HIV-1 is the causative agent of acquired immunodeficiency syndrome (AIDS), a condition in humans in which progressive failure of the immune system leads to the development of severe opportunistic infections and unusual malignant disorders (1). Infection occurs through the transfer of blood, semen, vaginal fluid, or breast milk, in which HIV-1 can be present as both free virus particles and/or within infected cells. The virus infects vital cells of the immune system, including CD4⁺ T-helper lymphocytes, macrophages, and dendritic cells, all of which are key to the development and orchestration of the immune response (2). This results in the targeted depletion of CD4⁺ T cells, the main function of which is to promote cytotoxic T-lymphocyte dependant killing of cells expressing foreign antigens and up regulate antibody production by B-lymphocytes. When CD4⁺ T cells numbers decline below a critical level (400/μl of blood) cell-mediated immunity is compromised and the body becomes progressively more susceptible to opportunistic infections (3). The first step of the HIV-1 replication cycle, attachment and entry into host cells, occurs through specific interactions between gp120, the glycoprotein which constitutes the surface unit of HIV-1 envelope spikes (Env) and the primary cellular receptor, CD4. This promotes further contacts between gp120 and members of the chemokine receptor family, among which CCR5 and CXCR4 are the most physiologically relevant and ultimately lead to the fusion of the viral and the host cell membranes (4). HIV-1 preferentially uses CCR5 during the acute phase of infection but, later in the course of HIV-1 infection progressing to AIDS, HIV-1 variants frequently appear that become adapted to utilize CXCR4.

Before encountering permissive CD4⁺ cells the virus may interact with several other alternative receptors, often referred as “attachment receptors” (5) such as Galactoside Ceramide (GalCer), present at the surface of epithelial cells, Mannose-Binding Lectin (MBL), Dendritic Cell Specific ICAM-3-Grabbing Non-integrin (DC-SIGN), or Heparan Sulfate Proteoglycans (HSPGs), the latter being present at the surface of virtually all cell types. Although these interactions generally do not permit infection per se, they can importantly affect mucosal cells (the portal through which HIV-1 enters in the body) attachment and transport across epithelial layers, tropism, tissue invasion, or cellular entry (6, 7). This review will discuss some aspects of the HIV-1-HSPG interaction and will describe how the biochemical characterization of this interaction led to the engineering of a new class of potential attachment and entry inhibitors.

Heparan sulfate proteoglycans are glycoproteins carrying one or more covalently bound heparan sulfate (HS) chains, a large anionic polysaccharide of the glycosaminoglycan (GAG) family, characterized by astonishing structural diversity and interactive properties. These complex molecules are widely distributed within tissues, and can be found at the cell surface such as the syndecans and glypicans, within the extracellular matrix such as agrin, perlecan, or type XVIII collagen, or intracellularly such as serglycin (8). Being predominantly and ubiquitously present in the extracellular milieu, these macromolecules are unsurprisingly playing essential roles in a vast number of biological processes occurring at the cell–cell and cell–matrix interface.

Over the past two decades, HSPGs have been found indeed to bind to a multitude of protein ligands, including cytokines, chemokines, morphogens, growth factors, adhesion and matrix molecules, receptors, enzymes, plasma proteins, etc. (8, 9). These interactions, which usually involve the HS chains, serve a large number of purposes. Functionally, HS has been known to affect the local concentration, the compartmentalization, the stability, the structure, and/or the activity of its ligands. Protein-HS interactions thus play critical roles, for example, in mediating the formation of chemokine gradients along which cells can migrate directionally (10–12), in providing a scaffold onto which two adjacent proteins such as growth factor-receptor complexes can interact (13), in protecting cytokines against proteolysis (14), in inducing protein conformational changes (15), in controlling or in restricting the diffusion of its ligands (16–18) thereby generating a local concentration of a given protein. As it will be described below, many microbial pathogens hijack HSPGs and take advantage of their interactive properties for their adhesion to host tissues and invasion of host cells. From a structural view point HSPGs’ multiple binding activities are believed to be closely related to the extended structural variability of the HS chain. It is a long (20–150 nm) and linear polysaccharide made of a repeating units of a 1 → 4 linked disaccharide motifs, comprising a glucuronic acid (GlcA) or its C-5 epimer, an iduronic acid (IdoA), and a N-acetyl- or N-sulfated-glucosamine (GlcNac or GlcNS), either or both of which may be O-sulfated at different positions. Variation in length, sulfation, and glucuronate/iduronate ratio, which occur in restricted domains of usually three to six disaccharides along the chain (Figure 1), generates a very large polydispersity and, as such, provides distinct docking sites for the various ligands of the polysaccharide (19, 20).

