Di Simone C, Zandonatti M A, Buchmeier M J
Department of Neuropharmacology, Scripps Research Institute, La Jolla, California 92037.
Virology. 1994 Feb;198(2):455-65. doi: 10.1006/viro.1994.1057.
Membrane fusion activity of the glycoprotein (GP) complex of LCMV was determined using the R18 fluorescent dequenching assay. Utilization of an endosomal entry route by LCMV and acidic activation of the membrane fusion activity were indicated by the inhibition of LCMV infection at an early stage by the lysosomal weak base chloroquine and the ionophore monensin. When LCMV was mixed with R18-labeled liposomes with a lipid composition mimicking that of the endosome, dequenching occurred only at acidic pH (< or = 6.0). The measured dequenching was inhibited by protease treatment of the LCMV, indicative of protein-mediated membrane fusion. The initial rate of fusion was measured at pH values between 5.3 and 7.0 and was found to decrease rapidly and linearly between pH 5.3 (0.177%/sec) and pH 6.0 (0.027%/sec). Binding and fusion of R18-labeled LCMV with BHK cells were also examined. No difference was found between R18-labeled and unlabeled LCMV in binding to or infection of BHK cells. Dequenching was observed in labeled LCMV endocytosed by BHK cells. With BHK cells neither LCMV fusion with the plasma membrane nor LCMV-induced cell-cell fusion was observed, even under acidic conditions, and examination of the sequence of LCMV GP did not reveal a likely candidate sequence for a "fusion peptide." The binding of conformationally dependent monoclonal antibodies to GP was measured at neutral and acidic pH in order to seek evidence of pH-dependent conformational change in GP. Dissociation of the GP-1 from the complex was measured by sucrose gradients run on purified virus. These experiments revealed that after exposure to acid pH the LCMV glycoprotein spike complex underwent irreversible conformational change in which GP-1 was dissociated from the virion, conformational epitopes on GP-1 were lost, and sequestered epitopes on GP-2 became exposed. Further, LCMV infectivity was irreversibly inactivated by exposure to acidic pH (< 6.0), likely due to the loss of GP-1 and conformation changes in GP-2.
利用R18荧光猝灭法测定淋巴细胞脉络丛脑膜炎病毒（LCMV）糖蛋白（GP）复合物的膜融合活性。溶酶体弱碱氯喹和离子载体莫能菌素在早期对LCMV感染的抑制作用表明LCMV利用内体进入途径并激活膜融合活性。当LCMV与具有模仿内体脂质组成的R18标记脂质体混合时，猝灭仅在酸性pH（≤6.0）下发生。通过蛋白酶处理LCMV可抑制测得的猝灭，这表明是蛋白质介导的膜融合。在pH值介于5.3和7.0之间测量融合的初始速率，发现在pH 5.3（0.177%/秒）至pH 6.0（0.027%/秒）之间迅速且呈线性下降。还检测了R18标记的LCMV与BHK细胞的结合及融合情况。在与BHK细胞的结合或感染方面，未发现R18标记的LCMV与未标记的LCMV之间存在差异。在被BHK细胞内吞的标记LCMV中观察到猝灭。对于BHK细胞，即使在酸性条件下，也未观察到LCMV与质膜的融合或LCMV诱导的细胞-细胞融合，并且对LCMV GP序列的检测未发现可能的“融合肽”候选序列。在中性和酸性pH下测量构象依赖性单克隆抗体与GP的结合，以寻找GP中pH依赖性构象变化的证据。通过对纯化病毒进行蔗糖梯度离心来测量GP-1从复合物中的解离。这些实验表明，暴露于酸性pH后，LCMV糖蛋白刺突复合物发生不可逆的构象变化，其中GP-1从病毒粒子上解离，GP-1上的构象表位丢失，GP-2上被隔离的表位暴露。此外，暴露于酸性pH（<6.0）会使LCMV感染性不可逆地失活，这可能是由于GP-1的丢失和GP-2的构象变化所致。
