HNP-1 prevents HIV-1 detachment and interferes with virus endocytosis.

(A) A diagram of pseudoviruses co-labeled with Ecliptic pHluorin fused to the ICAM-1 transmembrane domain (EcpH-TM, green) and Gag-mCherry (red) at neutral and low pH. (B) HIV-1 endocytosis assay using pseudoviruses co-labeled with EcpH-TM/Gag-mCherry. HXB2 pseudoviruses were pre-bound to TZM-bl entry in the cold (first panel), and virus entry was allowed to occur by incubating for 1 h at 37°C in the absence (second panel) or in the presence of 8.7 µM HNP-1 (third panel) or 20 µM HNP-1 in HBSS/10% serum (fourth panel). (C) Quantification of the effect of HNP-1 on uptake and dissociation of HXB2 pseudoviruses pre-bound to TZM-bl cells in the cold. The total amount of cell-associated viruses was assessed based on the Gag-mCherry signal from all particles in the image field (dark red bars). The drop in the overall mCherry signal following the incubation in the absence of defensin (second dark red bar) is primarily due to virus dissociation from cells, as described in [40], [86]. HNP-1 prevented the loss of the virus from the cell surface, both in the presence and in the absence of serum. Virus entry into acidic endosomes resulted in quenching of the EcpH signal and reduction of the EcpH/mCherry (green/red) fluorescence ratio (green-red gradient bars). Data points are means and SEM from 5–11 image fields each containing 30–40 cells. ***, P