Siglec-1 Is a Novel Dendritic Cell Receptor That Mediates HIV-1 Trans-Infection Through Recognition of Viral Membrane Gangliosides

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of immunity against pathogens. However, HIV-1 spread is strongly enhanced in clusters of DCs and CD4⁺ T cells. Uninfected DCs capture HIV-1 and mediate viral transfer to bystander CD4⁺ T cells through a process termed trans-infection. Initial studies identified the C-type lectin DC-SIGN as the HIV-1 binding factor on DCs, which interacts with the viral envelope glycoproteins. Upon DC maturation, however, DC-SIGN is down-regulated, while HIV-1 capture and trans-infection is strongly enhanced via a glycoprotein-independent capture pathway that recognizes sialyllactose-containing membrane gangliosides. Here we show that the sialic acid-binding Ig-like lectin 1 (Siglec-1, CD169), which is highly expressed on mature DCs, specifically binds HIV-1 and vesicles carrying sialyllactose. Furthermore, Siglec-1 is essential for trans-infection by mature DCs. These findings identify Siglec-1 as a key factor for HIV-1 spread via infectious DC/T-cell synapses, highlighting a novel mechanism that mediates HIV-1 dissemination in activated tissues.     

Dendritic cell-mediated trans-enhancement of human immunodeficiency virus type 1 infectivity is independent of DC-SIGN

Dendritic cells (DCs) enhance human immunodeficiency virus type 1 (HIV-1) infection of CD4(+) T lymphocytes in trans. The C-type lectin DC-SIGN, expressed on DCs, binds to the HIV-1 envelope glycoprotein gp120 and confers upon some cell lines the capacity to enhance trans-infection. Using a short hairpin RNA approach, we demonstrate that DC-SIGN is not required for efficient trans-enhancement by DCs. In addition, the DC-SIGN ligand mannan and an anti-DC-SIGN antibody did not inhibit DC-mediated enhancement. HIV-1 particles were internalized and were protected from protease treatment following binding to DCs, but not from binding to DC-SIGN-expressing Raji cells. Thus, DC-SIGN is not required for DC-mediated trans-enhancement of HIV infectivity.     

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.     

DC-SIGN interactions with human immunodeficiency virus type 1 and 2 and simian immunodeficiency virus

Dendritic cells (DCs) efficiently bind and transmit human immunodeficiency virus (HIV) to cocultured T cells and so may play an important role in HIV transmission. DC-SIGN, a novel C-type lectin that is expressed in DCs, has recently been shown to bind R5 HIV type 1 (HIV-1) strains and a laboratory-adapted X4 strain. To characterize the interaction of DC-SIGN with primate lentiviruses, we investigated the structural determinants of DC-SIGN required for virus binding and transmission to permissive cells. We constructed a panel of DC-SIGN mutants and established conditions which allowed comparable cell surface expression of all mutants. We found that R5, X4, and R5X4 HIV-1 isolates as well as simian immunodeficiency and HIV-2 strains bound to DC-SIGN and could be transmitted to CD4/coreceptor-positive cell types. DC-SIGN contains a single N-linked carbohydrate chain that is important for efficient cell surface expression but is not required for DC-SIGN-mediated virus binding and transmission. In contrast, C-terminal deletions removing either the lectin binding domain or the repeat region abrogated DC-SIGN function. Trypsin-EDTA treatment inhibited DC-SIGN mediated infection, indicating that virus was maintained at the surface of the DC-SIGN-expressing cells used in this study. Finally, quantitative fluorescence-activated cell sorting analysis of AU1-tagged DC-SIGN revealed that the efficiency of virus transmission was strongly affected by variations in DC-SIGN expression levels. Thus, variations in DC-SIGN expression levels on DCs could greatly affect the susceptibility of human individuals to HIV infection.     

