ABSTRACT
The CC-chemokine receptor CCR5 mediates fusion and entry of the most commonly transmitted human immunodeficiency virus type 1 (HIV-1) strains. We have isolated six new anti-CCR5 murine monoclonal antibodies (MAbs), designated PA8, PA9, PA10, PA11, PA12, and PA14. A panel of CCR5 alanine point mutants was used to map the epitopes of these MAbs and the previously described MAb 2D7 to specific amino acid residues in the N terminus and/or second extracellular loop regions of CCR5. This structural information was correlated with the MAbs' abilities to inhibit (i) HIV-1 entry, (ii) HIV-1 envelope glycoprotein-mediated membrane fusion, (iii) gp120 binding to CCR5, and (iv) CC-chemokine activity. Surprisingly, there was no correlation between the ability of a MAb to inhibit HIV-1 fusion-entry and its ability to inhibit either the binding of a gp120-soluble CD4 complex to CCR5 or CC-chemokine activity. MAbs PA9 to PA12, whose epitopes include residues in the CCR5 N terminus, strongly inhibited gp120 binding but only moderately inhibited HIV-1 fusion and entry and had no effect on RANTES-induced calcium mobilization. MAbs PA14 and 2D7, the most potent inhibitors of HIV-1 entry and fusion, were less effective at inhibiting gp120 binding and were variably potent at inhibiting RANTES-induced signaling. With respect to inhibiting HIV-1 entry and fusion, PA12 but not PA14 was potently synergistic when used in combination with 2D7, RANTES, and CD4-immunoglobulin G2, which inhibits HIV-1 attachment. The data support a model wherein HIV-1 entry occurs in three stages: receptor (CD4) binding, coreceptor (CCR5) binding, and coreceptor-mediated membrane fusion. The antibodies described will be useful for further dissecting these events.
Subject(s)
Chemokines, CC/metabolism , HIV Envelope Protein gp120/metabolism , HIV-1/metabolism , Membrane Fusion , Receptors, CCR5/metabolism , Alanine/genetics , Alanine/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , CD4-Positive T-Lymphocytes/immunology , Chemokine CCL5/immunology , Epitope Mapping , Humans , Mutagenesis , Receptors, CCR5/genetics , Receptors, CCR5/immunology , Signal Transduction , TransfectionSubject(s)
Chromosome Mapping/methods , Chromosomes, Human , Karyotyping/methods , Animals , Burkitt Lymphoma , Cell Separation/instrumentation , Cell Separation/methods , Cell Transformation, Neoplastic , DNA Primers , Flow Cytometry/instrumentation , Flow Cytometry/methods , Gene Library , Humans , Polymerase Chain Reaction/methods , Rats , Translocation, Genetic , Tumor Cells, CulturedABSTRACT
We have studied the breadth and potency of the inhibitory actions of the CC chemokines macrophage inhibitory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES against macrophage-tropic (M-tropic) primary isolates of human immunodeficiency virus type 1 (HIV-1) and of the CXC chemokine stromal cell-derived factor 1alpha against T-cell-tropic (T-tropic) isolates, using mitogen-stimulated primary CD4+ T cells as targets. There was considerable interisolate variation in the sensitivity of HIV-1 to chemokine inhibition, which was especially pronounced for the CC chemokines and M-tropic strains. However, this variation was not obviously dependent on the genetic subtype (A through F) of the virus isolates. Peripheral blood mononuclear cell donor-dependent variation in chemokine inhibition potency was also observed. Among the CC chemokines, the rank order for potency (from most to least potent) was RANTES, MIP-1beta, MIP-1alpha. Some M-tropic isolates, unexpectedly, were much more sensitive to RANTES than to MIP-1beta, whereas other isolates showed sensitivities comparable to those of these two chemokines. Down-regulation of the CCR5 and CXCR4 receptors occurred in cells treated with the cognate chemokines and probably contributes to anti-HIV-1 activity. Thus, for CCR5, the rank order for down-regulation was also RANTES, MIP-1beta, MIP-1alpha.
Subject(s)
Chemokines, CXC , Chemokines/pharmacology , HIV-1/drug effects , Anti-HIV Agents/pharmacology , Binding, Competitive , CD4-Positive T-Lymphocytes/virology , Chemokine CCL3 , Chemokine CCL4 , Chemokine CCL5/pharmacology , Chemokine CXCL12 , Down-Regulation , HIV Envelope Protein gp120/metabolism , HIV-1/genetics , HIV-1/physiology , Humans , In Vitro Techniques , Macrophage Inflammatory Proteins/pharmacology , Macrophages/virology , Receptors, CCR5/metabolism , Receptors, CXCR4/metabolism , Recombinant Proteins/pharmacology , Virus Replication/drug effectsABSTRACT
Centromere activation, an important mechanism in karyotype evolution, is occasionally observed in some human chromosome rearrangements. We report a possible occurrence of centromere activation in a marker chromosome containing an atypical centromere associated with an inverted duplication of the region 14q32 --> qter. The marker chromosome's reduced centromere lacks both the alpha and beta satellite sequences usually found at normal centromeres. In an attempt to identify the centromeric sequences, the marker chromosome was flow-sorted and amplified by a degenerate oligonucleotide primer polymerase chain reaction. Reverse chromosome painting experiments showed that the marker chromosome contains sequences that are unique to the distal region of chromosome 14, as well as a low copy number of (centromeric) sequences that are also highly represented in the centromeres of chromosomes 18 and 19. These data suggest the activation of a novel centromere in the 14q32 --> qter region, very likely consequent to the duplication of the region itself.
