Evidence for Common Structural Determinants of Human Immunodeficiency Virus Type 1 Coreceptor Activity Provided through Functional Analysis of CCR5/CXCR4 Chimeric Coreceptors

Human immunodeficiency virus type 1 (HIV-1) infection in vivo is dependent upon the interaction of the viral envelope glycoprotein gp120 with CC chemokine receptor 5 (CCR5) or CXC chemokine receptor 4 (CXCR4). To study the determinants of the gp120-coreceptor association, we generated a set of chimeric HIV-1 coreceptors which express all possible combinations of the four extracellular domains of CCR5 and CXCR4. Stable U87 astroglioma cell lines expressing CD4 and individual chimeric coreceptor proteins were tested against a variety of R5, X4, and R5X4 envelope glycoproteins and virus strains for their ability to support HIV-1-mediated cell fusion and infection, respectively. Each of the cell lines promoted fusion with cells expressing an HIV envelope glycoprotein, except for U87.CD4.5455, which presents the first extracellular loop (ECL1) and flanking sequences of CXCR4 in the context of CCR5. However, all of the chimeric coreceptors allowed productive infection by one or more of the viral strains tested. Viral phenotype was a predictive factor for the observed activity of the chimeric molecules; X4 and R5X4 HIV strains utilized a majority of the chimeras, while R5 strains were limited in their ability to infect cells expressing these chimeric molecules. The expression of CCR5 ECL2 within the CXCR4 backbone supported infection by an R5 primary isolate, but no chimeras bearing the N terminus of CCR5 exhibited activity with R5 strains. Remarkably, the introduction of any CXCR4 domain into the CCR5 backbone was sufficient to allow utilization by multiple X4 strains. However, critical determinants within ECL2 and/or ECL3 of CXCR4 were apparent for all X4 viruses upon replacement of these domains in CXCR4 with CCR5 sequences. Unexpectedly, chimeric coreceptor-facilitated entry was blocked in all cases by the presence of the CXCR4-specific inhibitor AMD3100. Our data provide proof that CCR5 contains elements that support usage by X4 viral strains and demonstrate that the gp120 interaction sites of CCR5 and CXCR4 are structurally related.     

T134, a Small-Molecule CXCR4 Inhibitor, Has No Cross-Drug Resistance with AMD3100, a CXCR4 Antagonist with a Different Structure

T22, an analog of polyphemusin II (18 amino acid residues), was found to block T-tropic human immunodeficiency virus type 1 (HIV-1) entry into target cells as a CXCR4 inhibitor. We synthesized T134, a small analog (14 amino acid residues) of T22 with reduced positive charges. T134 exhibited highly potent activity and significantly less cytotoxicity in comparison to that of T22. T134 prevents the anti-CXCR4 monoclonal antibody from binding to peripheral blood mononuclear cells but has no effect on the binding of anti-CCR5 monoclonal antibodies. Since T134 inhibits the binding of stromal cell-derived factor-1 (SDF-1) to MT-4 cells, it seems that T134 prevents HIV-1 entry by binding to CXCR4. The bicyclam AMD3100 has also been shown to block HIV-1 entry via CXCR4 but not via CCR5. Both T134 and AMD3100 are CXCR4 antagonists and low-molecular-weight compounds but have different structures. Our results indicate that T134 is active against wild-type T-tropic HIV-1 strains and against AMD3100-resistant strains.     

The Second Extracellular Loop of CXCR4 Determines Its Function as a Receptor for Feline Immunodeficiency Virus