DC-SIGN points the way to a novel mechanism for HIV-1 transmission

Dendritic cell (DC)-specific intercellular adhesion molecule 3 (ICAM-3) grabbing nonintegrin (DC-SIGN), a recently discovered type II transmembrane protein on DCs with a C-type lectin extracellular domain, is capable of binding ICAM-3 on resting T cells in the secondary lymphoid organs, providing the initial contact between these cells during the establishment of cell-mediated immunity. DC-SIGN also binds the HIV-1 envelope glycoprotein gp120 but does not function as a receptor for viral entry into DCs. Instead, DC-SIGN allows DCs in the peripheral mucosa to carry HIV-1 through the lymphatics in a "Trojan horse" fashion, where it is eventually delivered to the T cells. Also, the period of infectivity of HIV-1 is increased by several days as a result of DC-SIGN-gp120 binding, allowing for efficient trans-infection of T cells on DC arrival. The discovery of a cluster of related genes colocalized with DC-SIGN on chromosome 19p13.2-3, all displaying complex alternative splicing patterns, has led to a reexamination of the mechanisms underlying both the interactions between antigen-presenting cells (APCs) and T cells and the pathogenesis of HIV-1 infection.     

Langerin is a natural barrier to HIV-1 transmission by Langerhans cells

Human immunodeficiency virus-1 (HIV-1) is primarily transmitted sexually. Dendritic cells (DCs) in the subepithelium transmit HIV-1 to T cells through the C-type lectin DC-specific intercellular adhesion molecule (ICAM)-3-grabbing nonintegrin (DC-SIGN). However, the epithelial Langerhans cells (LCs) are the first DC subset to encounter HIV-1. It has generally been assumed that LCs mediate the transmission of HIV-1 to T cells through the C-type lectin Langerin, similarly to transmission by DC-SIGN on dendritic cells (DCs). Here we show that in stark contrast to DC-SIGN, Langerin prevents HIV-1 transmission by LCs. HIV-1 captured by Langerin was internalized into Birbeck granules and degraded. Langerin inhibited LC infection and this mechanism kept LCs refractory to HIV-1 transmission; inhibition of Langerin allowed LC infection and subsequent HIV-1 transmission. Notably, LCs also inhibited T-cell infection by viral clearance through Langerin. Thus Langerin is a natural barrier to HIV-1 infection, and strategies to combat infection must enhance, preserve or, at the very least, not interfere with Langerin expression and function.     

Colorectal Mucus Binds DC-SIGN and Inhibits HIV-1 Trans-Infection of CD4+ T-Lymphocytes

Bodily secretions, including breast milk and semen, contain factors that modulate HIV-1 infection. Since anal intercourse caries one of the highest risks for HIV-1 transmission, our aim was to determine whether colorectal mucus (CM) also contains factors interfering with HIV-1 infection and replication. CM from a number of individuals was collected and tested for the capacity to bind DC-SIGN and inhibit HIV-1 cis- or trans-infection of CD4⁺ T-lymphocytes. To this end, a DC-SIGN binding ELISA, a gp140 trimer competition ELISA and HIV-1 capture/ transfer assays were utilized. Subsequently we aimed to identify the DC-SIGN binding component through biochemical characterization and mass spectrometry analysis. CM was shown to bind DC-SIGN and competes with HIV-1 gp140 trimer for binding. Pre-incubation of Raji-DC-SIGN cells or immature dendritic cells (iDCs) with CM potently inhibits DC-SIGN mediated trans-infection of CD4⁺ T-lymphocytes with CCR5 and CXCR4 using HIV-1 strains, while no effect on direct infection is observed. Preliminary biochemical characterization demonstrates that the component seems to be large (>100kDa), heat and proteinase K resistant, binds in a α1–3 mannose independent manner and is highly variant between individuals. Immunoprecipitation using DC-SIGN-Fc coated agarose beads followed by mass spectrometry indicated lactoferrin (fragments) and its receptor (intelectin-1) as candidates. Using ELISA we showed that lactoferrin levels within CM correlate with DC-SIGN binding capacity. In conclusion, CM can bind the C-type lectin DC-SIGN and block HIV-1 trans-infection of both CCR5 and CXCR4 using HIV-1 strains. Furthermore, our data indicate that lactoferrin is a DC-SIGN binding component of CM. These results indicate that CM has the potential to interfere with pathogen transmission and modulate immune responses at the colorectal mucosa.     

