Identification of stimulating and inhibitory epitopes within the heat shock protein 70 molecule that modulate cytokine production and maturation of dendritic cells

The 70-kDa microbial heat shock protein (mHSP70) has a profound effect on the immune system, interacting with the CD40 receptor on DC and monocytes to produce cytokines and chemokines. The mHSP70 also induces maturation of dendritic cells (DC) and thus acts as an alternative ligand to CD40L on T cells. In this investigation, we have identified a cytokine-stimulating epitope (peptide 407-426), by activating DC with overlapping synthetic peptides (20-mers) derived from the sequence of mHSP70. This peptide also significantly enhances maturation of DC stimulated by mHSP70 or CD40L. The epitope is located at the base of the peptide-binding groove of HSP70 and has five critical residues. Furthermore, an inhibitory epitope (p457-496) was identified downstream from the peptide-binding groove that inhibits cytokine production and maturation of DC stimulated by HSP70 or CD40L. The p38 MAP kinase phosphorylation is critical in the alternative CD40-HSP70 pathway and is inhibited by p457-496 but enhanced by p407-426.     

Stimulation of cell surface CCR5 and CD40 molecules by their ligands or by HSP70 up-regulates APOBEC3G expression in CD4(+) T cells and dendritic cells

Apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like-3G (A3G) is an intracellular innate antiviral factor that deaminates retroviral cytidine to uridine. In an attempt to harness the anti-HIV effect of A3G, we searched for an agent that would up-regulate A3G and identify the receptors involved. Stimulation of cell surface CCR5 with CCL3 and CD40 with CD40L or both molecules with microbial 70-kDa heat shock protein (HSP)70 up-regulated A3G mRNA and protein expression in human CD4(+) T cells and monocyte-derived dendritic cells (DC), demonstrated by real-time PCR and Western blots, respectively. The specificity of CCR5 and CD40 stimulation was established by inhibition with TAK 779 and mAb to CD40, as well as using human embryonic kidney 293 cells transfected with CCR5 and CD40, respectively. A dose-dependent increase of A3G in CCL3- or HSP70-stimulated CD4(+) T cells was associated with inhibition in HIV-1 infectivity. To differentiate between the inhibitory effect of HSP70-induced CCR5 binding and that of A3G, GFP-labeled pseudovirions were used to infect human embryonic kidney 293 cells, which showed inhibition of pseudovirion uptake, consistent with A3G being responsible for the inhibitory effect. Ligation of cell surface CCR5 receptors by CCL3 or CD40 by CD40L activated the ERK1/2 and p38 MAPK signaling pathways that induced A3G mRNA expression and production of the A3G protein. These in vitro results were corroborated by in vivo studies in rhesus macaques in which A3G was significantly up-regulated following immunization with SIVgp120 and p27 linked to HSP70. This novel preventive approach may in addition to adaptive immunity use the intracellular innate antiviral effect of A3G.     

A Synthetic Peptide Fragment Derived from RANTES is a Potent Inhibitor of HIV-1 Infectivity Despite a Surprising Lack of CCR5 Receptor Affinity

CCR5 (CC-chemokine receptor 5) is a key co-receptor, in concert with CD4, for infectivity of HIV-1 (human immunodeficiency virus type-1) into healthy human cells, and RANTES, an endogenous ligandfor CCR5, is a potent inhibitor of HIV-1 infectivity. In this structure-activity relationship (SAR) study, peptide fragments derived from RANTES were designed, synthesized and evaluated fortheir ability to inhibit HIV-1 infectivity. The goal was to determine the effect of peptide length on anti-HIV activity and to obtain an optimally sized RANTES peptide probe for further SAR studies. The analogue Ac[Ala^10,11]RANTES-(1–14)NH₂, AA14, was identified as an effective inhibitor of HIV-1 infectivity at 10 nM but despite the functional activity, surprisingly it did not exhibit any notable affinity for the CCR5 chemokine receptor. Further, increasing peptide size enhanced neither the inhibition of HIV-1 infectivity nor CCR5 receptor affinity. As a potent inhibitor of HIV-1 infectivity,the lead analogue most likely utilizes a different (and currentlyunknown) mechanism than interaction with CCR5 for anti-HIV activity.     

