Markosyan Ruben M, Miao Chunhui, Zheng Yi-Min, Melikyan Gregory B, Liu Shan-Lu, Cohen Fredric S
Rush University Medical Center, Department of Molecular Biophysics and Physiology, Chicago, Illinois, United States of America.
University of Missouri School of Medicine, Bond Life Sciences Center, Department of Molecular Microbiology and Immunology, Columbia, Missouri, United States of America.
PLoS Pathog. 2016 Jan 5;12(1):e1005373. doi: 10.1371/journal.ppat.1005373. eCollection 2016 Jan.
Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge-a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry.
埃博拉病毒（EBOV）是一种高致病性丝状病毒，可在人类和动物中引起出血热。目前，人们对EBOV如何在内体膜内融合其包膜膜以引发感染了解甚少。我们成功测量了由EBOV融合蛋白GP介导的细胞-细胞融合，通过细胞质和膜染料的转移进行检测。一种小分子融合抑制剂、一种中和抗体以及已知可降低病毒感染的EBOV GP突变，都能大大降低融合。通过监测小分子水性染料在细胞间的重新分布以及通过电容测量，我们发现EBOV GP介导的融合孔不易扩大——这与其他病毒融合蛋白的行为有显著差异。EBOV GP必须被晚期内体驻留的组织蛋白酶B或L切割才能具备融合能力。细胞表面表达的GP的切割似乎发生在内体中，组织蛋白酶抑制剂、提高内体pH值的试剂或顺向转运抑制剂所施加的融合阻断证明了这一点。用重组可溶性组织蛋白酶B或嗜热菌蛋白酶处理效应细胞，后者可将GP切割成活性形式，增加了融合程度，这表明一部分表面表达的GP未被切割。虽然短暂暴露于酸性pH值可提高融合速率，但在中性pH值下也会发生融合。重要的是，在组织蛋白酶活性被阻断且EBOV GP被嗜热菌蛋白酶切割的实验中，融合程度与外部pH值无关。这些结果表明，低pH值通过组织蛋白酶众所周知的pH依赖性活性促进融合；切割后的EBOV GP诱导的融合是一个从根本上独立于pH值的过程。细胞-细胞融合系统揭示了EBOV进入的一些以前未被认识到的特征，这些特征在内体进入的背景下难以轻易阐明。