Binding of the mannose-specific lectin, griffithsin, to HIV-1 gp120 exposes the CD4-binding site

The glycans on HIV-1 gp120 play an important role in shielding neutralization-sensitive epitopes from antibody recognition. They also serve as targets for lectins that bind mannose-rich glycans. In this study, we investigated the interaction of the lectin griffithsin (GRFT) with HIV-1 gp120 and its effects on exposure of the CD4-binding site (CD4bs). We found that GRFT enhanced the binding of HIV-1 to plates coated with anti-CD4bs antibodies b12 and b6 or the CD4 receptor mimetic CD4-IgG2. The average enhancement of b12 or b6 binding was higher for subtype B viruses than for subtype C, while for CD4-IgG2, it was similar for both subtypes, although lower than observed with antibodies. This GRFT-mediated enhancement of HIV-1 binding to b12 was reflected in synergistic neutralization for 2 of the 4 viruses tested. The glycan at position 386, which shields the CD4bs, was involved in both GRFT-mediated enhancement of binding and neutralization synergism between GRFT and b12. Although GRFT enhanced CD4bs exposure, it simultaneously inhibited ligand binding to the coreceptor binding site, suggesting that GRFT-dependent enhancement and neutralization utilize independent mechanisms. This study shows for the first time that GRFT interaction with gp120 exposes the CD4bs through binding the glycan at position 386, which may have implications for how to access this conserved site.     

Host-soluble galectin-1 promotes HIV-1 replication through a direct interaction with glycans of viral gp120 and host CD4

Sexual transmission of HIV-1 requires virus adsorption to a target cell, typically a CD4(+) T lymphocyte residing in the lamina propria, beneath the epithelium. To escape the mucosal clearance system and reach its target cells, HIV-1 has evolved strategies to circumvent deleterious host factors. Galectin-1, a soluble lectin found in the underlayers of the epithelium, increases HIV-1 infectivity by accelerating its binding to susceptible cells. By comparison, galectin-3, a family member expressed by epithelial cells and part of the mucosal clearance system, does not perform similarly. We show here that galectin-1 directly binds to HIV-1 in a β-galactoside-dependent fashion through recognition of clusters of N-linked glycans on the viral envelope gp120. Unexpectedly, this preferential binding of galectin-1 does not rely on the primary sequence of any particular glycans. Instead, glycan clustering arising from the tertiary structure of gp120 hinders its binding by galectin-3. Increased polyvalency of a specific ligand epitope is a common strategy for glycans to increase their avidity for lectins. In this peculiar occurrence, glycan clustering is instead exploited to prevent binding of gp120 by galectin-3, which would lead to a biological dead-end for the virus. Our data also suggest that galectin-1 binds preferentially to CD4, the host receptor for gp120. Together, these results suggest that HIV-1 exploits galectin-1 to enhance gp120-CD4 interactions, thereby promoting virus attachment and infection events. Since viral adhesion is a rate-limiting step for HIV-1 entry, modulation of the gp120 interaction with galectin-1 could thus represent a novel approach for the prevention of HIV-1 transmission.     

High mannose-binding lectin with preference for the cluster of alpha1-2-mannose from the green alga Boodlea coacta is a potent entry inhibitor of HIV-1 and influenza viruses

The complete amino acid sequence of a lectin from the green alga Boodlea coacta (BCA), which was determined by a combination of Edman degradation of its peptide fragments and cDNA cloning, revealed the following: 1) B. coacta used a noncanonical genetic code (where TAA and TAG codons encode glutamine rather than a translation termination), and 2) BCA consisted of three internal tandem-repeated domains, each of which contains the sequence motif similar to the carbohydrate-binding site of Galanthus nivalis agglutinin-related lectins. Carbohydrate binding specificity of BCA was examined by a centrifugal ultrafiltration-HPLC assay using 42 pyridylaminated oligosaccharides. BCA bound to high mannose-type N-glycans but not to the complex-type, hybrid-type core structure of N-glycans or oligosaccharides from glycolipids. This lectin had exclusive specificity for α1-2-linked mannose at the nonreducing terminus. The binding activity was enhanced as the number of terminal α1-2-linked mannose substitutions increased. Mannobiose, mannotriose, and mannopentaose were incapable of binding to BCA. Thus, BCA preferentially recognized the nonreducing terminal α1-2-mannose cluster as a primary target. As predicted from carbohydrate-binding propensity, this lectin inhibited the HIV-1 entry into the host cells at a half-maximal effective concentration of 8.2 nm. A high association constant (3.71 × 10(8) M(-1)) of BCA with the HIV envelope glycoprotein gp120 was demonstrated by surface plasmon resonance analysis. Moreover, BCA showed the potent anti-influenza activity by directly binding to viral envelope hemagglutinin against various strains, including a clinical isolate of pandemic H1N1-2009 virus, revealing its potential as an antiviral reagent.     