A Synthetic Peptide Fragment Derived from RANTES is a Potent Inhibitor of HIV-1 Infectivity Despite a Surprising Lack of CCR5 Receptor Affinity

Summary CCR5 (CC-chemokine receptor 5) is a key co-receptor, in concert with CD4, for infectivity of HIV-1 (human immunodeficiency virus type-1) into healthy human cells, and RANTES, an endogenous ligand for CCR5, is a potent inhibitor of HIV-1 infectivity. In this structure-activity relationship (SAR) study, peptide fragments derived from RANTES were designed, synthesized and evaluated for their ability to inhibit HIV-1 infectivity. The goal was to determine the effect of peptide length on anti-HIV activity and to obtain an optimally sized RANTES peptide probe for further SAR studies. The analogue Ac[Ala^10,11]RANTES-(1–14)NH₂, AA14, was identified as an effective inhibitor of HIV-1 infectivity at 10 nM but despite the functional activity, surprisingly it did not exhibit any notable affinity for the CCR5 chemokine receptor. Further, increasing peptide size enhanced neither the inhibition of HIV-1 infectivity nor CCR5 receptor affinity. As a potent inhibitor of HIV-1 infectivity, the lead analogue most likely utilizes a different (and currently unknown) mechanism than interaction with CCR5 for anti-HIV activity.     

Inhibition of HIV-1 infection by synthetic peptides derived CCR5 fragments

HIV-1 infection requires interaction of viral envelope protein gp160 with CD4 and a chemokine receptor, CCR5 or CXCR4 as entry coreceptor. We designed HIV-inhibitory peptides targeted to CCR5 using a novel computer program (ANTIS), which searched all possible sense-antisense amino acid pairs between proteins. Seven AHBs were found in CCR5 receptor. All AHB peptides were synthesized and tested for their ability to prevent HIV-1 infection to human T cells. A peptide fragment (LC5) which is a part of the CCR5 receptor corresponding to the loop between the fifth and sixth transmembrane regions (amino acids 222-240) proved to inhibit HIV-1IIIB infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. LC5 effectively indicated dose-dependent manner, and the suppression was enhanced additively by T20 peptide, which inhibits infection in vitro by disrupting the gp41 conformational changes necessary for membrane fusion. Thus, these results indicate that CCR5-derived AHB peptides could provide a useful tool to define the mechanism(s) of HIV infection, and may provide insight which will contribute to the development of an anti-HIV-1 reagent.     

Design and synthesis of small molecule-sulfotyrosine mimetics that inhibit HIV-1 entry

In the absence of a cure or vaccine for HIV/AIDS, small molecule inhibitors remain an attractive choice for antiviral therapeutics. Recent structural and functional studies of the HIV-1 surface envelope glycoprotein gp120 have revealed sites of vulnerability that can be targeted by small molecule and peptide inhibitors, thereby inhibiting HIV-1 infection. Here we describe a series of small molecule entry inhibitors that were designed to mimic the sulfated N-terminal peptide of the HIV-1 coreceptor CCR5. From a panel of hydrazonothiazolyl pyrazolinones, we demonstrate that compounds containing naphthyl di- and tri-sulfonic acids inhibit HIV-1 infection in single round infectivity assays with the disulfonic acids being the most potent. Molecular docking supports the observed structure activity relationship, and SPR confirmed binding to gp120. In infectivity assays treatment with a representative naphthyl disulfonate and a disulfated CCR5 N-terminus peptide results in competitive inhibition, with combination indices >2. In total this work shows that gp120 and HIV-1 infection can be inhibited by small molecules that mimic the function of, and are competitive with the natural sulfated CCR5 N-terminus.     

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.     

