HIV-1 escape from the CCR5 antagonist maraviroc associated with an altered and less-efficient mechanism of gp120-CCR5 engagement that attenuates macrophage tropism

Maraviroc (MVC) inhibits the entry of human immunodeficiency virus type 1 (HIV-1) by binding to and modifying the conformation of the CCR5 extracellular loops (ECLs). Resistance to MVC results from alterations in the HIV-1 gp120 envelope glycoproteins (Env) enabling recognition of the drug-bound conformation of CCR5. To better understand the mechanisms underlying MVC resistance, we characterized the virus-cell interactions of gp120 from in vitro-generated MVC-resistant HIV-1 (MVC-Res Env), comparing them with those of gp120 from the sensitive parental virus (MVC-Sens Env). In the absence of the drug, MVC-Res Env maintains a highly efficient interaction with CCR5, similar to that of MVC-Sens Env, and displays a relatively modest increase in dependence on the CCR5 N terminus. However, in the presence of the drug, MVC-Res Env interacts much less efficiently with CCR5 and becomes critically dependent on the CCR5 N terminus and on positively charged elements of the drug-modified CCR5 ECL1 and ECL2 regions (His88 and His181, respectively). Structural analysis suggests that the Val323 resistance mutation in the gp120 V3 loop alters the secondary structure of the V3 loop and the buried surface area of the V3 loop-CCR5 N terminus interface. This altered mechanism of gp120-CCR5 engagement dramatically attenuates the entry of HIV-1 into monocyte-derived macrophages (MDM), cell-cell fusion activity in MDM, and viral replication capacity in MDM. In addition to confirming that HIV-1 escapes MVC by becoming heavily dependent on the CCR5 N terminus, our results reveal novel interactions with the drug-modified ECLs that are critical for the utilization of CCR5 by MVC-Res Env and provide additional insights into virus-cell interactions that modulate macrophage tropism.     

Mapping the determinants of the CCR5 amino-terminal sulfopeptide interaction with soluble human immunodeficiency virus type 1 gp120-CD4 complexes

CD4 and CCR5 mediate fusion and entry of R5 human immunodeficiency virus type 1 (HIV-1) strains. Sulfotyrosine and other negatively charged residues in the CCR5 amino-terminal domain (Nt) are crucial for gp120 binding and viral entry. We previously showed that a soluble gp120-CD4 complex specifically binds to a peptide corresponding to CCR5 Nt residues 2 to 18, with sulfotyrosines in positions 10 and 14. This sulfopeptide also inhibits soluble gp120-CD4 binding to cell surface CCR5 as well as infection by an R5 virus. Here we show that residues 10 to 18 constitute the minimal domain of the CCR5 Nt that is able to specifically interact with soluble gp120-CD4 complexes. In addition to sulfotyrosines in positions 10 and 14, negatively charged residues in positions 11 and 18 participate in this interaction. Furthermore, the CCR5 Nt binds to a CD4-induced surface on gp120 that is composed of conserved residues in the V3 loop stem and the C4 domain. Binding of gp120 to cell surface CCR5 is further influenced by residues in the crown of the V3 loop, C1, C2, and C3. Our data suggest that gp120 docking to CCR5 is a multistep process involving several independent regions of the envelope glycoprotein and the coreceptor.     

Alternative coreceptor requirements for efficient CCR5- and CXCR4-mediated HIV-1 entry into macrophages

