Allsopp T E, Scallan M F, Williams A, Fazakerley J K
Department of Veterinary Pathology, University of Edinburgh, EH9 1QH, UK.
Cell Death Differ. 1998 Jan;5(1):50-9. doi: 10.1038/sj.cdd.4400298.
Multicellular organisms can employ a number of defences to combat viral replication, the most dramatic being implementation of a cell autonomous apoptotic process. The overall cost to the viability of an organism of losing infected cells by apoptosis may be small if the dying cells can be substituted. In contrast, suicide of irreplaceable cells such as highly specialised neurons may have a more dramatic, even fatal consequence. Previous in vitro approaches to understanding whether neurotropic viruses cause neurons to apoptose have utilised transformed cell lines. These are not in the appropriate state of differentiation to provide an accurate indication of events in vivo. We have chosen to characterise the ability of a model CNS disease-causing virus, Semliki Forest virus (SFV), to infect and trigger apoptosis in primary cultures of nerve growth factor (NGF)-dependent sensory neurons. These cells are known to die when deprived of NGF and constitute a useful indicator of apoptosis. We observe that infection causes cell death which bears the morphological hallmarks of apoptosis, this occurs even in the present of survival promoting NGF and is concomitant with new virus production. Using the TUNEL (transferase dUTP nick end labelling) technique we show that SFV-induced apoptosis involves DNA fragmentation and requires caspase (CED-3/ICE cysteine protease) activation, as does apoptosis induced by NGF-deprivation. Extensive areas of apoptosis, as defined using a combination of ultrastructural analysis and TUNEL occur in infected neonatal mouse brains. The novel evidence that infection of primary neurons with SFV induces apoptosis with activation of one or more caspases defines a system for the further anlaysis of apoptosis regulation in physiologically relevant neurons.
多细胞生物可以采用多种防御机制来对抗病毒复制，其中最显著的是实施细胞自主凋亡过程。如果凋亡的感染细胞能够被替代，那么通过凋亡失去这些细胞对生物体生存能力造成的总体代价可能较小。相比之下，像高度特化的神经元这样不可替代的细胞自杀可能会产生更显著甚至致命的后果。以往用于了解嗜神经病毒是否会导致神经元凋亡的体外研究方法采用的是转化细胞系。这些细胞系处于不适当的分化状态，无法准确反映体内的实际情况。我们选择对一种引发中枢神经系统疾病的模型病毒——辛德毕斯病毒（SFV）感染并触发神经生长因子（NGF）依赖性感觉神经元原代培养物凋亡的能力进行表征。已知这些细胞在缺乏NGF时会死亡，是凋亡的一个有用指标。我们观察到感染会导致具有凋亡形态特征的细胞死亡，即使在存在促进存活的NGF的情况下也是如此，并且这与新病毒的产生同时发生。使用TUNEL（脱氧尿苷三磷酸末端转移酶介导的缺口末端标记）技术，我们发现SFV诱导的凋亡涉及DNA片段化，并且需要半胱天冬酶（CED-3/ICE半胱氨酸蛋白酶）激活，NGF缺乏诱导的凋亡也是如此。利用超微结构分析和TUNEL相结合的方法确定，感染的新生小鼠大脑中出现了广泛的凋亡区域。用SFV感染原代神经元可诱导凋亡并激活一种或多种半胱天冬酶，这一全新证据为进一步分析生理相关神经元中的凋亡调控定义了一个系统。