Recombinant extracellular domains of tetraspanin proteins are potent inhibitors of the infection of macrophages by human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) infection of human macrophages can be inhibited by antibodies which bind to the tetraspanin protein CD63, but not by antibodies that bind to other members of the tetraspanin family. This inhibitory response was limited to CCR5 (R5)-tropic virus and was only observed using macrophages, but not T cells. Here, we show that recombinant soluble forms of the large extracellular domain (EC2) of human tetraspanins CD9, CD63, CD81, and CD151 produced as fusion proteins with glutathione S-transferase (GST) can all potently and completely inhibit R5 HIV-1 infection of macrophages with 50% inhibitory concentration values of 0.11 to 1.2 nM. Infection of peripheral blood mononuclear cells could also be partly inhibited, although higher concentrations of EC2 proteins were required. Inhibition was largely coreceptor independent, as macrophage infections by virions pseudotyped with CXCR4 (X4)-tropic HIV-1 or vesicular stomatitis virus (VSV)-G glycoproteins were also inhibited, but was time dependent, since addition prior to or during, but not after, virus inoculation resulted in potent inhibition. Incubation with tetraspanins did not decrease CD4 or HIV-1 coreceptor expression but did block virion uptake. Colocalization of fluorescently labeled tetraspanin EC2 proteins and HIV-1 virions within, and with CD4 and CXCR4 at the cell surfaces of, macrophages could be detected, and internalized tetraspanin EC2 proteins were directed to vesicular compartments that contained internalized dextran and transferrin. Collectively, the data suggest that the mechanism of inhibition of HIV-1 infection by tetraspanins is at the step of virus entry, perhaps via interference with binding and/or the formation of CD4-coreceptor complexes within microdomains that are required for membrane fusion events.     

Regulation of human immunodeficiency virus type 1 infection, beta-chemokine production, and CCR5 expression in CD40L-stimulated macrophages: immune control of viral entry

Mononuclear phagocytes (MP) and T lymphocytes play a pivotal role in the host immune response to human immunodeficiency virus type 1 (HIV-1) infection. Regulation of such immune responses can be mediated, in part, through the interaction of the T-lymphocyte-expressed molecule CD40 ligand (CD40L) with its receptor on MP, CD40. Upregulation of CD40L on CD4+ peripheral blood mononuclear cells during advanced HIV-1 disease has previously been reported. Based on this observation, we studied the influence of CD40L-CD40 interactions on MP effector function and viral regulation in vitro. We monitored productive viral infection, cytokine and beta-chemokine production, and beta-chemokine receptor expression in monocyte-derived macrophages (MDM) after treatment with soluble CD40L. Beginning 1 day after infection and continuing at 3-day intervals, treatment with CD40L inhibited productive HIV-1 infection in MDM in a dose-dependent manner. A concomitant and marked upregulation of beta-chemokines (macrophage inhibitory proteins 1alpha and 1beta and RANTES [regulated upon activation normal T-cell expressed and secreted]) and the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) was observed in HIV-1-infected and CD40L-treated MDM relative to either infected or activated MDM alone. The addition of antibodies to RANTES or TNF-alpha led to a partial reversal of the CD40L-mediated inhibition of HIV-1 infection. Surface expression of CD4 and the beta-chemokine receptor CCR5 was reduced on MDM in response to treatment with CD40L. In addition, treatment of CCR5- and CD4-transfected 293T cells with secretory products from CD40L-stimulated MDM prior to infection with a CCR5-tropic HIV-1 reporter virus led to inhibition of viral entry. In conclusion, we demonstrate that CD40L-mediated inhibition of viral entry coincides with a broad range of MDM immune effector responses and the down-modulation of CCR5 and CD4 expression.     