The feline homolog of the α-chemokine receptor CXCR4 has recently been shown to support cell-cell fusion mediated by CXCR4-dependent strains of human immunodeficiency virus (HIV) and strains of feline immunodeficiency virus (FIV) that have been selected for growth in the Crandell feline kidney (CrFK) cell line. In this report we demonstrate that expression of CXCR4 alone is sufficient to render cells from diverse species permissive for fusion with FIV-infected cells, suggesting that CXCR4 is the sole receptor for CrFK-tropic strains of FIV, analogous to CD4-independent strains of HIV-2. To identify the regions of CXCR4 involved in fusion mediated by FIV, we screened panels of chimeric CXCR4 molecules for the ability to support fusion with FIV-infected cells. Human CXCR4 supported fusion more efficiently than feline CXCR4 and feline/human CXCR4 chimeras, suggesting that the second and third extracellular loops of human CXCR4 contain a critical determinant for receptor function. Rat/human CXCR4 chimeras suggested that the second extracellular loop contained the principal determinant for receptor function; however, chimeras constructed between human CXCR2 and CXCR4 revealed that the first and third loops of CXCR4 contribute to the FIV Env binding site, as replacement of these domains with the corresponding domains of CXCR2 rendered the molecule nonfunctional in fusion assays. Mutation of the DRY motif and the C-terminal cytoplasmic tail of CXCR4 did not affect the ability of the molecule to support fusion, suggesting that neither signalling via G proteins nor receptor internalization was required for fusion mediated by FIV; similarly, truncation of the N terminus of CXCR4 did not affect the function of the molecule as a receptor for FIV. CXCR4-transfected feline cells were rendered permissive for infection with both the CrFK-tropic PET isolate of FIV and the CXCR4-dependent RF strain of HIV-1, and susceptibility to infection correlated well with ability to support fusion. The data suggest that the second extracellular loop of CXCR4 is the major determinant of CXCR4 usage by FIV.     

Conformational study of a highly specific CXCR4 inhibitor, T140, disclosing the close proximity of its intrinsic pharmacophores associated with strong anti-HIV activity

We report the solution structure of T140, a truncated polyphemusin peptide analogue that efficiently inhibits infection of target cells by T-cell line-tropic strains of HIV-1 through its specific binding to a chemokine receptor, CXCR4. Nuclear magnetic resonance analysis and molecular dynamic calculations revealed that T140 has a rigidly structured conformation constituted by an antiparallel beta-sheet and a type II' beta-turn. A protuberance is formed on one side of the beta-sheet by the side-chain functional groups of the three amino acid residues (L-3-(2-naphthyl)alanine, Tyr5 and Arg14), each of which is indispensable for strong anti-HIV activity. These findings provide a rationale to dissect the structural basis for the ability of this compound to block the interaction between CXCR4 and envelope glycoproteins from T-tropic strains of HIV-1.     

Identification of gp120 binding sites on CXCR4 by using CD4-independent human immunodeficiency virus type 2 Env proteins

Human immunodeficiency virus (HIV) and simian (SIV) immunodeficiency virus entry is mediated by binding of the viral envelope glycoprotein (Env) to CD4 and chemokine receptors, CCR5 and/or CXCR4. CD4 induces extensive conformational changes that expose and/or induce formation of a chemokine receptor binding site on gp120. CD4-independent Env's of HIV type 1 (HIV-1), HIV-2, and SIV have been identified that exhibit exposed chemokine receptor binding sites and can bind directly to CCR5 or CXCR4 in the absence of CD4. While many studies have examined determinants for gp120-CCR5 binding, analysis of gp120-CXCR4 binding has been hindered by the apparently lower affinity of this interaction for X4-tropic HIV-1 isolates. We show here that gp120 proteins from two CD4-independent HIV-2 Env's, VCP and ROD/B, bind directly to CXCR4 with an apparently high affinity. By use of CXCR4 N-terminal deletion constructs, CXCR4-CXCR2 chimeras, and human-rat CXCR4 chimeras, binding determinants were shown to reside in the amino (N) terminus, extracellular loop 2 (ECL2), and ECL3. Alanine-scanning mutagenesis of charged residues, tyrosines, and phenylalanines in extracellular CXCR4 domains implicated multiple amino acids in the N terminus (E14/E15, D20, Y21, and D22), ECL2 (D187, R188, F189, Y190, and D193), and ECL3 (D262, E268, E277, and E282) in binding, although minor differences were noted between VCP and ROD/B. However, mutations in CXCR4 that markedly reduced binding did not necessarily hinder cell-cell fusion by VCP or ROD/B, especially in the presence of CD4. These gp120 proteins will be useful in dissecting determinants for CXCR4 binding and Env triggering and in evaluating pharmacologic inhibitors of the gp120-CXCR4 interaction.     

