Blumenthal R, Bali-Puri A, Walter A, Covell D, Eidelman O
Section of Membrane Structure and Function, National Cancer Institute, Bethesda, Maryland 20892.
J Biol Chem. 1987 Oct 5;262(28):13614-9.
We have studied fusion between membranes of vesicular stomatitis virus (VSV) and Vero cells using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We could identify the two pathways of fusion by the kinetics of R18 dequenching, effects of inhibitors, temperature dependence, and dependence on osmotic pressure. Fusion at the plasma membrane began immediately after lowering the pH below 6 and showed an approximately exponential time course, whereas fusion via the endocytic pathway (pH 7.4) became apparent after a time delay of about 2 min. Fusion via the endocytic pathway was attenuated by treating cells with metabolic inhibitors and agents that raise the pH of the endocytic vesicle. A 10-fold excess of unlabeled virus arrested R18VSV entry via the endocytic pathway, whereas R18 dequenching below pH 6 (fusion at the plasma membrane) was not affected by the presence of unlabeled virus. The temperature dependence for fusion at pH 7.4 (in the endosome) was much steeper than that for fusion at pH 5.9 (with the plasma membrane). Fusion via the endocytic pathway was attenuated at hypo-osmotic pressures, whereas fusion at the plasma membrane was not affected by this treatment. The pH profile of Vero-VSV fusion at the plasma membrane, as measured by the dequenching method, paralleled that observed for VSV-induced cell-cell fusion. Fusion was blocked by adding neutralizing antibody to the Vero-VSV complexes. Activation of the fusion process by lowering the pH was reversible, in that the rate of fusion was arrested by raising the pH back to 7.4. The observation that pH-dependent fusion occurred at similar rates with fragments and with intact cells indicates that pH, voltage, or osmotic gradients are not required for viral fusion.
我们利用基于十八烷基罗丹明（R18）荧光自猝灭解除的脂质混合测定法，研究了水泡性口炎病毒（VSV）与Vero细胞之间的膜融合。我们可以通过R18去猝灭动力学、抑制剂的作用、温度依赖性以及对渗透压的依赖性来确定两种融合途径。质膜处的融合在pH降至6以下后立即开始，并呈现出近似指数的时间进程，而通过内吞途径（pH 7.4）的融合在约2分钟的延迟后才变得明显。用代谢抑制剂和提高内吞小泡pH值的试剂处理细胞会减弱通过内吞途径的融合。未标记病毒过量10倍会阻止R18 VSV通过内吞途径进入，而pH低于6时的R18去猝灭（质膜处的融合）不受未标记病毒存在的影响。pH 7.4（在内体中）时融合的温度依赖性比pH 5.9（与质膜融合）时陡峭得多。低渗压力下通过内吞途径的融合会减弱，而质膜处的融合不受此处理的影响。通过去猝灭方法测量的Vero-VSV在质膜处融合的pH谱与VSV诱导的细胞-细胞融合所观察到的情况相似。向Vero-VSV复合物中加入中和抗体可阻断融合。通过降低pH激活融合过程是可逆的，即通过将pH值回升至7.4可阻止融合速率。pH依赖性融合在片段和完整细胞中以相似速率发生这一观察结果表明，病毒融合不需要pH、电压或渗透压梯度。
