Background:  The natural CCR5 ligands LD78a (also termed MIP-1) and LD78 represent highly related isoforms. LD78 is reportedly more potent in cellular calcium influx, chemotaxis induction, and the activity against HIV-1 than LD78. It is thought that interactions of the NH₂ terminus of LD78 (LD78 as well) and the second extracellular loop of CCR5 are crucial for the initiation of signal transduction.

Methods: We generated 6 LD78 variants, each with an amino acid substituted to Ala at the NH₂ terminus of LD78, and examined the structure-activity relationships. Cellular calcium mobilization and chemotaxis assays were performed. Inhibitory activity of LD78 variants against the replication of HIV-1_BaL and HIV-1_JRFL was determined by evaluating the reduction of p24 antigen production by the virus in PHA-PBM. The heterologous displacement study with LD78 variants was performed using CCR5-Molt4 cells and an anti-CCR5 murine monoclonal antibody 2D7 known to inhibit the binding of LD78 to CCR5. The level of CCR5 down-regulation by LD78 variants was determined using fluorescence-activated cell sorting analysis.

Results:  There was no significant difference in eliciting calcium influx and chemotaxis among the variants examined, except for LD78_T9A. The comparative order for HIV-1 replication inhibition was: LD78_P8A > LD78_D6A > LD78_WT, LD78_L3A > LD78_T7A, LD78_P2A > LD78_T9A. In heterologous displacement assays of LD78 variants using CCR5-Molt4 cells and 2D7, the comparative order for 2D7 displacement was: LD78_P8A, LD78_D6A > LD78_WT, LD78_L3A > LD78_T7A > LD78_P2A > LD78_T9A. The comparative order for CCR5 down-regulation induction was comparable to that for 2D7 displacement.

Conclusions:  The present data suggest that Asp-6, Pro-8, and Thr-9 are critical for LD78 binding to CCR5 and inhibition of R5 HIV-1 replication, and that LD78 binding to CCR5, regardless of affinity, is sufficient for LD78^’s initial signal transduction, while the greater magnitude of binding is required for the greater anti-HIV-1 activity. The data also suggest that LD78 variants with appropriate amino acid substitution(s) such as LD78_D6A and LD78_P8A may serve as effective chemokine-based anti-HIV-1 therapeutics while preserving LD7