Unique Monoclonal Antibody Recognizing the Third Extracellular Loop of CXCR4 Induces Lymphocyte Agglutination and Enhances Human Immunodeficiency Virus Type 1-Mediated Syncytium Formation and Productive Infection

To increase insight into the structural basis of CXCR4 utilization in human immunodeficiency virus type 1 (HIV-1) infection, a new generation of three monoclonal antibodies (MAbs) was developed in WKA rats. The A80 MAb, which binds an epitope in the third extracellular loop (ECL3) of CXCR4, has unique biologic properties that provide novel insights into CXCR4 function. This agent enhanced syncytium formation in activated human peripheral blood mononuclear cells (PBMC) infected with X4 or R5 and CEM cells infected with X4 HIV-1 strains. Exposure to A80 increased the productive infection of activated CD4(+) T cells and CEM cells with R5 and X4 viruses, respectively. This antibody uniquely induced agglutination of PBMC and CEM cells but did not activate calcium mobilization. Agglutination induced by A80 was inhibited by stromal cell-derived factor 1, T22, and phorbol 12-myristate 13-acetate but was not significantly altered by pretreatment of cells with pertussis toxin, wortmannin, or MAbs to LFA-1, ICAM-1, ICAM-2, and ICAM-3. The binding of the A145 and A120 MAbs was mapped to the N-terminal extracellular domain and a conformational epitope involving ECL1 and ECL2, respectively. Both of these MAbs inhibited HIV-1 infection and lacked the novel properties of A80. These results suggest a new role for CXCR4 in homologous lymphocyte adhesion that is ligand independent and in HIV-1 infection.     

The CXC chemokine receptor 4 ligands ubiquitin and stromal cell-derived factor-1α function through distinct receptor interactions

Recently, we identified extracellular ubiquitin as an endogenous CXC chemokine receptor (CXCR) 4 agonist. However, the receptor selectivity and molecular basis of the CXCR4 agonist activity of ubiquitin are unknown, and functional consequences of CXCR4 activation with ubiquitin are poorly defined. Here, we provide evidence that ubiquitin and the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α do not share CXCR7 as a receptor. We further demonstrate that ubiquitin does not utilize the typical two-site binding mechanism of chemokine-receptor interactions, in which the receptor N terminus is important for ligand binding. CXCR4 activation with ubiquitin and SDF-1α lead to similar Gα(i)-responses and to a comparable magnitude of phosphorylation of ERK-1/2, p90 ribosomal S6 kinase-l and Akt, although phosphorylations occur more transiently after activation with ubiquitin. Despite the similarity of signal transduction events after activation of CXCR4 with both ligands, ubiquitin possesses weaker chemotactic activity than SDF-lα in cell migration assays and does not interfere with productive entry of HIV-1 into P4.R5 multinuclear activation of galactosidase indicator cells. Unlike SDF-1α, ubiquitin lacks interactions with an N-terminal CXCR4 peptide in NMR spectroscopy experiments. Binding and signaling studies in the presence of antibodies against the N terminus and extracellular loops 2/3 of CXCR4 confirm that the ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain, whereas blockade of extracellular loops 2/3 prevents receptor binding and activation. Our findings define ubiquitin as a CXCR4 agonist, which does not interfere with productive cellular entry of HIV-1, and provide new mechanistic insights into interactions between CXCR4 and its natural ligands.     

Coreceptor tropism in human immunodeficiency virus type 1 subtype D: high prevalence of CXCR4 tropism and heterogeneous composition of viral populations

In human immunodeficiency virus type 1 (HIV-1) subtype B, CXCR4 coreceptor use ranges from approximately 20% in early infection to approximately 50% in advanced disease. Coreceptor use by non-subtype B HIV is less well characterized. We studied coreceptor tropism of subtype A and D HIV-1 collected from 68 pregnant, antiretroviral drug-naive Ugandan women (HIVNET 012 trial). None of 33 subtype A or 10 A/D-recombinant viruses used the CXCR4 coreceptor. In contrast, nine (36%) of 25 subtype D viruses used both CXCR4 and CCR5 coreceptors. Clonal analyses of the nine subtype D samples with dual or mixed tropism revealed heterogeneous viral populations comprised of X4-, R5-, and dual-tropic HIV-1 variants. In five of the six samples with dual-tropic strains, V3 loop sequences of dual-tropic clones were identical to those of cocirculating R5-tropic clones, indicating the presence of CXCR4 tropism determinants outside of the V3 loop. These dual-tropic variants with R5-tropic-like V3 loops, which we designated "dual-R," use CCR5 much more efficiently than CXCR4, in contrast to dual-tropic clones with X4-tropic-like V3 loops ("dual-X"). These observations have implications for pathogenesis and treatment of subtype D-infected individuals, for the association between V3 sequence and coreceptor tropism phenotype, and for understanding potential mechanisms of evolution from exclusive CCR5 use to efficient CXCR4 use by subtype D HIV-1.     