Macrophage tropism of human immunodeficiency virus type 1 (HIV-1) is distinct from coreceptor specificity of the viral envelope glycoproteins (Env), but the virus-cell interactions that contribute to efficient HIV-1 entry into macrophages, particularly via CXCR4, are not well understood. Here, we characterized a panel of HIV-1 Envs that use CCR5 (n = 14) or CXCR4 (n = 6) to enter monocyte-derived macrophages (MDM) with various degrees of efficiency. Our results show that efficient CCR5-mediated MDM entry by Env-pseudotyped reporter viruses is associated with increased tolerance of several mutations within the CCR5 N terminus. In contrast, efficient CXCR4-mediated MDM entry was associated with reduced tolerance of a large deletion within the CXCR4 N terminus. Env sequence analysis and structural modeling identified amino acid variants at positions 261 and 263 within the gp41-interactive region of gp120 and a variant at position 326 within the gp120 V3 loop that were associated with efficient CXCR4-mediated MDM entry. Mutagenesis studies showed that the gp41 interaction domain variants exert a significant but strain-specific influence on CXCR4-mediated MDM entry, suggesting that the structural integrity of the gp120-gp41 interface is important for efficient CXCR4-mediated MDM entry of certain HIV-1 strains. However, the presence of Ile326 in the gp120 V3 loop stem, which we show by molecular modeling is located at the gp120-coreceptor interface and predicted to interact with the CXCR4 N terminus, was found to be critical for efficient CXCR4-mediated MDM entry of divergent CXCR4-using Envs. Together, the results of our study provide novel insights into alternative mechanisms of Env-coreceptor engagement that are associated with efficient CCR5- and CXCR4-mediated HIV-1 entry into macrophages.     

A functional, discontinuous HIV-1 gp120 C3/C4 domain-derived, branched, synthetic peptide that binds to CD4 and inhibits MIP-1alpha chemokine binding.

This paper describes a branched synthetic peptide [3.7] that incorporates sequence discontinuous residues of HIV-1 gp120 constant regions. The approach was to bring together residues of gp120 known to interact with human cell membranes such that the peptide could fold to mimic the native molecule. The peptide incorporates elements of both the conserved CD4 and CCR5 binding sites. The 3.7 peptide, which cannot be produced by conventional genetic engineering methods, is recognized by antiserum raised to native gp120. The peptide also binds to CD4 and competitively inhibits binding of QS4120 an antibody directed against the CDR2 region of CD4. When preincubated with the CD4+ve MM6 macrophage cell line, which expresses mRNA for the CCR3 and CCR5 chemokine receptors, both 3.7 and gp120 inhibit binding of the chemokine MIP-1alpha. The peptide also inhibits infection of primary macrophages by M-tropic HIV-1. Thus, 3.7 is a prototype candidate peptide for a vaccine against HIV-1 and represents a novel approach to the rational design of peptides that can mimic complex sequence discontinuous ligand binding sites of clinically relevant proteins.     

Interaction of RANTES with syndecan-1 and syndecan-4 expressed by human primary macrophages

Interaction of RANTES with its membrane ligands or receptors transduces multiple intracellular signals. Whether RANTES uses proteoglycans (PGs) belonging to the syndecan family to attach to primary cells expressing RANTES G-protein-coupled receptors (GPCRs) was investigated. We demonstrate that RANTES specifically binds to high and low affinity binding sites on human monocyte-derived macrophages (MDM). We show by co-immunoprecipitation experiments that RANTES is associated on these cells with syndecan-1 and syndecan-4, but neither with syndecan-2 nor with betaglycan, in addition to CD44 and its GPCRs, CCR5 and CCR1. Glycosaminidases pre-treatment of the monocyte derived-macrophages strongly decreases the binding of RANTES to syndecan-1 and syndecan-4 and also to CCR5, and abolishes RANTES binding to CD44. This suggests that glycosaminoglycans (GAGs) are involved in RANTES binding to the PGs and that such bindings facilitate the subsequent interaction of RANTES with CCR5, on the MDM, characterized by low membrane expression of CCR5. The role of these interactions in the pathophysiology of RANTES deserves further study.     

