Phenotypic knockout of HIV type 1 chemokine coreceptor CCR-5 by intrakines as potential therapeutic approach for HIV-1 infection

A genetic defect in a CC-chemokine receptor (CCR)-5, the principal coreceptor for the macrophage-tropic HIV type 1 (HIV-1), recently was found to naturally protect CCR-5-defective, but healthy, individuals from HIV-1 infection. In this study, we mimic the natural resistance of the CCR-5-defective individuals by designing a strategy to phenotypically knock out CCR-5. The inactivation of the CCR-5 coreceptor is accomplished by targeting a modified CC-chemokine to the endoplasmic reticulum to block the surface expression of newly synthesized CCR-5. The lymphocytes transduced to express the intracellular chemokine, termed “intrakine,” were found to be viable and resistant to macrophage-tropic HIV-1 infection. Thus, this gene-based intrakine strategy targeted at the conserved cellular receptor for the prevention of HIV-1 entry should have significant advantages over currently described approaches for HIV-1 therapy.

The neutral glycosphingolipid globotriaosylceramide promotes fusion mediated by a CD4-dependent CXCR4-utilizing HIV type 1 envelope glycoprotein

Previously, we showed that the addition of human erythrocyte glycosphingolipids (GSLs) to nonhuman CD4+ or GSL-depleted human CD4+ cells rendered those cells susceptible to HIV-1 envelope glycoprotein-mediated cell fusion. Individual components in the GSL mixture were isolated by fractionation on a silica-gel column and incorporated into the membranes of CD4+ cells. GSL-supplemented target cells were then examined for their ability to fuse with TF228 cells expressing HIV-1LAI envelope glycoprotein. We found that one GSL fraction, fraction 3, exhibited the highest recovery of fusion after incorporation into CD4+ nonhuman and GSL-depleted HeLa-CD4 cells and that fraction 3 contained a single GSL fraction. Fraction 3 was characterized by MS, NMR spectroscopy, enzymatic analysis, and immunostaining with an antiglobotriaosylceramide (Gb3) antibody and was found to be Gal(α1→4)Gal(β1→4)Glc-Cer (Gb3). The addition of fraction 3 or Gb3 to GSL-depleted HeLa-CD4 cells recovered fusion, but the addition of galactosylceramide, glucosylceramide, the monosialoganglioside, GM3, lactosylceramide, globoside, the disialoganglioside, GD3, or α-galactosidase A-digested fraction 3 had no effect. Our findings show that the neutral GSL, Gb3, is required for CD4/CXCR4-dependent HIV-1 fusion.

Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor activity

The C-C chemokine receptor 5 (CCR5) plays a crucial role in facilitating the entry of macrophage-tropic strains of the HIV-1 into cells, but the mechanism of this phenomenon is completely unknown. To explore the role of CCR5-derived signal transduction in viral entry, we introduced mutations into two cytoplasmic domains of CCR5 involved in receptor-mediated function. Truncation of the terminal carboxyl-tail to eight amino acids or mutation of the highly conserved aspartate-arginine-tyrosine, or DRY, sequence in the second cytoplasmic loop of CCR5 effectively blocked chemokine-dependent activation of classic second messengers, intracellular calcium fluxes, and the cellular response of chemotaxis. In contrast, none of the mutations altered the ability of CCR5 to act as an HIV-1 coreceptor. We conclude that the initiation of signal transduction, the prototypic function of G protein coupled receptors, is not required for CCR5 to act as a coreceptor for HIV-1 entry into cells.

A binding pocket for a small molecule inhibitor of HIV-1 entry within the transmembrane helices of CCR5

HIV-1 entry into CD4+ cells requires the sequential interactions of the viral envelope glycoproteins with CD4 and a coreceptor such as the chemokine receptors CCR5 and CXCR4. A plausible approach to blocking this process is to use small molecule antagonists of coreceptor function. One such inhibitor has been described for CCR5: the TAK-779 molecule. To facilitate the further development of entry inhibitors as antiviral drugs, we have explored how TAK-779 acts to prevent HIV-1 infection, and we have mapped its site of interaction with CCR5. We find that TAK-779 inhibits HIV-1 replication at the membrane fusion stage by blocking the interaction of the viral surface glycoprotein gp120 with CCR5. We could identify no amino acid substitutions within the extracellular domain of CCR5 that affected the antiviral action of TAK-779. However, alanine scanning mutagenesis of the transmembrane domains revealed that the binding site for TAK-779 on CCR5 is located near the extracellular surface of the receptor, within a cavity formed between transmembrane helices 1, 2, 3, and 7.