Binding of human immunodeficiency virus type 1 to immature dendritic cells can occur independently of DC-SIGN and mannose binding C-type lectin receptors via a cholesterol-dependent pathway

Interactions of human immunodeficiency virus type 1 (HIV-1) with immature dendritic cells (DC) are believed to be multifactorial and involve binding to the CD4 antigen, DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), mannose binding C-type lectin receptors (MCLR), and heparan sulfate proteoglycans (HSPG). In this study we assessed the relative contributions of these previously defined virus attachment factors to HIV binding and accumulation in DC and the subsequent transfer of the bound virus particle to CD4(+) T cells. Using competitive inhibitors of HIV-1 attachment to DC, we have identified the existence of DC-SIGN-, MCLR-, and HSPG-independent mechanism(s) of HIV attachment and internalization. Furthermore, virus particles bound by DC independently of CD4, DC-SIGN, MCLR, and HSPG are efficiently transmitted to T cells. Treatment of virus particles with the protease subtilisin or treatment of immature DC with trypsin significantly reduced virus binding, thus demonstrating the role of HIV envelope glycoprotein interactions with unidentified DC-surface factor(s). Finally, this DC-mediated virus binding and internalization are dependent on lipid rafts. We propose that pathways to HIV-1 attachment and uptake in DC exhibit functional redundancy; that is, they are made up of multiple independent activities that can, at least in part, compensate for one another.     

Siglecs facilitate HIV-1 infection of macrophages through adhesion with viral sialic acids

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) infects macrophages effectively, despite relatively low levels of cell surface-expressed CD4. Although HIV-1 infections are defined by viral tropisms according to chemokine receptor usage (R5 and X4), variations in infection are common within both R5- and X4-tropic viruses, indicating additional factors may contribute to viral tropism. METHODOLOGY AND PRINCIPAL FINDINGS: Using both solution and cell surface binding experiments, we showed that R5- and X4-tropic HIV-1 gp120 proteins recognized a family of I-type lectin receptors, the Sialic acid-binding immunoglobulin-like lectins (Siglec). The recognition was through envelope-associated sialic acids that promoted viral adhesion to macrophages. The sialic acid-mediated viral-host interaction facilitated both R5-tropic pseudovirus and HIV-1(BaL) infection of macrophages. The high affinity Siglec-1 contributed the most to HIV-1 infection and the variation in Siglec-1 expression on primary macrophages from different donors was associated statistically with sialic acid-facilitated viral infection. Furthermore, envelope-associated sialoglycan variations on various strains of R5-tropic viruses also affected infection. CONCLUSIONS AND SIGNIFICANCE OF THE FINDINGS: Our study showed that sialic acids on the viral envelope facilitated HIV-1 infection of macrophages through interacting with Siglec receptors, and the expression of Siglec-1 correlated with viral sialic acid-mediated host attachment. This glycan-mediated viral adhesion underscores the importance of viral sialic acids in HIV infection and pathogenesis, and suggests a novel class of antiviral compounds targeting Siglec receptors.     