The crown and stem of the V3 loop play distinct roles in human immunodeficiency virus type 1 envelope glycoprotein interactions with the CCR5 coreceptor

Human immunodeficiency virus type 1 envelope glycoprotein gp120 interacts with CD4 and the CCR5 coreceptor in order to mediate viral entry. A CD4-induced surface on gp120, primarily composed of residues in the V3 loop and the C4 domain, interacts with CCR5. In the present study, we generated envelope glycoproteins comprising chimeric V3 loops and/or V3 loops with deletions and studied their binding to CCR5 amino-terminal domain (Nt)-based sulfopeptides and cell surface CCR5, as well as their ability to mediate viral entry. We thus delineated two functionally distinct domains of the V3 loop, the V3 stem and the V3 crown. The V3 stem alone mediates soluble gp120 binding to the CCR5 Nt. In contrast, both the V3 stem and crown are required for soluble gp120 binding to cell surface CCR5. Within the context of a virion, however, the V3 crown alone determines coreceptor usage. Our data support a two-site gp120-CCR5 binding model wherein the V3 crown and stem interact with distinct regions of CCR5 in order to mediate viral entry.     

Structure and Dynamics of the gp120 V3 Loop That Confers Noncompetitive Resistance in R5 HIV-1JR-FL to Maraviroc

Maraviroc, an (HIV-1) entry inhibitor, binds to CCR5 and efficiently prevents R5 human immunodeficiency virus type 1 (HIV-1) from using CCR5 as a coreceptor for entry into CD4⁺ cells. However, HIV-1 can elude maraviroc by using the drug-bound form of CCR5 as a coreceptor. This property is known as noncompetitive resistance. HIV-1_V3-M5 derived from HIV-1_JR-FLan is a noncompetitive-resistant virus that contains five mutations (I304V/F312W/T314A/E317D/I318V) in the gp120 V3 loop alone. To obtain genetic and structural insights into maraviroc resistance in HIV-1, we performed here mutagenesis and computer-assisted structural study. A series of site-directed mutagenesis experiments demonstrated that combinations of V3 mutations are required for HIV-1_JR-FLan to replicate in the presence of 1 µM maraviroc, and that a T199K mutation in the C2 region increases viral fitness in combination with V3 mutations. Molecular dynamic (MD) simulations of the gp120 outer domain V3 loop with or without the five mutations showed that the V3 mutations induced (i) changes in V3 configuration on the gp120 outer domain, (ii) reduction of an anti-parallel β-sheet in the V3 stem region, (iii) reduction in fluctuations of the V3 tip and stem regions, and (iv) a shift of the fluctuation site at the V3 base region. These results suggest that the HIV-1 gp120 V3 mutations that confer maraviroc resistance alter structure and dynamics of the V3 loop on the gp120 outer domain, and enable interactions between gp120 and the drug-bound form of CCR5.     

Binding of the CC-chemokine RANTES to syndecan-1 and syndecan-4 expressed on HeLa cells

It is believed that proteoglycans influence biological properties of chemokines. We show that the CC chemokine RANTES binds not only to high-affinity binding sites on CCR5-positive HeLa cells but also to low-affinity binding sites on HeLa cells expressing or lacking RANTES G protein-coupled receptors. Coimmunoprecipitation studies demonstrate that RANTES forms complexes with glycanated syndecan (SD)-1 and -4, in addition to CCR5 on the CCR5-positive HeLa cells. Moreover, confocal microscopy analysis shows the colocalization of RANTES with SD-1 and -4. Glycosaminoglycans removal from the cells by glycosaminidases treatment prevented RANTES binding to SD-1 and -4 and decreased RANTES binding to CCR5 on the CCR5-positive HeLa cells. Removal of glycosaminoglycans by glycosaminidases treatment of the complexes, RANTES/SD-1/SD-4/+/-CCR5, immobilized on beads, reversed SD-1 and -4 bindings. Therefore, RANTES bindings to SD-1 and -4 depend on glycosaminoglycans and facilitate RANTES interaction with CCR5. Extracting plasma membrane cholesterol abolished the coimmunoprecipitation of SD-1 with RANTES, suggesting that rafts are involved in RANTES association to SD-1. Confocal microscopy analysis as well as coimmunoprecipitation experiments show a RANTES-independent heteromeric complex on the CCR5-positive HeLa cells, SD-1, SD-4, and CCR5. This complex is likely a functional unit in which proteoglycans may modulate RANTES binding to CCR5.     

