Peptide mimotopes selected with HIV-1-blocking monoclonal antibodies against CCR5 represent motifs specific for HIV-1 entry.
CCR5 is a chemokine receptor that mediates entry of human immunodeficiency virus-1 (HIV-1). Two monoclonal antibodies (mAbs) that block HIV-1 entry, 3A9 and 5C7, were used to select peptide mimotopes of sequences on CCR5 from phage displayed peptide libraries. The selected mimotofpes comprised motifs at the N-terminus and on the first and third extracellular loops (ECL1 and ECL3) of CCR5. Amino acids in these motifs were exchanged for alanines by site-directed mutagenesis (sdm) in the cDNA for human CCR5. Ensuing effects on antibody binding to CCR5, cellular entry of HIV-1 and chemokine-induced signalling were analysed by transfection of mutant cDNAs into HEK293.CD4 cells. For both mAbs, fluorescence-activated cell sorting analysis was used to define overlapping conformational epitopes on CCR5 at the N-terminus, on ECL1 and ECL3. Mutation of the N-terminal motif 10YD11 prevented HIV-1 entry into transfected cells as judged by single round infection assays with R5 and R5X4 HIV-1 isolates, as did mutation of the motif 96FG97 in ECL1, whereas mutation of the motif 274RLD276 in ECL3 had only a minor effect. None of the motifs in CCR5 relevant to HIV-1 entry disrupted chemokine-induced signalling. Thus, peptide mimotopes of conformational contact sites of CCR5 with the paratope of mAbs 3A9 and 5C7 represent sites on CCR5 that are essential for HIV-1 entry. Structural knowledge of these mimotopes could help elucidate the nature of the interaction between CCR5 and HIV-1, and thus the derivation of specific inhibitors of entry of HIV-1 into susceptible cells without interference with chemokine signalling.
Königs
Christoph
C
Georg-Speyer-Haus, Institute for Biomedical Research, Frankfurt am Main, Germany.
Pustowka
Anette
A
Irving
James
J
Kessel
Christoph
C
Klich
Katharina
K
Wegner
Valérie
V
Rowley
Merrill J
MJ
Mackay
Ian R
IR
Kreuz
Wolfhart
W
Griesinger
Christian
C
Dietrich
Ursula
U
D020816:Amino Acid Motifs / Q000235:genetics / Q000276:immunology; D019138:Antibodies, Blocking / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody / Q000276:immunology; D002478:Cells, Cultured; D018604:Epitope Mapping; D005434:Flow Cytometry; D015497:HIV-1 / Q000276:immunology* / Q000378:metabolism / Q000502:physiology; D006801:Humans; D008954:Models, Biological; D018716:Molecular Mimicry / Q000502:physiology*; D009154:Mutation; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification*; D019713:Receptors, CCR5 / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D053586:Virus Internalization
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D019713:Receptors, CCR5