guanosine-triphosphate and HIV-Infections

CC chemokine receptor 5 (CCR5) is a G-protein-coupled receptor for the chemokines CCL3, -4, and -5 and a coreceptor for entry of R5-tropic strains of human immunodeficiency virus type 1 (HIV-1) into CD4(+) T-cells. We investigated the mechanisms whereby nonpeptidic, low molecular weight CCR5 ligands block HIV-1 entry and infection. Displacement binding assays and dissociation kinetics demonstrated that two of these molecules, i.e. TAK779 and maraviroc (MVC), inhibit CCL3 and the HIV-1 envelope glycoprotein gp120 binding to CCR5 by a noncompetitive and allosteric mechanism, supporting the view that they bind to regions of CCR5 distinct from the gp120- and CCL3-binding sites. We observed that TAK779 and MVC are full and weak inverse agonists for CCR5, respectively, indicating that they stabilize distinct CCR5 conformations with impaired abilities to activate G-proteins. Dissociation of [(125)I]CCL3 from CCR5 was accelerated by TAK779, to a lesser extent by MVC, and by GTP analogs, suggesting that inverse agonism contributes to allosteric inhibition of the chemokine binding to CCR5. TAK779 and MVC also promote dissociation of [(35)S]gp120 from CCR5 with an efficiency that correlates with their ability to act as inverse agonists. Displacement experiments revealed that affinities of MVC and TAK779 for the [(35)S]gp120-binding receptors are in the same range (IC(50) ∼6.4 versus 22 nm), although we found that MVC is 100-fold more potent than TAK779 for inhibiting HIV infection. This suggests that allosteric CCR5 inhibitors not only act by blocking gp120 binding but also alter distinct steps of CCR5 usage in the course of HIV infection.

Topics: Allosteric Regulation; Chemokine CCL3; Cyclohexanes; Guanosine Triphosphate; HEK293 Cells; HIV Envelope Protein gp120; HIV Infections; HIV-1; Humans; Ligands; Maraviroc; Protein Binding; Receptors, CCR5; Triazoles

Topics: Adenine; Adenosine Diphosphate; Adenosine Triphosphate; Adult; CD4-Positive T-Lymphocytes; Female; Guanine; Guanosine Diphosphate; Guanosine Triphosphate; HIV Infections; HIV Seropositivity; HIV-1; Humans; Lymphocyte Subsets; Male; Middle Aged; Purine Nucleotides; T-Lymphocyte Subsets

We have constructed the recombinant baculovirus which expresses the human immunodeficiency virus type 1 negative factor (nef) gene. Spodoptera frugiperda cells infected with the recombinant virus produced a 27-kDa protein which reacted with rabbit antisera raised against a carboxy-terminal synthetic peptide of the Nef protein by immunoblot analysis. Labeling experiment showed that the recombinant Nef protein was myristoylated. The recombinant Nef protein was purified to near homogeneity by DEAE-Sephacel, phenyl-Sepharose 4B, blue-Sepharose, and Sephadex G-150 column chromatography. No detectable GTP binding activity was observed in the purified recombinant Nef product.

Topics: Animals; Baculoviridae; Cloning, Molecular; Gene Products, nef; GTP-Binding Proteins; Guanosine Triphosphate; HIV Antibodies; HIV Antigens; HIV Infections; HIV Seropositivity; HIV-1; Humans; Moths; Myristic Acid; Myristic Acids; nef Gene Products, Human Immunodeficiency Virus; Protein Processing, Post-Translational; Recombinant Proteins