Tat Protein Induces Human Immunodeficiency Virus Type 1 (HIV-1) Coreceptors and Promotes Infection with both Macrophage-Tropic and T-Lymphotropic HIV-1 Strains

Chemokine receptors CCR5 and CXCR4 are the primary fusion coreceptors utilized for CD4-mediated entry by macrophage (M)- and T-cell line (T)-tropic human immunodeficiency virus type 1 (HIV-1) strains, respectively. Here we demonstrate that HIV-1 Tat protein, a potent viral transactivator shown to be released as a soluble protein by infected cells, differentially induced CXCR4 and CCR5 expression in peripheral blood mononuclear cells. CCR3, a less frequently used coreceptor for certain M-tropic strains, was also induced. CXCR4 was induced on both lymphocytes and monocytes/macrophages, whereas CCR5 and CCR3 were induced on monocytes/macrophages but not on lymphocytes. The pattern of chemokine receptor induction by Tat was distinct from that by phytohemagglutinin. Moreover, Tat-induced CXCR4 and CCR5 expression was dose dependent. Monocytes/macrophages were more susceptible to Tat-mediated induction of CXCR4 and CCR5 than lymphocytes, and CCR5 was more readily induced than CXCR4. The concentrations of Tat effective in inducing CXCR4 and CCR5 expression were within the picomolar range and close to the range of extracellular Tat observed in sera from HIV-1-infected individuals. The induction of CCR5 and CXCR4 expression correlated with Tat-enhanced infectivity of M- and T-tropic viruses, respectively. Taken together, our results define a novel role for Tat in HIV-1 pathogenesis that promotes the infectivity of both M- and T-tropic HIV-1 strains in primary human leukocytes, notably in monocytes/macrophages.     

Effect of mutations in the second extracellular loop of CXCR4 on its utilization by human and feline immunodeficiency viruses

CCR5 and CXCR4 are the principal CD4-associated coreceptors used by human immunodeficiency virus type 1 (HIV-1). CXCR4 is also a receptor for the feline immunodeficiency virus (FIV). The rat CXCR4 cannot mediate infection by HIV-1NDK or by FIVPET (both cell line-adapted strains) because of sequence differences with human CXCR4 in the second extracellular loop (ECL2). Here we made similar observations for HIV-189.6 (a strain also using CCR5) and for a primary HIV-1 isolate. It showed the role of ECL2 in the coreceptor activity of CXCR4 for different types of HIV-1 strains. By exchanging ECL2 residues between human and rat CXCR4, we found that several amino acid differences contributed to the inactivity of the rat CXCR4 toward HIV-189.6. In contrast, its inactivity toward HIV-1NDK seemed principally due to a serine at position 193 instead of to an aspartic acid (Asp193) in human CXCR4. Likewise, a mutation of Asp187 prevented usage of CXCR4 by FIVPET. Different mutations of Asp193, including its replacement by a glutamic acid, markedly reduced or suppressed the activity of CXCR4 for HIV-1NDK infection, indicating that the negative charge was not the only requirement. Mutations of Asp193 and of arginine residues (Arg183 and Arg188) of CXCR4 reduced the efficiency of HIV-1 infection for all HIV-1 strains tested. Other ECL2 mutations tested had strain-specific effects or no apparent effect on HIV-1 infection. The ECL2 mutants allowed us to identify residues contributing to the epitope of the 12G5 monoclonal antibody. Overall, residues with different charges and interspersed in ECL2 seem to participate in the coreceptor activity of CXCR4. This suggests that a conformational rather than linear epitope of ECL2 contributes to the HIV-1 binding site. However, certain HIV-1 and FIV strains seem to require the presence of a particular ECL2 residue.     