Microvirin,a Novel α(1,2)-Mannose-specific Lectin Isolated from Microcystis aeruginosa,Has Anti-HIV-1 Activity Comparable with That of Cyanovirin-N but a Much Higher Safety Profile

Microvirin (MVN), a recently isolated lectin from the cyanobacterium Microcystis aeruginosa PCC7806, shares 33% identity with the potent anti-human immunodeficiency virus (HIV) protein cyanovirin-N (CV-N) isolated from Nostoc ellipsosporum, and both lectins bind to similar carbohydrate structures. MVN is able to inhibit infection by a wide variety of HIV-1 laboratory-adapted strains and clinical isolates of different tropisms and subtypes in peripheral blood mononuclear cells. MVN also inhibits syncytium formation between persistently HIV-1-infected T cells and uninfected CD4⁺ T cells and inhibits DC-SIGN-mediated HIV-1 binding and transmission to CD4⁺ T cells. Long term passaging of HIV-1 exposed to dose-escalating concentrations of MVN resulted in the selection of a mutant virus with four deleted high mannose-type glycans in the envelope gp120. The MVN-resistant virus was still highly sensitive to various other carbohydrate binding lectins (e.g. CV-N, HHA, GNA, and UDA) but not anymore to the carbohydrate-specific 2G12 monoclonal antibody. Importantly, MVN is more than 50-fold less cytotoxic than CV-N. Also in sharp contrast to CV-N, MVN did not increase the level of the activation markers CD25, CD69, and HLA-DR in CD4⁺ T lymphocytes, and subsequently, MVN did not enhance viral replication in pretreated peripheral blood mononuclear cells. Therefore, MVN may qualify as a useful lectin for potential microbicidal use based on its broad and potent antiviral activity and virtual lack of any stimulatory properties and cellular toxicity.     

Absence of association between mannose-binding lectin gene polymorphisms and HIV-1 infection in a Colombian population

Mannose-binding lectin (MBL) is a calcium-dependent lectin shown to play an important role in innate immunity to infection by activating the classical complement pathway and phagocytosis. In vitro studies have shown that MBL is able to bind to the gp120 HIV-1 surface antigen, and variants of the gene are associated with increased risk of HIV infection among Scandinavians. We investigated the association of genetic MBL variants and HIV-1 infection in 278 Colombian HIV-infected and control individuals. MBL genotype frequencies were similar for both groups, and no association was detected between MBL alleles B, C, and D and susceptibility to HIV-1 infection ( P=1.0). Since there is a well-documented link between the tested MBL alleles and very low MBL serum concentration, these results do not support the hypothesis that MBL levels are a risk factor for HIV-1 infection in Colombia.     

Dendritic Cell Immunoreceptor Is a New Target for Anti-AIDS Drug Development: Identification of DCIR/HIV-1 Inhibitors

The HIV-1 pandemic continues to expand while no effective vaccine or cure is yet available. Existing therapies have managed to limit mortality and control viral proliferation, but are associated with side effects, do not cure the disease and are subject to development of resistance. Finding new therapeutic targets and drugs is therefore crucial. We have previously shown that the dendritic cell immunoreceptor (DCIR), a C-type lectin receptor expressed on dendritic cells (DCs), acts as an attachment factor for HIV-1 to DCs and contributes to HIV-1 transmission to CD4⁺ T lymphocytes (CD4TL). Directly involved in HIV-1 infection, DCIR is expressed in apoptotic or infected CD4TL and promotes trans-infection to bystander cells. Here we report the 3D modelling of the extracellular domain of DCIR. Based on this structure, two surface accessible pockets containing the carbohydrate recognition domain and the EPS binding motif, respectively, were targeted for screening of chemicals that will disrupt normal interaction with HIV-1 particle. Preliminary screening using Raji-CD4-DCIR cells allowed identification of two inhibitors that decreased HIV-1 attachment and propagation. The impact of these inhibitors on infection of DCs and CD4TL was evaluated as well. The results of this study thus identify novel molecules capable of blocking HIV-1 transmission by DCs and CD4TL.     

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.     

HIV envelope binding by macrophage-expressed gp340 promotes HIV-1 infection

The scavenger receptor cysteine-rich protein gp340 functions as part of the host innate immune defense system at mucosal surfaces. In the genital tract, its expression by cervical and vaginal epithelial cells promotes HIV trans-infection and may play a role in sexual transmission. Gp340 is an alternatively spliced product of the deleted in malignant brain tumors 1 (DMBT1) gene. In addition to its innate immune system activity, DMBT1 demonstrates instability in multiple types of cancer and plays a role in epithelial cell differentiation. We demonstrate that monocyte-derived macrophages express gp340 and that HIV-1 infection is decreased when envelope cannot bind it. Inhibition of infection occurred at the level of fusion of M-, T-, and dual-tropic envelopes. Additional HIV-1 envelope binding molecules, such as dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), mannose-binding lectin, and heparan sulfate, enhance the efficiency of infection of the cells that express them by increasing the local concentration of infectious virus. Our data suggest that gp340, which is expressed by macrophages in vivo, may function to enhance infection in much the same manner. Its expression on tissue macrophages and epithelial cells suggests important new opportunities for HIV-1 pathogenesis investigation and therapy.     