CD4-independent infection of astrocytes by human immunodeficiency virus type 1: requirement for the human mannose receptor

Human immunodeficiency virus type 1 (HIV-1) infection occurs in the central nervous system and causes a variety of neurobehavioral and neuropathological disorders. Both microglia, the residential macrophages in the brain, and astrocytes are susceptible to HIV-1 infection. Unlike microglia that express and utilize CD4 and chemokine coreceptors CCR5 and CCR3 for HIV-1 infection, astrocytes fail to express CD4. Astrocytes express several chemokine coreceptors; however, the involvement of these receptors in astrocyte HIV-1 infection appears to be insignificant. In the present study using an expression cloning strategy, the cDNA for the human mannose receptor (hMR) was found to be essential for CD4-independent HIV-1 infectivity. Ectopic expression of functional hMR rendered U87.MG astrocytic cells susceptible to HIV-1 infection, whereas anti-hMR serum and hMR-specific siRNA blocked HIV-1 infection in human primary astrocytes. In agreement with these findings, hMR bound to HIV-1 virions via the abundant and highly mannosylated sugar moieties of HIV-1 envelope glycoprotein gp120 in a Ca(2+)-dependent fashion. Moreover, hMR-mediated HIV-1 infection was dependent upon endocytic trafficking as assessed by transmission electron microscopy, as well as inhibition of viral entry by endosomo- and lysosomotropic drugs. Taken together, these results demonstrate the direct involvement of hMR in HIV-1 infection of astrocytes and suggest that HIV-1 interaction with hMR plays an important role in HIV-1 neuropathogenesis.     

Stimulation of Th1-polarizing cytokines,C-C chemokines,maturation of dendritic cells,and adjuvant function by the peptide binding fragment of heat shock protein 70

The peptide binding C-terminal portion of heat shock protein (HSP)70 (aa 359-610) stimulates human monocytes to produce IL-12, TNF-alpha, NO, and C-C chemokines. The N-terminal, ATPase portion (HSP70(1-358)) failed to stimulate any of these cytokines or chemokines. Both native and the truncated HSP70(359-610) stimulation of chemokine production is mediated by the CD40 costimulatory molecule. Maturation of dendritic cells was induced by stimulation with native HSP70, was not seen with the N-terminal HSP70(1-358), but was enhanced with HSP70(359-610), as demonstrated by up-regulation of CD83, CCR7, CD86, CD80, and HLA class II. In vivo studies in macaques showed that immunization with HSP70(359-610) enhances the production of IL-12 and RANTES. Immunization with peptide-bound HSP70(359-610) in mice induced higher serum IgG2a and IgG3 Abs than the native HSP70-bound peptide. This study suggests that the C-terminal, peptide-binding portion of HSP70 is responsible for stimulating Th1-polarizing cytokines, C-C chemokines, and an adjuvant function.     

G protein-dependent CCR5 signaling is not required for efficient infection of primary T lymphocytes and macrophages by R5 human immunodeficiency virus type 1 isolates

The requirement of human immunodeficiency virus (HIV)-induced CCR5 activation for infection by R5 HIV type 1 (HIV-1) strains remains controversial. Ectopic CCR5 expression in CD4(+)-transformed cells or pharmacological inhibition of G(alpha)i proteins coupled to CCR5 left unsolved whether CCR5-dependent cell activation is necessary for the HIV life cycle. In this study, we investigated the role played by HIV-induced CCR5-dependent cell signaling during infection of primary CD4-expressing leukocytes. Using lentiviral vectors, we restored CCR5 expression in T lymphocytes and macrophages from individuals carrying the homozygous 32-bp deletion of the CCR5 gene (ccr5 Delta32/Delta32). Expression of wild-type (wt) CCR5 in ccr5 Delta32/Delta32 cells permitted infection by R5 HIV isolates. We assessed the capacity of a CCR5 derivative carrying a mutated DRY motif (CCR5-R126N) in the second intracellular loop to work as an HIV-1 coreceptor. The R126N mutation is known to disable G protein coupling and agonist-induced signal transduction through CCR5 and other G protein-coupled receptors. Despite its inability to promote either intracellular calcium mobilization or cell chemotaxis, the inactive CCR5-R126N mutant provided full coreceptor function to several R5 HIV-1 isolates in primary cells as efficiently as wt CCR5. We conclude that in a primary, CCR5-reconstituted CD4(+) cell environment, G protein signaling is dispensable for R5 HIV-1 isolates to actively infect primary CD4(+) T lymphocytes or macrophages.     