Molecular cloning and characterization of a murine pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin

Pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 (PBSF/SDF-1) is a member of the CXC group of chemokines that is initially identified as a bone marrow stromal cell-derived factor and as a pre-B-cell stimulatory factor. Although most chemokines are thought to be inducible inflammatory mediators, PBSF/SDF-1 is essential for perinatal viability, B lymphopoiesis, bone marrow myelopoiesis, and cardiac ventricular septal formation, and it has chemotactic activities on resting lymphocytes and monocytes. In this paper, we have isolated a cDNA that encodes a seven transmembrane-spanning-domain receptor, designated pre-B-cell-derived chemokine receptor (PB-CKR) from a murine pre-B-cell clone, DW34. The deduced amino acid sequence has 90% identity with that of a HUMSTSR/fusin, a human immunodeficiency virus 1 (HIV-1) entry coreceptor. However, the second extracellular region has lower identity (67%) compared with HUMSTSR/fusin. PB-CKR is expressed during embryo genesis and in many organs and T cells of adult mice. Murine PBSF/SDF-1 induced an increase in intracellular free Ca2+ in DW34 cells and PB-CKR-transfected Chinese hamster ovary (CHO) cells, suggesting that PB-CKR is a functional receptor for murine PBSF/SDF-1. Murine PBSF/SDF-1 also induced Ca2+ influx in fusin-transfected CHO cells. On the other hand, considering previous results that HIV-1 does not enter murine T cells that expressed human CD4, PB-CKR may not support HIV-1 infection. Thus, PB-CKR will be an important tool for functional mapping of HIV-1 entry coreceptor fusin and for understanding the function of PBSF/SDF-1 further.

Substance P antagonist (CP-96,345) inhibits HIV-1 replication in human mononuclear phagocytes

Substance P (SP) is a potent modulator of neuroimmunoregulation. We recently reported that human immune cells express SP and its receptor. We have now investigated the possible role that SP and its receptor plays in HIV infection of human mononuclear phagocytes. SP enhanced HIV replication in human blood-isolated mononuclear phagocytes, whereas the nonpeptide SP antagonist (CP-96,345) potently inhibited HIV infectivity of these cells in a concentration-dependent fashion. CP-96,345 prevented the formation of typical giant syncytia induced by HIV Bal strain replication in these cells. This inhibitory effect of CP-96,345 was because of the antagonism of neurokinin-1 receptor, a primary SP receptor. Both CP-96,345 and anti-SP antibody inhibited SP-enhanced HIV replication in monocyte-derived macrophages (MDM). Among HIV strains tested (both prototype and primary isolates), only the R5 strains (Bal, ADA, BL-6, and CSF-6) that use the CCR5 coreceptor for entry into MDM were significantly inhibited by CP-96,345; in contrast, the X4 strain (UG024), which uses CXCR4 as its coreceptor, was not inhibited. In addition, the M-tropic ADA (CCR5-dependent)-pseudotyped HIV infection of MDM was markedly inhibited by CP-96,345, whereas murine leukemia virus-pseudotyped HIV was not affected, indicating that the major effect of CP-96,345 is regulated by Env-determined early events in HIV infection of MDM. CP-96,345 significantly down-regulated CCR5 expression in MDM at both protein and mRNA levels. Thus, SP–neurokinin-1 receptor interaction may play an important role in the regulation of CCR5 expression in MDM, affecting the R5 HIV strain infection of MDM.

Étude du rôle des régions variables 4 et 5 dans les changements de conformation de la gp120 du VIH-1

Le VIH infecte les cellules par fusion de sa membrane avec la membrane de la cellule cible. Cette fusion est effectuée par les glycoprotéines de l'enveloppe (Env) qui sont synthétisées en tant que précurseur, gp160, qui est ensuite clivé en gp120 et gp41. La protéine gp41 est la partie transmembranaire du complexe de l'enveloppe et l’ancre à la particule virale alors que la gp120 assure la liaison au récepteur cellulaire CD4 et corécepteur CCR5 ou CXCR4. Ces interactions successives induisent des changements de conformation d’Env qui alimentent le processus d'entrée du virus conduisant finalement à l'insertion du peptide de fusion de la gp41 dans la membrane de la cellule cible. La sous-unité extérieure gp120 contient cinq régions variables (V1 à V5), dont trois (V1, V2 et V3) étant capables d’empêcher l’adoption spontanée de la conformation liée à CD4. Cependant, le rôle de régions variables V4 et V5 vis-à-vis de ces changements de conformation reste inconnu. Pour étudier leur effet, des mutants de l'isolat primaire de clade B YU2, comprenant une délétion de la V5 ou une mutation au niveau de tous les sites potentiels de N-glycosylation de la V4 (PNGS), ont été générés. L'effet des mutations sur la conformation des glycoprotéines d'enveloppe a été analysé par immunoprécipitation et résonance de plasmon de surface avec des anticorps dont la liaison dépend de la conformation adopté par la gp120. Ni le retrait des PNGS de la V4 ni la délétion de V5 n’a affecté les changements conformationnels d’Env tels que mesurés par ces techniques, ce qui suggère que les régions variables V1, V2 et V3 sont les principaux acteurs dans la prévention de l’adoption de la conformation lié de CD4 d’Env.

