HNP-1 stabilizes HIV-1 pre-hairpin intermediates.

Top: A kinetic model of HIV-cell fusion. Progression of virus fusion through the surface accessible steps – receptor binding (V/CD4), coreceptor binding (V/CD4/CoR) and endocytosis (VE) – is measured by adding specific inhibitors at different time intervals. Here, kCD4, kCoR and kE are the effective rate constants (kF is the rate constant for HIV-endosome fusion (VF) that is not resolved by the current approach). The steps that form/expose pre-hairpin intermediates (PHI) on the cell surface are highlighted in blue, while intracellular PHIs, which are no longer accessible to added inhibitors, are highlighted in orange (a vertical dashed line separates internalized viruses from external viruses). C52L is carried over by the PHIs into endosomes (curved red arrow) where it blocks subsequent fusion. The virus is assumed to inactivate at every step of the fusion reaction through detachment from cells and by undergoing non-productive endocytosis (not shown for visual clarity). The inactivation rate constant ki was estimated based on the rate of non-productive endocytosis measured in [10]. An alternative pathway for HIV-1 escape from C52L by fusing with the plasma membrane is shown by a dashed arrow and is colored gray. In this case, the kinetics of the V/CD4/CoR escape from C52L is described by kF. (A–D) HXB2 pseudoviruses (A, B) or BaL pseudoviruses (C, D) were pre-bound to TZM-bl cells in the cold and allowed to undergo fusion for 90 min at 37°C, either in the absence (A, C) or in the presence (B, D) of 7.3 µM HNP-1 in HBSS supplemented with 10% human serum. At indicated time points, fully inhibitory concentrations of HIV-1 fusion inhibitors, BMS-806, AMD3100, TAK-779 or C52L, were added, and incubation was continued till the 90 min point. The resulting virus fusion was measured by the BlaM assay, as described in Materials and Methods. Data points are means and SEM from 2 independent experiments performed in triplicate. Solid lines are obtained by curve-fitting using the three-step kinetic model (see also [10]). The calculated effective rate constants are given in Table 1. Insets in panels B and D show the effect of 7.3 µM HNP-1 in serum-containing medium on HXB2 and BaL fusion, respectively. *, P<0.04 (two-tailed t-test).