Macrophage inflammatory protein 1alpha inhibits postentry steps of human immunodeficiency virus type 1 infection via suppression of intracellular cyclic AMP

Primary isolates of human immunodeficiency virus type 1 (HIV-1) predominantly use chemokine receptor CCR5 to enter target cells. The natural ligands of CCR5, the beta-chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES, interfere with HIV-1 binding to CCR5 receptors and decrease the amount of virions entering cells. Although the inhibition of HIV-1 entry by beta-chemokines is well documented, their effects on postentry steps of the viral life cycle and on host cell components that control the outcome of infection after viral entry are not well defined. Here, we show that all three beta-chemokines, and MIP-1alpha in particular, inhibit postentry steps of the HIV-1 life cycle in primary lymphocytes, presumably via suppression of intracellular levels of cyclic AMP (cAMP). Productive HIV-1 infection of primary lymphocytes requires cellular activation. Cell activation increases intracellular cAMP, which is required for efficient synthesis of proviral DNA during early steps of viral infection. Binding of MIP-1alpha to cognate receptors decreases activation-induced intracellular cAMP levels through the activation of inhibitory G proteins. Furthermore, inhibition of one of the downstream targets of cAMP, cAMP-dependent PKA, significantly inhibits synthesis of HIV-1-specific DNA without affecting virus entry. These data reveal that beta-chemokine-mediated inhibition of virus replication in primary lymphocytes combines inhibitory effects at the entry and postentry levels and imply the involvement of beta-chemokine-induced signaling in postentry inhibition of HIV-1 infection.     

Multiple restrictions of human immunodeficiency virus type 1 in feline cells

The productive replication of human immunodeficiency virus type 1 (HIV-1) occurs exclusively in defined cells of human or chimpanzee origin, explaining why heterologous animal models for HIV replication, pathogenesis, vaccination, and therapy are not available. This lack of an animal model for HIV-1 studies prompted us to examine the susceptibility of feline cells in order to evaluate the cat (Felis catus) as an animal model for studying HIV-1. Here, we report that feline cell lines harbor multiple restrictions with respect to HIV-1 replication. The feline CD4 receptor does not permit virus infection. Feline T-cell lines MYA-1 and FeT-1C showed postentry restrictions resulting in low HIV-1 luciferase reporter activity and low expression of viral Gag-Pol proteins when pseudotyped vectors were used. Feline fibroblastic CrFK and KE-R cells, expressing human CD4 and CCR5, were very permissive for viral entry and HIV-long terminal repeat-driven expression but failed to support spreading infection. KE-R cells displayed a profound block with respect to release of HIV-1 particles. In contrast, CrFK cells allowed very efficient particle production; however, the CrFK cell-derived HIV-1 particles had low specific infectivity. We subsequently identified feline apolipoprotein B-editing catalytic polypeptide 3 (feAPOBEC3) proteins as active inhibitors of HIV-1 particle infectivity. CrFK cells express at least three different APOBEC3s: APOBEC3C, APOBEC3H, and APOBEC3CH. While the feAPOBEC3C did not significantly inhibit HIV-1, the feAPOBEC3H and feAPOBEC3CH induced G to A hypermutations of the viral cDNA and reduced the infectivity approximately 10- to approximately 40-fold.     

Identification of a linear peptide recognized by monoclonal antibody 2D7 capable of generating CCR5-specific antibodies with human immunodeficiency virus-neutralizing activity