Synthesis and evaluation of pseudopeptide analogues of a specific CXCR4 inhibitor, T140: the insertion of an (E)-alkene dipeptide isostere into the betaII'-turn moiety

A 14-residue peptide, T140, strongly inhibits the T-cell line-tropic HIV-1 (X4-HIV-1) infection, since this peptide functions as a specific antagonist against a chemokine receptor, CXCR4. T140 takes an antiparallel beta-sheet structure with a type II' beta-turn. In the present paper, we have designed and synthesized several T140 analogues, in which an (E)-alkene dipeptide isostere was inserted into the type II' beta-turn moiety, as a bridging study to develop nonpeptidic CXCR4 inhibitors. It has been proven that the turn region of T140 can be replaced by the above surrogate with the maintenance of strong anti-HIV activity.     

Identification of conserved and variable structures in the human immunodeficiency virus gp120 glycoprotein of importance for CXCR4 binding

CD4 and the chemokine receptors, CXCR4 and CCR5, serve as receptors for human immunodeficiency virus type 1 (HIV-1). Binding of the HIV-1 gp120 envelope glycoprotein to the chemokine receptors normally requires prior interaction with CD4. Mapping the determinants on gp120 for the low-affinity interaction with CXCR4 has been difficult due to the nonspecific binding of this viral glycoprotein to cell surfaces. Here we examine the binding of a panel of gp120 mutants to paramagnetic proteoliposomes displaying CXCR4 on their surfaces. We show that the gp120 beta19 strand and third variable (V3) loop contain residues important for CXCR4 interaction. Basic residues from both elements, as well as a conserved hydrophobic residue at the V3 tip, contribute to CXCR4 binding. Removal of the gp120 V1/V2 variable loops allows the envelope glycoprotein to bind CXCR4 in a CD4-independent manner. These results indicate that although some variable gp120 residues contribute to the specific binding to CCR5 or CXCR4, gp120 elements common to CXCR4- or CCR5-using strains are involved in the interaction with both coreceptors.     

Structure-function study and anti-HIV activity of synthetic peptide analogues derived from viral chemokine vMIP-II

The viral macrophage inflammatory protein II (vMIP-II) shows a broad spectrum interaction with both CC and CXC chemokine receptors including CCR5 and CXCR4, two principal coreceptors for the cellular entry of human immunodeficiency virus type 1 (HIV-1). Recently, we have shown that a synthetic peptide derived from the N-terminus of vMIP-II, designated as V1, is a potent antagonist of CXCR4 but not CCR5 [Zhou, N., et al. (2000) Biochemistry 39, 3782-3787]. In this study, we synthesized a series of new peptides derived from other regions of vMIP-II and characterized their binding activities with both CXCR4 and CCR5. The results provided further support for the notion that the N-terminus of vMIP-II is the major determinant for CXCR4 recognition and that vMIP-II probably interacts with other chemokine receptors such as CCR5 with different sequence and conformational determinants. To understand the structure-function relationship of V1 peptide, its solution conformation was studied using circular dichroism spectroscopy, which showed a random conformation similar to that of the corresponding N-terminus in native vMIP-II. In addition, we synthesized a series of mutant analogues of V1 containing alanine, glycine, or phenylalanine substitution at various positions. Residues Val-1, Arg-7, and Lys-9 of V1 peptide were found to be critical for receptor interaction, because single alanine replacement at these positions dramatically decreased peptide binding to CXCR4. In contrast, alanine or phenylalanine substitution at Cys-11 led to significant enhancement in peptide affinity for CXCR4. Finally, we showed that V1 peptide inhibits HIV-1 replication in CXCR4(+) T-cell lines. These studies provide new insights into the structure-function relationship of V1 peptide and demonstrate that this peptide may be a lead for the development of therapeutic agents.     

Neomycin B-arginine conjugate, a novel HIV-1 Tat antagonist: synthesis and anti-HIV activities.

