We have previously shown that aptamer B4, which shares critical structural motifs with aptamer B40, competes for binding to gp120 with the CD4i MAb 17b (8
). Taken together with the CD4 dependence of efficient binding of aptamer B40 to the core gp120 construct described above, this pointed strongly to the possibility that the aptamer binding site was close to that of CCR5. To test this, we carried out a BIAcore surface plasmon resonance competition binding analysis using the 22-mer tyrosine-sulfated peptide (S-peptide) corresponding to the N terminus of CCR5 that acts as an essential component of the coreceptor for HIV-1 on the host cell (2
), as well as an unsulfated version of the same peptide (C-peptide) and sCD4, as alternate immobilized binding partners for gp120 (Fig. ). As controls, RANTES, a physiological ligand for both CCR3 and CCR5 (3
), and the anti-CCR5 MAb 3A9, but not monocyte chemoattractant protein 1, a ligand for CCR2 and CCR4, bound to the CCR5 peptides, as expected (data not shown).
FIG. 3. Effect of preincubation with aptamer or sCD4 on gp120 binding to immobilized receptors. BIAcore sensorgram overlays show the competition binding of (A) monomeric Ba-L gp120, (B) gp120-sCD4 complex, (C) gp120-aptamer B40 complex, and (D) gp120-sCD4-aptamer (more ...)
Monomeric Ba-L gp120 bound well to immobilized CD4 but weakly to the S-peptide and negligibly to the C-peptide (Fig. ). However, preincubating gp120 with CD4 increased binding to both CCR5 peptides by more than 2.5-fold (Fig. ), confirming the CD4-induced binding property of the peptides to gp120 (14
). Moreover, the approximately threefold difference in binding of both gp120 and gp120-sCD4 complex to the S-peptide versus C-peptide confirm that the tyrosine sulfate moieties contribute substantially to the association of the gp120-sCD4 complexes, as previously reported (13
). As expected, the gp120-sCD4 complex did not bind to immobilized CD4 (Fig. ). When the gp120-17b MAb complex was injected, it bound only to the immobilized CD4 and not to the CCR5 peptides (data not shown), also as expected. The gp120-aptamer B40 complex also bound to the immobilized CD4 but not to the CCR5 peptides (Fig. ). The ternary complexes, gp120-sCD4-17 MAb (data not shown) and gp120-sCD4-aptamer B40 (Fig. ), did not bind to either the immobilized CCR5 peptides or human CD4.
Taken together, these results show that the aptamer competes with the N-terminal ectodomain of CCR5 for binding to gp120 and hence the aptamer binding site (aptatope) must overlap with the basic, conserved, coreceptor-binding region on gp120. This is consistent with the inability of aptamer B40 to bind the gp120 of the X4 strain, HXB2 (data not shown) in contrast to aptamers raised against X4 strains (32
). The ability of aptamer B40 to bind to the coreceptor-binding site without prior ligation of CD4 by gp120, in contrast to the CD4 dependence of the binding of CD4i antibodies and of CCR5 itself, may result from a combination of its small size and high charge density and confer on it remarkable antiviral potential.