A tyrosine-sulfated peptide based on the N terminus of CCR5 interacts with a CD4-enhanced epitope of the HIV-1 gp120 envelope glycoprotein and inhibits HIV-1 entry

The sequential association of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 with CD4 and a seven-transmembrane segment coreceptor such as CCR5 or CXCR4 initiates entry of the virus into its target cell. The N terminus of CCR5, which contains several sulfated tyrosines, plays a critical role in the CD4-dependent association of gp120 with CCR5 and in viral entry. Here we demonstrate that a tyrosine-sulfated peptide based on the N terminus of CCR5, but not its unsulfated analogue, inhibits infection of macrophages and peripheral blood mononuclear cells by CCR5-dependent, but not CXCR4-dependent, HIV-1 isolates. The sulfated peptide also inhibited the association of CCR5-expressing cells with gp120-soluble CD4 complexes and, less efficiently, with MIP-1alpha. Moreover, this peptide inhibited the precipitation of gp120 by 48d and 23e antibodies, which recognize CD4-inducible gp120 epitopes, but not by several other antibodies that recognize proximal epitopes. The ability of the sulfated peptide to block 48d association with gp120 was dependent in part on seven tropism-determining residues in the third variable (V3) and fourth conserved (C4) domains of gp120. These data underscore the important role of the N-terminal sulfate moieties of CCR5 in the entry of R5 HIV-1 isolates and localize a critical contact between gp120 and CCR5.     

Macrophage tropism of human immunodeficiency virus type 1 isolates from brain and lymphoid tissues predicts neurotropism independent of coreceptor specificity

The viral determinants that underlie human immunodeficiency virus type 1 (HIV-1) neurotropism are unknown, due in part to limited studies on viruses isolated from brain. Previous studies suggest that brain-derived viruses are macrophage tropic (M-tropic) and principally use CCR5 for virus entry. To better understand HIV-1 neurotropism, we isolated primary viruses from autopsy brain, cerebral spinal fluid, blood, spleen, and lymph node samples from AIDS patients with dementia and HIV-1 encephalitis. Isolates were characterized to determine coreceptor usage and replication capacity in peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages (MDM), and microglia. Env V1/V2 and V3 heteroduplex tracking assay and sequence analyses were performed to characterize distinct variants in viral quasispecies. Viruses isolated from brain, which consisted of variants that were distinct from those in lymphoid tissues, used CCR5 (R5), CXCR4 (X4), or both coreceptors (R5X4). Minor usage of CCR2b, CCR3, CCR8, and Apj was also observed. Primary brain and lymphoid isolates that replicated to high levels in MDM showed a similar capacity to replicate in microglia. Six of 11 R5 isolates that replicated efficiently in PBMC could not replicate in MDM or microglia due to a block in virus entry. CD4 overexpression in microglia transduced with retroviral vectors had no effect on the restricted replication of these virus strains. Furthermore, infection of transfected cells expressing different amounts of CD4 or CCR5 with M-tropic and non-M-tropic R5 isolates revealed a similar dependence on CD4 and CCR5 levels for entry, suggesting that the entry block was not due to low levels of either receptor. Studies using TAK-779 and AMD3100 showed that two highly M-tropic isolates entered microglia primarily via CXCR4. These results suggest that HIV-1 tropism for macrophages and microglia is restricted at the entry level by a mechanism independent of coreceptor specificity. These findings provide evidence that M-tropism rather than CCR5 usage predicts HIV-1 neurotropism.     

V3 loop truncations in HIV-1 envelope impart resistance to coreceptor inhibitors and enhanced sensitivity to neutralizing antibodies

The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (DeltaV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds.     