Attachment to host tissues is a critical step for most pathogens’ invasion and dissemination. It is therefore not very remarkable that due to its wide expression and large interactive properties, HS is used by many pathogens for that purpose (22–26). These include parasites, for example Plasmodium falciparum (27) bacteria, such as Pseudomonas aeruginosa (28), Borrelia burdorferi (29), or Mycobacterium tuberculosis (30), and many viruses, amongst which are found B (31), C (32), delta (33), and E (34) hepatitis viruses, Human Papillomavirus (35), Herpes viruses (36), HTLV-1 (37), or HIV-1 (38). Several lines of evidence have identified HS as an initial receptor for viral infection. Firstly, many capsid or envelope viral proteins bind to HS, secondly, elimination of cell surface HS is usually associated with increased cell resistance to infection that results from a reduction in the virus’ ability to bind to the cell surface (39), thirdly, soluble HS or HS like molecules, including heparin, a chemically related GAG and dextran sulfate, inhibit viral attachment and subsequent entry in cell culture experiments (40–42). Finally, it has been described that a number of viruses undergo cell culture adaptation changes resulting in an increased binding to HS (43–46). Together this suggests that selection during cell culture of mutants that bind HS with high affinity confer a selective advantage to the viruses. It has thus been thought that HS could facilitate concentration of the viral particles at the cell surface, restricting their diffusion to the quasi-two-dimensional network of polysaccharides around the cell and as such enhances the probability of access to specific entry receptors. HS can also capture viral particles at the surface of non-permissive cells, and then mediate in trans infection by presenting these viruses to attachment and entry receptors on permissive cells (47). HS binding can also go well beyond the simple attachment mechanism and can play a more direct role in cellular entry. This has been demonstrated in particular for HSV, a virus whose entry into epithelial cells involves several glycoproteins of the envelope (48). While the viral envelope glycoproteins gB and gC participate in the initial cell attachment through binding to HS, the gD, which binds to herpes virus entry mediator (HVEM) or to nectin, triggers fusion between host and viral membranes, but can also promote viral entry by interacting with a specific HS motif comprising a 3-O-sulfated glucosamine residue (49). Interestingly, a 3-O-sulfonated HS octasaccharide, produced by chemical means, was shown to inhibit the HSV-1 host-cell interaction (50), suggesting the use of HS derived molecules as therapeutic tools against viral pathogens.

HIV-1 is transmitted by viral exposure at the mucosal surfaces, which can occur in the genital tract (semen, blood) the intestinal tract (semen, blood, breast milk), or through the placenta (maternal blood), or the bloodstream (blood products). Within the context of vaginal or rectal transmission, HIV-1 must first cross a normally protective mucosal epithelium to reach the underlying dendritic cells, macrophages, and T cells which all express the virus primary receptor, CD4 and at least one of its two co-receptors, CCR5 or CXCR4, and are thus the three major cellular targets of HIV-1. In these early events of transmission, before specific cell infection, HS has been shown to play important roles in viral adsorption and dissemination. In the semen first, which is the main vector for HIV-1 dissemination, and which contains both free virions and infected leukocytes, it has been found that spermatozoa can capture HIV-1 in a HS dependant manner. Such spermatozoa-attached viruses are efficiently transmitted to dendritic cells, macrophages, and T cells (51) to which access could be made possible through mucosal microabrasions or through dendritic cell projections that extend to the luminal surface of the mucosa.