CCR5 is the major coreceptor for human immunodeficiency virus (HIV) infection. The murine monoclonal antibody (MAb) 2D7, which recognizes a conformation-dependent epitope in the second extracellular loop of CCR5, is one of the most potent inhibitors of R5 virus cell entry. However, attempts to humanize 2D7 for in vivo human use have been unsuccessful so far. A filamentous phage library expressing random peptides was used to identify a peptide mimitope that is recognized by MAb 2D7. A synthetic peptide containing this sequence (2D7-2SK) bound to MAb 2D7 with high affinity and reversed its HIV type 1 (HIV-1) fusion inhibitory activity. The peptide contains sequence homologies to two distal regions of the second extracellular loop of human CCR5, both of which are required for MAb 2D7 binding. Rabbit anti-2D7-mimitope antibodies competed with MAb 2D7 for binding to the 2D7-2SK peptide in Biacore biosensor testing. Importantly, the rabbit anti-2D7-2SK antibodies bound to CCR5 on cells and specifically inhibited R5 (but not X4) envelope-mediated syncytium formation. These antibodies also neutralized infection of human peripheral blood mononuclear cells with R5 HIV isolates comparably to MAb 2D7. In summary, we have identified a novel peptide that closely mimics the MAb 2D7 epitope on CCR5. This peptide could be included as a potential vaccine candidate or to isolate 2D7-like human antibodies as entry inhibitors for R5 viruses.     

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.     

Multiple CCR5 conformations on the cell surface are used differentially by human immunodeficiency viruses resistant or sensitive to CCR5 inhibitors

Resistance to small-molecule CCR5 inhibitors arises when HIV-1 variants acquire the ability to use inhibitor-bound CCR5 while still recognizing free CCR5. Two isolates, CC101.19 and D1/85.16, became resistant via four substitutions in the gp120 V3 region and three in the gp41 fusion peptide (FP), respectively. The binding characteristics of a panel of monoclonal antibodies (MAbs) imply that several antigenic forms of CCR5 are expressed at different levels on the surfaces of U87-CD4-CCR5 cells and primary CD4(+) T cells, in a cell-type-dependent manner. CCR5 binding and HIV-1 infection inhibition experiments suggest that the two CCR5 inhibitor-resistant viruses altered their interactions with CCR5 in different ways. As a result, both mutants became generally more sensitive to inhibition by CCR5 MAbs, and the FP mutant is specifically sensitive to a MAb that stains discrete cell surface clusters of CCR5 that may correspond to lipid rafts. We conclude that some MAbs detect different antigenic forms of CCR5 and that inhibitor-sensitive and -resistant viruses can use these CCR5 forms differently for entry in the presence or absence of CCR5 inhibitors.     

CXCR4 and CCR5 regulation and expression patterns on T- and monocyte-macrophage cell lineages: implications for susceptibility to infection by HIV-1

Chemokine receptor expression may vary dramatically among cell subsets. Therefore, the stage of differentiation and the lineage of CD4 cells may profoundly affect their susceptibility to infection by human immunodeficiency virus type 1 (HIV-1). However, the mechanisms of coreceptor competition for association with HIV-1 glycoproteins remain unknown. Here, we propose mathematical models that address the interdependence of the concentrations of CD4 and CCR5 for efficient infection by M-tropic HIV-1 as well as additional complications originated by coreceptor competition caused by posttranslational modifications that positively or negatively affect the coreceptor ability to form complexes with CD4 and/or HIV-1 envelope. Furthermore, since CCR5 and CXCR4 expression on human leukocytes designate these cells as HIV-1 potential targets, the expression of the major HIV-1 coreceptors are also dynamically modeled/quantified as function of the stage of cell differentiation. Results show that although coreceptor competition degree has limited influence on R5 strain infectivity, the infectivity of CXCR4-using isolates strongly depends on the CD4 expression, according to the coreceptor competition model proposed in Lee et al. [J. Virol. 74(11) (2000) 5016]. Understanding the role of in vivo alterations in CD4, CCR5 and CXCR4 densities on HIV-1 cell entry may help the development of optimal control strategies for AIDS pathogenesis.     

Syntheses and SAR studies of 4-(heteroarylpiperdin-1-yl-methyl)-pyrrolidin-1-yl-acetic acid antagonists of the human CCR5 chemokine receptor

Efforts toward the exploration of the title compounds as CCR5 antagonists are disclosed. The basis for such work stems from the fact that cellular proliferation of HIV-1 requires the cooperative assistance of both CCR5 and CD4 receptors. The synthesis and SAR of pyrrolidineacetic acid derivatives as CCR5 antagonists displaying potent binding and antiviral properties in a HeLa cell-based HIV-1 infectivity assay are discussed.