Soluble mimics of a chemokine receptor: chemokine binding by receptor elements juxtaposed on a soluble scaffold

Despite the broad biological importance of G protein-coupled receptors (GPCRs), ligand recognition by GPCRs remains poorly understood. To explore the roles of GPCR extracellular elements in ligand binding and to provide a tractable system for structural analyses of GPCR/ligand interactions, we have developed a soluble protein that mimics ligand recognition by a GPCR. This receptor analog, dubbed CROSS5, consists of the N-terminal and third extracellular loop regions of CC chemokine receptor 3 (CCR3) displayed on the surface of a small soluble protein, the B1 domain of Streptococcal protein G. CROSS5 binds to the CCR3 ligand eotaxin with a dissociation equilibrium constant of 2.9 +/- 0.8 microM and competes with CCR3 for eotaxin binding. Control proteins indicate that juxtaposition of both CCR3 elements is required for optimal binding to eotaxin. Moreover, the affinities of CROSS5 for a series of eotaxin mutants are highly correlated with the apparent affinities of CCR3 for the same mutants, demonstrating that CROSS5 uses many of the same interactions as does the native receptor. The strategy used to develop CROSS5 could be applied to many other GPCRs, with a variety of potential applications.     

Role of V3 independent domains on a dualtropic human immunodeficiency virus type 1 (HIV-1) envelope gp120 in CCR5 coreceptor utilization and viral infectivity

The molecular mechanism of human immunodeficiency virus type 1 (HIV-1) entry into cells involves specific interactions between the viral envelope glycoprotein gp120 and two target cell proteins, CD4 and either CCR5 or CXCR4 chemokine receptors. In order to delineate the functional role of HIV-1 gp120 subdomains of dualtropic strains in CCR5 coreceptor usage, we used a panel of chimeric viruses in which the V1/V2 and V3 domains of gp120 from the dualtropic HIV-1(KMT) isolate were introduced either alone or in combination into the T-tropic HIV-1(NL4-3) background. These chimeric constructs were employed in cell-cell fusion and cell-free virus infectivity assays using cell lines expressing CD4 and the CCR5 chemokine receptor. In both assays, the V3 domain of HIV-1(KMT) but not the V1/V2 domain proved to be the principal determinant of CCR5 coreceptor usage. However, in the cell-free viral infectivity assay although a chimeric virus with a combined V1/V2 and V3 domains of HIV-1(KMT) efficiently fused with coreceptor expressing cells, yet its infectivity was markedly diminished in CCR5 as well as CXCR4 expressing cells. Restoring a comparable level of infection of such chimeric virus required the C3-V5 domain from HIV-1(KMT) to be introduced. Our present findings confirmed that the V3 domain is the major determinant of fusion activity and cellular tropism, and demonstrated a dispensable role for the V1/V2 domain. In addition the C3-V5 domain appeared to play an important role in viral infectivity when the corresponding V1/V2 and V3 domains are present.     

A highly conserved arginine in gp120 governs HIV-1 binding to both syndecans and CCR5 via sulfated motifs

HIV-1 has maximized its utilization of syndecans. It uses them as in cis receptors to infect macrophages and as in trans receptors to infect T-lymphocytes. In this study, we investigated at a molecular level the mechanisms that control HIV-1-syndecan interactions. We found that a single conserved arginine (Arg-298) in the V3 region of gp120 governs HIV-1 binding to syndecans. We found that an amine group on the side chain of this residue is necessary for syndecan utilization by HIV-1. Furthermore, we showed that HIV-1 binds syndecans via a 6-O sulfation, demonstrating that this binding is not the result of random interactions between basic residues and negative charges, but the result of specific contacts between gp120 and a well defined sulfation in syndecans. Surprisingly, we found that Arg-298, which mediates HIV-1 binding to syndecans, also mediates HIV-1 binding to CCR5. We postulated that HIV-1 recognizes similar motifs on syndecans and CCR5. Supporting this hypothesis, we obtained several lines of evidence that suggest that the 6-O sulfation recognized by HIV-1 on syndecans mimics the sulfated tyrosines recognized by HIV-1 in the N terminus of CCR5. Our finding that CCR5 and syndecans are exploited by HIV-1 via a single determinant echoes the mechanisms by which chemokines utilize these two disparate receptors and suggests that the gp120/chemokine mimicry may represent a common strategy in microbial pathogenesis.     