The mechanisms used by free or cell associated virions to cross an otherwise healthy mucosal barrier are not well known and might differ depending on the tissue sites (for example vaginal or rectal epithelium) where infection occurs (52). HIV-1 can interact with the epithelial cells and can traverse the epithelium through transcytosis, endocytosis followed by exocytosis or by penetrating the gaps in between cells, gaining access to susceptible leukocytes that will further propagate and spread the infection (53, 54). In this context, it has been well known that epithelial cells express large amount of HSPGs which can sequester HIV particles. For example, attachment of HIV to an ectocervical epithelium-derived cell line can be inhibited both by heparinase (an enzyme that depolymerizes HS and removes it from the cell surface) or by soluble heparin. Interestingly, it has been found that cell surface bound HIV particles remain infectious for at least 6 days, and upon co-culture with CD4⁺ cells, can be efficiently transmitted to its target cells (55). It has been also reported that HSPG significantly contributes to both attachment to the apical pole of – and transcytosis through – an endometrial epithelium-derived cell line (7). Similarly, cell-free HIV particles have been shown to transcytose (although with low efficiency) through primary genital epithelial cells, a process that was dependant on syndecan, one of the major HSPGs expressed by epithelial cells (56).

Finally, HS was recently found to be indispensable for gp120-mediated induction of TLR signaling in intestinal and genital epithelial cells. In the gp120-TLR-HS complex, HS was critical to activate the intracellular NF-kB pathway which lead to downstream synthesis of proinflammatory cytokines and chemokines, and whose upregulation was associated with tight junction disruption and loss of barrier function (57). Therefore, beyond acting as an ancillary attachment receptor, HS can contribute to barrier loss and initiation of immune activation that could be the first step in the characteristic chronic immune activation of HIV-1 pathogenesis.

Primary infection can also develop from viral exposure in the oral cavity (mother to infant nursing or during oral intercourse), where the palatine tonsil is a replication site for HIV-1. Studies aiming at characterizing the expression pattern for HIV-1 ligands on human palatine tonsils have shown that HS was largely present on both the surface of the stratified squamous epithelium and on the reticulated epithelium lining the tonsillar crypts and where it is likely to provide stable binding for the virus, allowing it to penetrate beneath the luminal surface and encounter CD4⁺ cells (58). Finally, it has also been shown that HIV-1 infection of trophoblasts is independent of CD4 but, at least partly, relies on HSPG. Mother to child vertical transmission of the virus is a major cause of HIV-1 infection in infants, and direct infection of trophoblasts, the cells that form the placental barrier, may cause this transmission (59).

HIV-1 is also trapped very efficiently by endothelial cells, which usually express large amounts of HSPG. Syndecan-3, for example, which delineates the contour of endothelial cells in lymphoid tissue high endothelial venules, does not substitute for HIV-1 entry receptors but captures HIV-1 and presents it to passing permissive T cells, thus mediating an in trans mechanism of infection. Furthermore, whereas unbound viruses lose infectivity in 1 day, syndecan-attached HIV-1 remains infectious for up to 1 week (60).

Finally, HSPGs also significantly contributes to HIV-1 invasion in the brain and neurological complications that often characterize AIDS patients. Whereas HIV-1 can enter the central nervous system within infected CD4⁺T-cell and monocytes that traffic across the blood brain barrier, several in vitro and in vivo reports described that free HIV-1 can be taken up by brain endothelial cells in a HS dependant manner, internalized and exocytosed, as a way to cross the blood brain barrier before infection and replication can occur in central nervous system cells such as microglia and astrocytes (61–63).

