Specific depletion of P^k correlates with increased HIV-1 infection. CD4⁺ HeLa cells (clone 6C) were transfected daily with either control siRNA or P^k synthase (P^k-S) siRNA, and cultured for 72 hours to deplete P^k-S, and subsequently P^k. FACS analysis was performed and scatter plots of CD4⁺ HeLa cells labeled with VT1B-Alexa₄₅₈ were analyzed, where background was compensated to unstained controls. (A) Histogram plots representing percentage of cell populations expressing P^k. (B) TLC of total GSLs extracted from control and P^k-S siRNA-transfected CD4⁺ HeLa cells. Lane 1: GSL standards. Lane 2: Control siRNA-transfected cells. Lane 3: P^k-S siRNA-transfected cells. (C) VT1 overlay for P^k detection was carried out on TLC of total GSLs Lane 1: GSL standards. Lane 2: Control siRNA-transfected cells. Lane 3: P^k-S siRNA-transfected cells. (D) HIV-1_IIIB (MOI, 0.3) infection of control (control siRNAs)- or P^k-S siRNA-transfected CD4⁺ HeLa cells was measured by p24^gag production after 5 days of culture. Data are representative of the mean plus or minus SEM where n = 3 infection data points. *P < .05. This figure is representative of 3 independent experiments. (E) A schematic working model for P^k-induced protection against HIV-1 infection. HIV first binds to CD4, which exposes the GSL and chemokine coreceptor binding site within the V3 loop of HIV gp120. When P^k is highly expressed, it can successfully compete with chemokine coreceptor for the exposed sites within the V3 loop; thus, P^k interferes with the process of membrane fusion.