HIV-1 Envelope glycoprotein modulates CXCR4 clustering and dynamics on the T cell membrane

HIV-1 entry into susceptible cells requires the dynamic interaction of its envelope (Env) glycoprotein with the host cell receptor CD4 and a co-receptor, either CCR5 or CXCR4. While the molecular mechanisms of Env-receptor interactions that lead to membrane fusion have been extensively studied, the precise nanoscale redistribution of these co-receptors to viral binding sites remains unknown. In this study, we employed single-particle tracking total internal reflection fluorescence (SPT-TIRF) microscopy to quantitatively analyze nanoscale organizational changes of CXCR4 on CD4 ⁺ T cell surfaces following binding of X4-tropic HIV-1. Our data reveal that both recombinant X4-gp120 and virus-like particles (VLPs) expressing physiological levels of X4 Env proteins (gp120 and gp41) promote CXCR4 clustering, a phenomenon linked to cell infection. Furthermore, these ligands induced oligomerization of CXCR4 ^R334X , a naturally occurring mutant of CXCR4 associated with WHIM syndrome that fails to oligomerize upon CXCL12 binding but nonetheless supports HIV-1 infection. Our findings establish a connection between CXCR4 clustering and HIV-1 infection, enhancing our understanding of the initial events in viral attachment and entry. These results further suggest that HIV-1 infection necessitates a specific spatial arrangement of co-receptors, distinct from that induced by their natural chemokine ligands, highlighting the critical role of cell-surface receptor spatial organization in cellular function.