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Introduction:  HIV-1 phenotype is defined by tropism and co-receptor utilization. Use of CXCR4 (X4) co-receptor provides a biomarker for HIV-1 pathogenesis in vivo. Env gp120 V3 amino acid composition predicts X4 usage on lymphocytes, but its value as a major determinant for phenotype diminishes with regard to X4-mediated entry into monocyte-derived macrophages (MDM). We hypothesize that X4 entry into MDM requires discontinuous determinants in HIV-1 gp120, which differ from peripheral blood mononuclear cells and T-cell lines.

Methods:  Fifty HIV-1 infected children and young adults were prospectively enrolled to examine the relationship between viral phenotype and clinical outcomes. Chimeric envelopes (Env) were constructed with hypervariable domains from primary D-X4 viruses and HIV-1_LAI (T-X4). Single-cycle luciferase-tagged viruses (NL4-3.Luc.E^-R⁺) were pseudotyped with the chimeric Env to evaluate the contribution of each of the 5 hypervariable domains of gp120 to X4 usage on MDM.

Results:  D-X4 viruses were identified in 22 of 50 subjects, of whom 19 had CD4 T cells ≤ 15%. The relationship between CD4% and viral phenotype was significant (p < 0.001, Chi square). V3 domains from D-X4 isolates were sufficient for X4 entry into T-cell lines and PBMC, but insufficient to mediate entry into MDM. Systematic exchange of hypervariable domains between D-X4 and T-X4 Env indicated that V1 and V3 comprise a core region of Env that mediated efficient use of CXCR4 on MT-2 cells and peripheral blood mononuclear cells. However, efficient entry into MDM required additional determinants from V2 and V5. The novel features of D-X4 Env from primary viruses in our study were found in V2, which had net positive charges, and in V1, which included unusual proline or cysteine residues localized in the region of length polymorphism. D-X4 V1 or V2 domains differed from both M-R5 or T-X4 domains, but were shared with HIV-1_89.6 (D-R5X4) V1 or V2 domains.

Conclusion:  Our results suggest that cell-type-specific regulation of viral entry is comprised of a discontinuous interaction of V1, V2, and V3 hypervariable loops and elements of CD4-binding. Determinants of receptor affinity are significant for development of new treatment strategies, including therapeutic vaccines that control replication of D-X4 viruses to prevent evolution of more pathogenic D-X4 viruses and delay disease progression.

Keywords: Dual tropism; envelope; macrophages