Although this review is devoted to HIV-1 attachment and entry, it is worth noting that, in addition to gp120, several other HIV proteins regulating various aspects of the virus life cycle also appeared to bind cellular HS after being released from HIV-infected cells. These protein-HS interactions contribute to trigger a variety of biological effects related to AIDS-associated pathologies. This includes p17, the matrix protein (64) which up-regulates cytokine production thus deregulating the functions of many immune cells; TAT, the transacting activator of transcription (65) which exerts angiogenic, cell proliferation, chemoinvasion activities and induces peripheral neuropathies, immune suppression, and tumorigenesis; and finally Vpr, the Viral protein R (66), which induces cell cycle arrest and apoptosis.

Altogether, regarding attachment and entry, these studies showed that HSPG serves a number of purposes during the early steps of HIV-1 dissemination, from capturing and presenting in trans free virions to replicative cells, to permitting the transfer of viral particles across epithelial or endothelial barriers (Figure 2). HSPG, when expressed by CD4⁺ permissive cells may also increase infectivity by favoring in cis viral particle concentration at the cell surface (see below).

A number of polyanions have been investigated for their ability to inhibit HIV-1 infection in clinical trials. In addition to suramin, dextran sulfate, and heparin, which were considered for systemic use, this includes many other sulfated/acidic compounds such as carrageenan, cellulose sulfate, polystyrene sulfonate or maleic acid, naphthalene sulfonate, and cellulose acetate phthalate, developed as anti HIV-1 microbicides. Unfortunately, none of these compounds proved to be effective in vivo presumably due to poor availability, toxicity, sequestration by plasma proteins, induced reduction in epithelial integrity and concomitant increases in permeability to HIV-1 particles (67, 68). Their variability, in terms of molecular weights and degrees of sulfation also made them difficult to standardize.

These studies thus showed that HS binding to CXCR4 tropic gp120 is constitutive (the V3 loop -which dominates the interaction- is surface exposed), and can be enhanced by CD4 (which, together with V3 loop reorganization, exposes a new HS binding domain), while it is entirely CD4-induced for CCR5 tropic gp120. Interestingly, all these HBDs are located close to each other, at the proximity of- or within- the co-receptor binding site and are collectively involved in the conformational changes induced upon interaction with CD4 and in co-receptor recognition. In particular, mutations of Arg-419, Lys-421, Lys-432 within the bridging sheet, and Arg298, Arg306, Arg308, within the V3 loop, which are targeted by heparin, decrease the ability of gp120 to interact with CXCR4. This strongly suggested that polyanionic compounds, in addition to prevent HIV-1 association to cell surface HSPG, could directly block co-receptor binding and thus inhibit entry. However, being cryptic on the HIV-1 associated gp120, the co-receptor binding site needs to be exposed to be efficiently targeted. To this aim, a new molecule composed of CD4 covalently linked to HS has been prepared. To render this molecule potentially druggable it was based on a small CD4 mimetic (rather than recombinant CD4) and a chemically synthesized HS dodecamer (rather than natural derived HS, whose almost infinite structural variety would have made impossible the obtention of a defined compound). This chemically defined glycoconjugate (termed mCD4-HS₁₂), whose size is 6000 Da, was shown to bind to gp120 through its mCD4 moiety and induce the structural modifications necessary to expose the co-receptor binding domain which therefore became available to be blocked by the HS₁₂ moiety (Figure 5). This compound thus successfully targets two critical and highly conserved domains of gp120; the CD4 and the co-receptor binding domains. From a biochemical point of view, this compound blocks the binding of the prototypic R5-gp120 (YU2), to both CD4 and mAb 17b (directed against the co-receptor binding domain), while it blocks that of the prototypic X4-gp120 (MN) to CD4, HS and mAb 17b, with low nanomolar affinity (99). This molecule also prevents gp120 association to purified native CCR5 and CXCR4 co-receptors (100) and consequently displays a strong antiviral activity against R5- and X4- HIV-1 as well as against dual tropic virus with IC₅₀ as low as 5 nM.