Role of Cell Surface Glycosaminoglycans of Human T Cells in Human Immunodeficiency Virus Type-1 (HIV-1) Infection

To investigate the role of cell surface glycosaminoglycans (GAGs), including heparan sulfate (HS), on HIV-1 infection in human T cells, HIV-1 binding and infection were determined after treatment of T-cell lines and CD4 + T cells from normal peripheral blood mononuclear cells (PBMC) with GAG-degrading enzyme or a GAG metabolic sulfation inhibitor. Heparitinase I (hep I) and sodium chlorate prevented binding of HIV-1/IIIB to MT-4 cells as revealed by indirect immunofluorescence procedures, thereby inhibiting infection. Hep I was less effective in the binding inhibition of the macrophage-tropic strain HIV-1/SF162 than that of the T-cell line-tropic strain HIV-1/IIIB. The binding of HIV-1/SF162 was about 100-fold less dependent on cell surface HS than HIV-1/IIIB. Human HTLV-I positive T-cell lines expressed more HS than HTLV-I negative T-cell lines or normal CD4 + T cells when stained with anti-HS mAbs against either native or heparitinase-treated HS. With the exception of endo-β-galactosidase (endo-β-gal), GAG-degrading enzymes, including hep I, chondroitinase ABC (chon ABC), chondroitinase AC II (chon AC II) and keratanase, did not prevent the binding of HIV-1/IIIB to CD4+ T cells from normal PBMC. These results indicate that the cell surface HS of human T cells participates in HIV-1 infection by facilitating HIV-1/IIIB binding to MT-4 cells. In particular, the sulfation of HS chains is critical. Since the expression of cell surface HS varies among T cells, which are not consistently sensitive to hep I treatment in HIV-1 binding inhibition, other GAG-like molecules may also be involved.     

13C-NMR study of the binding of [1-13C]galactose-labelled N-acetyllactosamine and [1-13C]galactose-enriched hen ovalbumin to soybean agglutinin

The binding of the tide compounds to soybean agglutinin was investigated using 13C-NMR spectroscopy. The equilibrium constant for the binding of N-acetyllactosamine was found to be smaller than that obtained for the binding of ovalbumin (1.1 X 10(3) vs. 7.4 X 10(3) M-1). Only two binding sites per lectin tetramer were determined for the binding of ovalbumin, which is half the number of binding sites reported for the binding of small ligands to the lectin. Steric interference between the bulky ovalbumin molecules is believed to be the reason for the observed decrease in the apparent number of binding sites on the lectin.     

Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein.

We have expressed the human immunodeficiency virus type 1 (HIV-1) gag polyprotein (Pr55gag) in bacteria under the control of the T7 phage gene 10 promoter. When the gene encoding the viral protease is included in cis, in the -1 reading frame, the expected proteolytic cleavage products MA and CA are produced. Disruption of the protease-coding sequence prevents proteolytic processing, and full-length polyprotein is produced. Pr55gag, separated from bacterial proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immobilized on nitrocellulose membranes, binds RNA containing sequences from the 5' end of the HIV-1 genome. This binding is tolerant of a wide range of pH and temperature but has distinct salt preferences. Conditions were identified which prevented nonspecific binding of RNA to bacterial proteins but still allowed binding to Pr55gag. Under these conditions, irrelevant RNA probes lacking HIV-1 sequences bound Pr55gag less efficiently. Quantitation of binding to Pr55gag by HIV-1 RNA probes with deletions mutations demonstrated that there are two regions lying within the HIV-1 gag gene which independently promote binding of RNA to Pr55gag.