Increased CXCR4-dependent HIV-1 Fusion in Activated T Cells: Role of CD4/CXCR4 Association

Activation of peripheral CD4 T cells resulted in augmented fusion with X4 human im- munodeficiency virus type 1 (HIV-1) envelope-ex- pressing cells without parallel increases in the sur- face expression of CD4 or CXC chemokine recep- tor 4 (CXCR4). Our study used biochemical methods and biological assays to correlate the in- creased fusion potential of activated T cells with changes in CXCR4 isoforms and CD4-CXCR4 as- sociation. Western blot analyses of CXCR4, precip- itated from resting T cells, identified several CXCR4 species with molecular weights of 47, 50, 62, and 98 kDa. After 24 h stimulation with phy- tohemagglutinin/interleukin-2, a marked reduction was seen in the 47-kDa, with a concomitant in- crease in the amounts of 50 and 62-64 kDa CXCR4. T cell activation also induced an increase in the coprecipitation of CXCR4 with CD4. The 62-kDa CXCR4 predominantly coprecipitated with CD4 and was shown to be ubiquitinated. Stripping of CD4 from the cell surface with pronase treat- ment prior to cell lysis only partially reduced co- precipitation of CD4 with the 62-kDa CXCR4, re- vealing a pool of intracellular CD4-CXCR4 com- plexes. Coprecipitation of CXCR4 with CD4 was reduced in activated cells treated with Brefeldin A and Monensin, suggesting that late endosomes play a role in intracellular association of CXCR4 with CD4. Confocal microscopy confirmed the colocal- ization of CD4 and CXCR4 within CD63 endo- cytic compartments. These findings demonstrated a correlation between the enhanced susceptibility of activated T cells to HIV-1 fusion and accumula- tion of ubiquitinated 62-64 kDa CXCR4 species, which preferentially associated with CD4. The CD4-CXCR4 complexes may shuttle between late endosomes and the cell surface. J. Leukoc. Biol. 78: 1306-1317; 2005.