ABSTRACT
CD4 and CCR5 mediate fusion and entry of R5 human immunodeficiency virus type 1 (HIV-1) strains. Sulfotyrosine and other negatively charged residues in the CCR5 amino-terminal domain (Nt) are crucial for gp120 binding and viral entry. We previously showed that a soluble gp120-CD4 complex specifically binds to a peptide corresponding to CCR5 Nt residues 2 to 18, with sulfotyrosines in positions 10 and 14. This sulfopeptide also inhibits soluble gp120-CD4 binding to cell surface CCR5 as well as infection by an R5 virus. Here we show that residues 10 to 18 constitute the minimal domain of the CCR5 Nt that is able to specifically interact with soluble gp120-CD4 complexes. In addition to sulfotyrosines in positions 10 and 14, negatively charged residues in positions 11 and 18 participate in this interaction. Furthermore, the CCR5 Nt binds to a CD4-induced surface on gp120 that is composed of conserved residues in the V3 loop stem and the C4 domain. Binding of gp120 to cell surface CCR5 is further influenced by residues in the crown of the V3 loop, C1, C2, and C3. Our data suggest that gp120 docking to CCR5 is a multistep process involving several independent regions of the envelope glycoprotein and the coreceptor.
Subject(s)
Amino Acids, Sulfur/chemistry , CD4 Antigens/metabolism , HIV Envelope Protein gp120/metabolism , HIV-1/physiology , Peptides/metabolism , Receptors, CCR5/chemistry , Amides/metabolism , Amino Acids, Sulfur/metabolism , Antibodies, Monoclonal/metabolism , Chemokines/metabolism , Enzyme-Linked Immunosorbent Assay/methods , HIV Envelope Protein gp120/genetics , Humans , Immunoglobulin G/metabolism , Peptides/chemistry , Quaternary Ammonium Compounds/metabolism , Receptors, CCR5/genetics , Receptors, CCR5/metabolism , Surface Plasmon ResonanceABSTRACT
The HIV-1 envelope glycoprotein gp120 interacts consecutively with CD4 and the CCR5 coreceptor to mediate the entry of certain HIV-1 strains into target cells. Acidic residues and sulfotyrosines in the amino-terminal domain (Nt) of CCR5 are crucial for viral fusion and entry. We tested the binding of a panel of CCR5 Nt peptides to different soluble gp120/CD4 complexes and anti-CCR5 mAbs. The tyrosine residues in the peptides were sulfated, phosphorylated, or unmodified. None of the gp120/CD4 complexes associated with peptides containing unmodified or phosphorylated tyrosines. The gp120/CD4 complexes containing envelope glycoproteins from isolates that use CCR5 as a coreceptor associated with Nt peptides containing sulfotyrosines but not with peptides containing sulfotyrosines in scrambled Nt sequences. Finally, only peptides containing sulfotyrosines inhibited the entry of an R5 isolate. Our data show that proper posttranslational modification of the CCR5 Nt is required for gp120 binding and viral entry. More importantly, the Nt domain determines the specificity of the interaction between CCR5 and gp120s from isolates that use this coreceptor.
Subject(s)
HIV Envelope Protein gp120/metabolism , HIV-1/metabolism , Receptors, CCR5/metabolism , Tyrosine/analogs & derivatives , Amino Acid Sequence , Antibodies, Monoclonal/pharmacology , CD4 Antigens/chemistry , CD4 Antigens/metabolism , Cell Line , Epitopes/metabolism , HIV Envelope Protein gp120/chemistry , HeLa Cells , Human T-lymphotropic virus 1/metabolism , Humans , Leukemia Virus, Murine/metabolism , Macromolecular Substances , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Protein Binding/drug effects , Protein Processing, Post-Translational , Protein Structure, Tertiary , Receptors, CCR5/chemistry , Surface Plasmon Resonance , Tyrosine/physiologyABSTRACT
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.
