Synchronized Retrovirus Fusion in Cells Expressing Alternative Receptor Isoforms Releases the Viral Core into Distinct Sub-cellular Compartments

Disparate enveloped viruses initiate infection by fusing with endosomes. However, the highly diverse and dynamic nature of endosomes impairs mechanistic studies of fusion and identification of sub-cellular sites supporting the nucleocapsid release. We took advantage of the extreme stability of avian retrovirus-receptor complexes at neutral pH and of acid-dependence of virus-endosome fusion to isolate the latter step from preceding asynchronous internalization/trafficking steps. Viruses were trapped within endosomes in the presence of NH4Cl. Removal of NH4Cl resulted in a quick and uniform acidification of all subcellular compartments, thereby initiating synchronous viral fusion. Single virus imaging demonstrated that fusion was initiated within seconds after acidification and often culminated in the release of the viral core from an endosome. Comparative studies of cells expressing either the transmembrane or GPI-anchored receptor isoform revealed that the transmembrane receptor delivered the virus to more fusion-permissive compartments. Thus the identity of endosomal compartments, in addition to their acidity, appears to modulate viral fusion. A more striking manifestation of the virus delivery to distinct compartments in the presence of NH4Cl was the viral core release into the cytosol of cells expressing the transmembrane receptor and into endosomes of cells expressing the GPI-anchored isoform. In the latter cells, the newly released cores exhibited restricted mobility and were exposed to a more acidic environment than the cytoplasm. These cores appear to enter into the cytosol after an additional slow temperature-dependent step. We conclude that the NH4Cl block traps the virus within intralumenal vesicles of late endosomes in cells expressing the GPI-anchored receptor. Viruses surrounded by more than one endosomal membrane release their core into the cytoplasm in two steps – fusion with an intralumenal vesicle followed by a yet unknown temperature-dependent step that liberates the core from late endosomes.

多种不同的有包膜病毒（enveloped virus）通过与内体（endosome）融合启动感染。然而，内体兼具高度多样性与动态变化的特性，这阻碍了病毒融合机制研究以及对支持核衣壳（nucleocapsid）释放的亚细胞位点的鉴定。我们利用禽逆转录病毒（avian retrovirus）-受体复合物在中性pH条件下的极端稳定性，以及病毒-内体融合的酸依赖性，将融合步骤与此前的异步内化/转运步骤分离开来。在氯化铵（NH₄Cl）存在的环境中，病毒被滞留在内体中。移除氯化铵后，所有亚细胞区域会快速且均一地发生酸化，从而同步启动病毒融合过程。单病毒成像实验显示，酸化后数秒内便会启动融合，且融合通常以病毒核心从内体中释放为最终结果。
针对分别表达跨膜受体（transmembrane receptor）与糖基磷脂酰肌醇（Glycosylphosphatidylinositol, GPI）锚定受体亚型的细胞所开展的对比研究表明，跨膜受体可将病毒运送至更易发生融合的内体区室。由此可见，除内体的酸化程度外，其固有特性似乎也可调控病毒融合过程。
在氯化铵存在的条件下，病毒被运送至不同区室的现象还有更显著的表现：在表达跨膜受体的细胞中，病毒核心被释放至细胞质（cytosol）；而在表达GPI锚定受体亚型的细胞中，病毒核心则被释放至内体内部。在后者这类细胞中，新释放的病毒核心活动受限，且所处环境的酸度高于细胞质。这类病毒核心似乎需要经过额外的、依赖温度的缓慢步骤，才能进入细胞质。
我们最终得出结论：在表达GPI锚定受体的细胞中，氯化铵的阻滞作用会将病毒滞留在晚期内体（late endosome）的腔内囊泡（intralumenal vesicle）中。被多层内体膜包裹的病毒，会通过两步将核心释放至细胞质：首先与腔内囊泡发生融合，随后再经过一个尚未明确的、依赖温度的步骤，将核心从晚期内体中释放出来。