CXCR4/SDF1 interaction inhibits the primordial to primary follicle transition in the neonatal mouse ovary

The molecular mechanisms behind the entry of the primordial follicle into the growing follicle pool remain poorly understood. To investigate this process further, a microarray-based comparison was undertaken between 2-day postpartum mouse ovaries consisting of primordial follicles/naked oocytes only and those with both primordial follicles and newly activated follicles (7-day postpartum). Gene candidates identified included the chemoattractive cytokine stromal derived factor-1 (SDF1) and its receptor CXCR4. SDF1 and CXCR4 have been implicated in a variety of physiological processes including the migration of embryonic germ cells to the gonads. SDF1-alpha expression increased with the developmental stage of the follicle. Embryonic expression was found to be dichotomous post-genii cell migration, with low expression in the female. Immunohistochemical studies nonetheless indicate that the autocrine pattern of expression ligand and receptor begins during embryonic life. Addition of recombinant SDF1-alpha to neonatal mouse ovaries in vitro resulted in significantly higher follicle densities than for control ovaries. TUNEL analysis indicated no detectable difference in populations of apoptotic cells of treated or control ovaries. Treated ovaries also contained a significantly lower percentage of activated follicles as determined by measurement of oocyte diameter and morphological analysis. Treatment of cultured ovaries with an inhibitor of SDF1-alpha, AMD3100, ablated the effect of SDF1-alpha. By retaining follicles in an unactivated state, SDF1/CXCR4 signaling may play an important role in maintaining the size and longevity of the primordial follicle pool. (c) 2006 Elsevier Inc. All rights reserved.

Modulation de l'activité transcriptionnelle des récepteurs des estrogènes par le récepteur de chimiokine CXCR4

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Dimérisation du récepteur de chimiokine CXCR4

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Modulation de l'activité transcriptionnelle des récepteurs des estrogènes par le récepteur de chimiokine CXCR4

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Dimérisation du récepteur de chimiokine CXCR4

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Développement et caractérisation de ligands du récepteur à chimiokine CXCR4

Le récepteur à chimiokine CXCR4 est à ce jour l’un des récepteurs couplés aux protéines G les plus étudiés. Le CXCL12, sa chimiokine endogène induit l’activation de plusieurs voies de signalisation cruciales à plusieurs processus physiologiques. Par ailleurs, dans de nombreux processus pathologiques comme le cancer, le récepteur CXCR4 et/ou son ligand endogène sont surexprimés et facilite la dissémination et le maintien de conditions favorables à la prolifération cancéreuse. Afin d’étudier le récepteur CXCR4 et sa signalisation, notre approche vise à développer des ligands ciblant le CXCR4 en se basant sur CXCL12. Par des études de relation structure-activité et du design rationnel, nous avons conçu des chimères du CXCR4. Ces outils pharmacologiques nous permettent de mieux extraire les déterminants structuraux impliqués dans l’activation du CXCR4 mais aussi d’étudier les voies de signalisation associées à ces nouvelles entités chimiques. Nos données de relation structure-activité ont permis de mettre en évidence deux positions clés sur le N-terminal de nos chimères, la position 3 et la position 7 cruciales pour l’affinité et l’efficacité respectivement. Nous avons pu moduler l’efficacité ainsi que l’affinité de nos chimères en introduisant des acides aminés non naturels capables de potentialiser l’effet pharmacologique. Nous avons également corrélé nos résultats de SAR avec de la dynamique moléculaire réalisée à partir des deux structures cristallographiques du CXCR4. Nos données de dynamique moléculaire montrent des différences structurales importantes au niveau des domaines transmembranaires 3 et 7 en présence ou non de nos chimères. Par ailleurs, nous avons développé un nouveau déterminant avec des propriétés pharmacocinétiques améliorées comparativement au déterminant d’affinité des chimères de première génération. La caractérisation de ce déterminant a par ailleurs révélé son caractère agoniste inverse. Tous ces résultats apportent des éléments clés pour un meilleur design de molécules à visée thérapeutique ciblant le CXCR4.