HIV-1 transactivating protein Tat is essential for virus replication and progression of HIV disease. HIV-1 Tat stimulates transactivation by binding to HIV-1 transactivator responsive element (TAR) RNA, and while secreted extracellularly, it acts as an immunosuppressor, an activator of quiescent T-cells for productive HIV-1 infection, and by binding to CXC chemokine receptor type 4 (CXCR4) as a chemokine analogue. Here we present a novel HIV-1 Tat antagonist, a neomycin B-hexaarginine conjugate (NeoR), which inhibits Tat transactivation and antagonizes Tat extracellular activities, such as increased viral production, induction of CXCR4 expression, suppression of CD3-activated proliferation of lymphocytes, and upregulation of the CD8 receptor. Moreover, Tat inhibits binding of fluoresceine isothiocyanate (FITC)-labeled NeoR to human peripheral blood mononuclear cells (PBMC), indicating that Tat and NeoR bind to the same cellular target. This is further substantiated by the finding that NeoR competes with the binding of monoclonal Abs to CXCR4. Furthermore, NeoR suppresses HIV-1 binding to cells. Importantly, NeoR accumulates in the cell nuclei and inhibits the replication of M- and T-tropic HIV-1 laboratory isolates (EC(50) = 0.8-5.3 microM). A putative model structure for the TAR-NeoR complex, which complies with available experimental data, is presented. We conclude that NeoR is a multitarget HIV-1 inhibitor; the structure, and molecular modeling and dynamics, suggest its binding to TAR RNA. NeoR inhibits HIV-1 binding to cells, partially by blocking the CXCR4 HIV-1 coreceptor, and it antagonizes Tat functions. NeoR is therefore an attractive lead compound, capable of interfering with different stages of HIV infection and AIDS pathogenesis.     

CXCR4-derived synthetic peptides inducing anti-HIV-1 antibodies

Despite almost 30 years since the identification of the human immunodeficiency virus type I (HIV-1), development of effective AIDS vaccines has been hindered by the high mutability of HIV-1. The HIV-1 co-receptors CCR5 and CXCR4 are genetically stable, but viral proteins may mutate rapidly during the course of infection. CXCR4 is a seven transmembrane G protein-coupled receptor, possessing an N-terminal region (NT) and three extracellular loops (ECL1-3). Previous studies have shown that the CXCR4-ED-derived peptides inhibit the entry of HIV-1 by interacting with gp120, an HIV-1 envelope glycoprotein. In the present study, antigenicity of CXCR4-derived peptides has been investigated and the anti-HIV-1 effects of induced antisera have been assessed. It was found that CXCR4-ED-derived antigen molecules immunize mice, showing that the linear peptides have higher antigenicity than the cyclic peptides. The L1- and L2-induced antisera inhibited the HIV-1 entry significantly, while anti-N1 antibodies have no inhibitory activity. This study produced promising examples for the design of AIDS vaccines which target the human protein and can overcome mutability of HIV-1.     

Migration of antigen-specific T cells away from CXCR4-binding human immunodeficiency virus type 1 gp120

Cell-mediated immunity depends in part on appropriate migration and localization of cytotoxic T lymphocytes (CTL), a process regulated by chemokines and adhesion molecules. Many viruses, including human immunodeficiency virus type 1 (HIV-1), encode chemotactically active proteins, suggesting that dysregulation of immune cell trafficking may be a strategy for immune evasion. HIV-1 gp120, a retroviral envelope protein, has been shown to act as a T-cell chemoattractant via binding to the chemokine receptor and HIV-1 coreceptor CXCR4. We have previously shown that T cells move away from the chemokine stromal cell-derived factor 1 (SDF-1) in a concentration-dependent and CXCR4 receptor-mediated manner. Here, we demonstrate that CXCR4-binding HIV-1 X4 gp120 causes the movement of T cells, including HIV-specific CTL, away from high concentrations of the viral protein. This migratory response is CD4 independent and inhibited by anti-CXCR4 antibodies and pertussis toxin. Additionally, the expression of X4 gp120 by target cells reduces CTL efficacy in an in vitro system designed to account for the effect of cell migration on the ability of CTL to kill their target cells. Recombinant X4 gp120 also significantly reduced antigen-specific T-cell infiltration at a site of antigen challenge in vivo. The repellant activity of HIV-1 gp120 on immune cells in vitro and in vivo was shown to be dependent on the V2 and V3 loops of HIV-1 gp120. These data suggest that the active movement of T cells away from CXCR4-binding HIV-1 gp120, which we previously termed fugetaxis, may provide a novel mechanism by which HIV-1 evades challenge by immune effector cells in vivo.     