Investigation of the gp120 binding to HS has revealed a contrasting situation that is far more complex than previously thought. HIV-1 that uses CXCR4 as an entry co-receptor features up to four HBDs on their gp120, including the V1/V2 and V3 loops and the co-receptor binding site, while those using CCR5 mostly display HS binding activity essentially within the conserved and characteristically basic co-receptor binding domain. In that context, it is worth noting that the N-terminus of CCR5 and CXCR4 contain sulfotyrosine, as do a number of antibodies, directed against the gp120 co-receptor binding domains (101), also indicating that the gp120 CD4 induced region can be liganded by sulfated moieties.

Characterization of these HBDs, in particular within the CD4 induced surface, whose cryptic nature limits its accessibility both temporally and spatially during infection, led to the engineering of a new class of compounds in which HS was covalently linked to mCD4. These compounds, conceptually distinct from any other existing HIV-1 inhibitors, function by simultaneously exposing and blocking the HIV-1 co-receptor binding site, and therefore inhibit binding of gp120 to both CD4 and CCR5/CXCR4. It thus efficiently inhibits viral replication by blocking entry, which is currently considered as a compelling target for controlling viral replication (102). This molecule also inhibits the binding of gp120 to HS and has thus the potential for preventing viral adsorption on mucosa or viral transport through the blood brain barrier. It could therefore be further developed for both prevention and therapy following topical and/or parenteral application. In this regard, it is worth noting that these compounds are much more defined and have a much more specific mode of action than the above described polyanions that have been investigated up to now.

Despite tremendous progresses made in the development of antiviral drugs (103), HIV-1 continues to be a major health concern and remains one of the leading causes of death worldwide, which necessitate the development of new antivirals. With regards to inhibitor development, it is worth noting that mCD4-HS₁₂ is bivalent. Multivalency has a number of functional advantages, such as achieving high affinity, and increasing strength and specificity for the binding site. It has been indeed found that the mCD4-HS₁₂ is, by far, more active than either its moieties alone, each reciprocally enhancing the blocking activity of the other in a cooperative manner (99). Targeting the gp120 co-receptor binding site, which although is well conserved across various HIV-1 strains exists in a dynamic equilibrium between its unliganded- and CD4-bound conformations, might thus be relatively challenging. From a structural point of view, it is interesting that HS is characterized by considerable internal motion and variation in its local three-dimensional structure. The IdoA, in particular, also exists in a dynamic equilibrium between a chair and a twisted skew-boat form, which may itself represent the average of a rapidly fluctuating ensemble of related structures. The conformation of HS also depends on its local sequence, the presence of poorly sulfated GlcA-GlcNAc domains giving rise to chain flexibility (104). It is thus tempting to suggest that HS is well designed to interact with an ensemble of conformationally dynamic structures such as that of the co-receptor binding domain of gp120, the high specificity of the conjugated bivalent compound being brought by the mCD4 moiety.

Currently, the HS₁₂ moiety of the molecule displays a regular and highly sulfated sequence, (2-O-sulfated iduronic acid linked to N- and 6-O-sulfated glucosamine), so that the exact sulfation pattern recognized by gp120 is hidden in the fully sulfated oligosaccharide. HS synthesis is notoriously difficult, and in view of its huge diversity (see Figure 1), the synthesis of a library addressing structure-activity relationships is not realistic. In an effort to pinpoint the sulfate groups that are functionally essential to gp120 binding, the HS was substituted with sulfotyrosine-containing HS mimetic peptide, the synthesis of which is much more straightforward, and more easily amenable to sequence-activity relationship investigation. This molecule compares very well with HS₁₂, and when conjugated to mCD4 broadly inhibits the replication of several HIV-1 strains with an IC₅₀ of 1 nM (100), thus, opening the route to future developments.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.