Loss of a single N-linked glycan allows CD4-independent human immunodeficiency virus type 1 infection by altering the position of the gp120 V1/V2 variable loops

The gp120 envelope glycoprotein of primary human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and the CCR5 chemokine receptor on the target cell. Previously, we adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for CD4-independent replication were limited to the V2 loop-V1/V2 stem. Here we show that elimination of a single glycosylation site at asparagine 197 in the V1/V2 stem is sufficient for CD4-independent gp120 binding to CCR5 and for HIV-1 entry into CD4-negative cells expressing CCR5. Deletion of the V1/V2 loops also allowed CD4-independent viral entry and gp120 binding to CCR5. The binding of the wild-type ADA gp120 to CCR5 was less dependent upon CD4 at 4 degrees C than at 37 degrees C. In the absence of the V1/V2 loops, neither removal of the N-linked carbohydrate at asparagine 197 nor lowering of the temperature increased the CD4-independent phenotypes. A CCR5-binding conformation of gp120, achieved by CD4 interaction or by modification of temperature, glycosylation, or variable loops, was preferentially recognized by the monoclonal antibody 48d. These results suggest that the CCR5-binding region of gp120 is occluded by the V1/V2 variable loops, the position of which can be modulated by temperature, CD4 binding, or an N-linked glycan in the V1/V2 stem.     

Analysis of the mechanism by which the small-molecule CCR5 antagonists SCH-351125 and SCH-350581 inhibit human immunodeficiency virus type 1 entry

Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the consecutive interaction of the envelope glycoprotein gp120 with CD4 and a coreceptor such as CCR5 or CXCR4. The CCR5 coreceptor is used by the most commonly transmitted HIV-1 strains that often persist throughout the course of infection. Compounds targeting CCR5-mediated entry are a novel class of drugs being developed to treat HIV-1 infection. In this study, we have identified the mechanism of action of two inhibitors of CCR5 function, SCH-350581 (AD101) and SCH-351125 (SCH-C). AD101 is more potent than SCH-C at inhibiting HIV-1 replication in primary lymphocytes, as well as viral entry and gp120 binding to cell lines. Both molecules also block the binding of several anti-CCR5 monoclonal antibodies that recognize epitopes in the second extracellular loop of CCR5. Alanine mutagenesis of the transmembrane domain of CCR5 suggests that AD101 and SCH-C bind to overlapping but nonidentical sites within a putative ligand-binding cavity formed by transmembrane helices 1, 2, 3, and 7. We propose that the binding of small molecules to the transmembrane domain of CCR5 may disrupt the conformation of its extracellular domain, thereby inhibiting ligand binding to CCR5.     

Mode of action for linear peptide inhibitors of HIV-1 gp120 interactions

The linear peptide 12p1 (RINNIPWSEAMM) was previously isolated from a phage display library and was found to inhibit interaction of HIV-1 gp120 with both CD4 and a CCR5 surrogate, mAb 17b [Ferrer, M., and Harrison, S. (1999) J. Virol. 73, 5795-5802]. In this work, we investigated the mechanism that leads to this dual inhibition of gp120 binding. We found that there is a direct interaction of 12p1 with gp120, which occurs with a binding stoichiometry of 1:1. The peptide inhibits binding of monomeric YU2 gp120 to both sCD4 and 17b at IC(50) values of 1.1 and 1.6 microM, respectively. The 12p1 peptide also inhibited the binding of these ligands to trimeric envelope glycoproteins, blocked the binding of gp120 to the native coreceptor CCR5, and specifically inhibited HIV-1 infection of target cells in vitro. Analyses of sCD4 saturation of monomeric gp120 in the presence or absence of a fixed concentration of peptide suggest that 12p1 suppression of CD4 binding to gp120 is due to allosteric inhibitory effects rather than competitive inhibition of CD4 binding. Using a panel of gp120 mutants that exhibit weakened inhibition by 12p1, the putative binding site of the peptide was mapped to a region immediately adjacent to, but distinguishable from, the CD4 binding footprint. In the case of the peptide, the effects of single-12p1 residue substitutions and various peptide truncations indicate that the side chain of Trp7 and other structural elements of 12p1 are critical for gp120 binding or efficient inhibition of binding of a ligand to gp120. Finally, 12p1 was unable to inhibit binding of sCD4 to a gp120 mutant that is believed to resemble the CD4-induced conformation of gp120. These results suggest that 12p1 preferentially binds gp120 prior to engagement of CD4; binding of the peptide to gp120 limits the interaction with ligands (CD4 and CCR5) that are generally crucial for viral entry. More importantly, these results indicate that 12p1 binds to a unique site that may prove to be a prototypic target for novel CD4-gp120 inhibitors.     