Evidence as a HIV-1 self-defense vaccine of cyclic chimeric dodecapeptide warped from undecapeptidyl arch of extracellular loop 2 in both CCR5 and CXCR4

Novel conformation-specific antibodies were raised against a cyclic chimeric dodecapeptidyl multiple antigen peptide (cCD-MAP) constructed with a spacer-armed Gly-Asp dipeptide and two pentapeptides (S(169)-Q(170)-K(171)-E(172)-G(173) of CCR5 and E(179)-A(180)-D(181)-D(182)-R(183) of CXCR4) which are components of the undecapeptidyl arch (UPA: from R(168) to C(178) in CCR5, from N(176) to C(186) in CXCR4) of extracellular loop 2 (ECL2) in chemokine receptors (CCR5 and CXCR4). Of the antibodies raised, one monoclonal antibody, CPMAb-I (IgMkappa), reacted with cCD-MAP, but not with the linear chimeric dodecapeptide-MAP. The antibody reacted with the cells separately expressing CCR5 or CXCR4, but not with those not expressing the coreceptors. Moreover, the antibody markedly suppressed infection by X4, R5, or R5X4 virus in a dose-dependent manner in a new phenotypic assay for drug susceptibility of HIV-1 using CCR5-expressing Hela/CD4(+) cell clone 1-10 (MAGIC-5). Moreover, CPMAb-I interfered with LAV-1(BRU) infection (m.o.i. = 0.01) of Molt4#8 cells cocultured with CPMAb-I-producing hybridoma in the transwell, and significantly interfered with neither chemotaxis nor calcium influx induced with stromal cell-derived factor 1 alpha (SDF-1alpha). Thus, the antibody raised against the cCD-MAP provides powerful protection or defense against HIV-1 infection. We therefore propose the cCD-MAP or its derivative immunogen as a novel candidate for an HIV-1 coreceptor-based self-defense vaccine.     

In vivo evolution of X4 human immunodeficiency virus type 1 variants in the natural course of infection coincides with decreasing sensitivity to CXCR4 antagonists

CXCR4-using (X4) human immunodeficiency virus type 1 (HIV-1) variants evolve from CCR5-restricted (R5) HIV-1 variants. Early after their first appearance in vivo, X4 HIV-1 variants additionally use CCR5. The ability to use CCR5 in addition to CXCR4 is generally lost late in infection. Here we studied whether this evolution of the coreceptor repertoire is also reflected in a changing sensitivity of X4 variants to CXCR4 antagonists such as peptide T22 and the synthetic compound AMD3100. We observed differences in the concentrations of CXCR4 antagonists needed to suppress replication of X4 HIV variants from different patients. In general, late X4 HIV variants were less sensitive to AMD3100 than were early R5X4 HIV variants. The differences between early R5X4 HIV variants and late X4 variants were less pronounced for T22-mediated inhibition. These results suggest an ongoing evolution of X4 virus variants toward more efficient usage of the cellular entry complex.     

Suppression of multiclade R5 and X4 human immunodeficiency virus type-1 infections by a coreceptor-based anti-HIV strategy

A cyclic chimeric dodecapeptide (cCD) mimicking the conformation-specific domains of CCR5 and CXCR4 was prepared in which Gly-Asp links the amino and carboxyl termini of two combined pentapeptides (S169-G173 of CCR5; E179-R183 of CXCR4) derived from human immunodeficiency virus type-1 (HIV-1) coreceptors. The immunization of Balb/c mice with cCD conjugated with a multiple-antigen peptide (cCD-MAP) induced seven cCD-specific monoclonal antibodies (mAbs, CPMAb-I to -VII) that reacted with native CCR5 and CXCR4. Among the tested mAbs, CPMAb-I and -II potently inhibited the infection of both the R5 and X4 laboratory strains. CPMAb-III and -VI were effective against only R5 laboratory strains, and also against some X4 and R5 primary isolates. CPMAb-IV and -V had potent antiviral activities against the R5 and X4 primary isolates. In particular, CPMAb-VII was protective against not only R5 and X4 laboratory strains, but also most of the R5 and X4 primary isolates. Moreover, cCD-MAP immunization also induced antibodies that were effective against R5 and X4 multiclade HIV-1 isolates in vitro in two of three cynomolgus monkeys. Taken together, the results suggest that cCD-MAP is a candidate multiclade immunogen that can be used to block multiclade R5 and X4 HIV-1 infections.