This proposal deals with the relative efficiency of co-receptor utilization for human immunodeficiency virus-type 1 (HIV-1) to enter and infect different target cells. Macrophage-tropic HIV uses both CD4 and the CCR5 chemokine receptor as co-receptors for virus entry. Individuals homozygous for a 32 base pair deletion in CCR5delta (CCR5delta 32/delta32) are resistant to HIV-1 infection. We have studied SCID mice repopulated with PBMC from CCR5delta 32/delta32, CCR5delta32/+. and normal +/+ donors. CCR5delta32/delta32 hu-PBL-SCID mice were resistant to infection with M-tropic virus, showed slower kinetics of infection with M/T- tropic virus, and had normal or accelerated kinetics of infection with T-tropic virus. CCR5delta 32/+ hu-PBL-SCID mice showed significantly delayed kinetics of virus replication after infection with M-tropic virus. These observations suggest that CCR5delta expression is both necessary and limiting for efficient primary transmission of HIV-1. We propose that the requirements for viral entry differ for macrophages and T cells, and for T cells at different stages of activation, and that expression of CCR5delta by these cells will explain the difference between in vitro models and the results in exposed uninfected individuals and the hu-PBL-SID model. We will perform experiments with additional CCR5delta 32+ donors in hu-PBL-SCID mice to confirm the kinetic delay in virus replication with M-tropic but not T-tropic HIV-1 isolates. We will examine CCR5 expression by RNASE protection assay (RPA) and antibody staining in CCR5 32/+ and CCR5+/+ T cell lines and T cells recovered from hu-PBL-SCID mice, and follow the kinetics of CCR5 expression after infection in vitro and in peritoneal lavage and lymph nodes of control and infected hy-PBL- SCID mice. We will perform in situ hybridization for CCR5 and HIV-1 RNA in lymph node section from infected hu-PBL-SCID mice to determine relative localization patterns. The relative receptor expression and usage in macrophages versus CD4 T cells will be determined in infectivity studies, and by assessing sensitivity of chemokines or chemoine antagonists. Infection with macrophage-versus T cell-derived HIV-1 will be compared. We will determine the relative co-receptor utilization in CD4 T cells at different stages of activation or activated in different ways (mitogen+ IL-2 vs. Anti-CD3 + anti-CD28; in vivo activated T cells), and also measure the membrane co-localization of CD4 and CCR5. Finally, we will use T cells lines form CCR5 32/32 individuals transfected with CCR5 or CMV US28 constructs to generate isogeneic lines differing in co-receptor expression to determine if direct infection is required for cell death.
