Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion.

The CCR5 chemokine receptor is a rhodopsin-like G protein-coupled receptor that mediates the effects of pro-inflammatory ß-chemokines. CCR5 is also the major co-receptor for entry of human immunodeficiency virus (HIV) into human cells. G protein-coupled receptors exist in ensembles of active and inactive conformations. Active receptor conformations can be stabilized by mutations. Although binding of the HIV envelope protein to CCR5 stimulates cellular signaling, the CCR5 conformation that induces fusion of the viral membrane with cellular membranes is not known. We mutated conserved amino acids to generate constitutively active CCR5 receptors, which are stabilized in active conformations, and tested the ability of constitutively active CCR5 receptors to mediate HIV envelope-directed membrane fusion. Mutation of the Asp³·49(¹²5) and Arg6·³²(²²5) residues of CCR5 did not cause constitutive activity, but Lys or Pro substitutions for Thr²·56(8²), in the TxP motif, caused high basal inositol phosphate signaling. Signaling did not increase in response to MIP-1ß, suggesting that the Thr²·56(8²) mutants were fully stabilized in active conformations. The Thr²·56(8²)Lys mutation severely decreased cell surface CCR5 expression. Combining the Thr²·56(8²)Lys mutation with an Arg6·³²(²²5)Gln mutation partially reversed the decrease in expression. Mutants with Thr²·56(8²)Lys substitutions were poor mediators of HIV envelope-directed membrane fusion, but mutants with the Thr²·65(8²)Pro substitution exhibited full co-receptor function. Our results suggest that the Thr²·65(8²)Lys and Thr²·65(8²)Pro mutations stabilize distinct constitutively active CCR5 conformations. Lys in position 2.65(82) stabilizes activated receptor conformations that appear to be constitutively internalized and do not induce envelope-dependent membrane fusion, whereas Pro stabilizes activated conformations that are not constitutively internalized and fully mediate envelope-directed membrane fusion.

High-affinity binding of southern African HIV type 1 subtype C envelope protein, gp120, to the CCR5 coreceptor.

HIV-1 subtype C is the fastest spreading subtype worldwide and predominantly uses the CCR5 coreceptor, showing minimal transition to the X4 phenotype. This raises the possibility that envelope proteins of HIV-1 subtype C have structural features that favor interaction with CCR5. Preference for CCR5 could arise from enhanced affinity of HIV-1 subtype C for CCR5. To test this, we have characterized the interaction of gp120 envelope proteins from HIV-1 subtype C clones with CD4 and CCR5. Recombinant gp120 proteins from isolates of HIV-1 subtypes B and C were expressed, purified, and assessed in a CD4 binding assay and a CCR5 chemokine competition binding assay. All gp120 proteins bound to CD4-expressing cells, except one, 97ZA347ts, which had Arg substituted for the Cys239 in the conserved C2 loop. Reconstitution of Cys239, using site-directed mutagenesis, restored CD4 binding, while introducing Arg or Ser into position 239 of the functional Du151 gp120 protein abrogated CD4 binding. This shows that the Cys228-Cys239 disulfide bond of gp120 is required for high-affinity binding to CD4. Recombinant gp120 proteins from two HIV-1 subtype B clones bound CCR5 in the presence of CD4, while gp120 from the X4-tropic, HxB2, clone did not bind CCR5. gp120 from two functional HIV-1 subtype C clones, Du151 and MOLE1, bound CCR5 with high affinity in the presence of CD4 and Du151 showed significant CCR5 binding in the absence of CD4. A gp120 from a nonfunctional subtype C clone had lower affinity for CCR5. These results indicate that HIV-1 subtype C proteins have high affinity for CCR5 with variable dependence on CD4.