The class A macrophage scavenger receptor type I (SR-AI) recognizes complement iC3b and mediates NF-κB activation

The macrophage scavenger receptor SR-AI binds to host tissue debris to perform clearance and it binds to bacteria for phagocytosis. In addition, SR-AI modulates macrophage activation through cell signaling. However, investigation of SR-AI signaling on macrophages is complicated due to its promiscuous ligand specificity that overlaps with other macrophage receptors. Therefore, we expressed SR-AI on HEK 293T cells to investigate its ligand binding and signaling. On 293T cells, SR-AI could respond to E. coli DH5α, leading to NF-κB activation and IL-8 production. However, this requires E. coli DH5α to be sensitized by fresh serum that is treated with heat-inactivation or complement C3 depletion. Anti-C3 antibody inhibits the binding of SR-AI to serum-sensitized DH5α and blocks DH5α stimulation of SR-AI signaling. Further analysis showed that SR-AI can directly bind to purified iC3b but not C3 or C3b. By mutagenesis, The SRCR domain of SR-AI was found to be essential in SR-AI binding to serum-sensitized DH5α. These results revealed a novel property of SR-AI as a complement receptor for iC3b-opsonized bacteria that can elicit cell signaling.     

INSL5 is a high affinity specific agonist for GPCR142 (GPR100)

Insulin-like peptide 5 (INSL5) is a peptide that belongs to the relaxin/insulin family, and its receptor has not been identified. In this report, we demonstrate that INSL5 is a specific agonist for GPCR142. Human INSL5 displaces the binding of (125)I-relaxin-3 to GPCR142 with a high affinity (K(i) = 1.5 nM). In a saturation binding assay, (125)I-INSL5 binds GPCR142 with a K(d) value of 2.5 nM. In functional guanosine (gamma-thio)-triphosphate binding and cAMP accumulation assays, INSL5 potently activates GPCR142 with EC(50) values of 1.3 and 1.2 nM, respectively. In addition, INSL5 stimulates Ca(2+) mobilization in HEK293 cells expressing GPCR142 and G alpha(16). Overall, INSL5 behaves as an agonist for GPCR142 similar to relaxin-3. However, unlike relaxin-3, which is also a potent agonist for GPCR135 and LGR7, INSL5 does not activate either GPCR135 or LGR7. INSL5 inhibits (125)I-relaxin-3 binding to GPCR135 with a low potency (K(i) = 500 nM). A functional assay shows that INSL5 (1 microm) is a weak antagonist for GPCR135. In addition, INSL5 (up to 1 microm) shows no affinity or activity at LGR7 or LGR8 either in a binding assay or a bio-functional assay. Previously, we have demonstrated that GPCR142 mRNA is expressed in peripheral tissues, particularly in the colon. Here we show that INSL5 mRNA is expressed in many peripheral tissues, similar to GPCR142. The high affinity interaction between INSL5 and GPCR142 coupled with their co-evolution and partially overlapping tissue expression patterns strongly suggest that INSL5 is an endogenous ligand for GPCR142.