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

顶部：HIV-细胞融合动力学模型。病毒融合通过膜表面可及步骤的进程——受体结合（V/CD4）、共受体结合（V/CD4/CoR）与内吞作用（VE）——可通过在不同时间节点添加特异性抑制剂进行量化测定。本模型中，kCD4、kCoR与kE为有效速率常数（kF为HIV-内体融合（VF）的速率常数，当前实验方法无法解析该步骤）。在细胞表面形成或暴露前发夹中间体（pre-hairpin intermediates, PHI）的步骤以蓝色标注；而细胞内的PHI因不再可被外加抑制剂结合，以橙色标注（垂直虚线将内化病毒与胞外病毒分隔开）。C52L可随PHI被转运至内体（红色弯曲箭头标注），并在此阻断后续融合过程。本模型假定病毒在融合反应的每一步中，均会通过脱离细胞或发生无效内吞（为可视化清晰未展示该过程）而失活。失活速率常数ki根据文献[10]中测定的无效内吞速率进行估算。HIV-1通过与细胞质膜融合从而逃逸C52L作用的替代途径以灰色虚线箭头标注。在此途径中，V/CD4/CoR复合物逃逸C52L的动力学过程由kF表征。(A-D) 将HXB2假病毒（A、B组）或BaL假病毒（C、D组）在低温条件下预结合至TZM-bl细胞，随后于37℃下进行90分钟的融合反应，反应体系分别为不含（A、C组）或含有（B、D组）7.3 μM HNP-1、添加10%人血清的汉克平衡盐溶液（Hank's Balanced Salt Solution, HBSS）。在指定时间节点，加入终浓度足以完全抑制HIV-1融合的抑制剂BMS-806、AMD3100、TAK-779或C52L，随后继续孵育至总时长90分钟。病毒融合程度通过BlaM检测法进行定量，具体操作参见材料与方法部分。数据点为2次独立重复实验（每组设3个复孔）的均值与标准误（standard error of the mean, SEM）。实线为通过三步动力学模型拟合得到的曲线（另参见文献[10]）。计算得到的有效速率常数详见表1。B、D组的内嵌图分别展示了含血清培养基中7.3 μM HNP-1对HXB2与BaL病毒融合的影响。*，P