Tooze J, Hollinshead M, Reis B, Radsak K, Kern H
European Molecular Biology Laboratory, Heidelberg, Germany.
Eur J Cell Biol. 1993 Feb;60(1):163-78.
We have investigated by electron microscopy the envelopment of progeny human cytomegalovirus particles and vaccinia virus particles. As host cells we used primary human foreskin fibroblasts, in which both viruses replicate, and also AtT20 cells in which vaccinia virus, but not human cytomegalovirus, replicates. As we show here, primary human foreskin fibroblasts contain tubular early endosomes like those in AtT20 cells and many other cells in culture. Our aim was to ascertain whether or not the cisternae which wrap the maturing progeny viral particles are related to tubular endosomes. When infected cells were incubated with the fluid phase endocytic tracer horseradish peroxidase (HRP) at appropriate times after infection (5-8 h post vaccinia infection and 72 h or 96 h post cytomegalovirus infection), we found that many of the partially or fully enveloped vaccinia and human cytomegalovirus particles in the cytoplasm had HRP reaction product in the lumen of their cisternal envelope. Examination of single and serial sections indicated that the cisternal envelopes of the progeny virions were derived from tubular endosomes. Pulse-chase experiments with HRP showed that the viral particles with envelopes derived from tubular endosomes were not directed to late endosomes and autophagic digestion but were released from the cells. Experiments with Brefeldin A established that the envelopment of viral particles by endosomal cisternae proceeded for at least 2 h in the absence of a Golgi apparatus. Our data indicate that vaccinia virus and human cytomegalovirus (and presumably other pox and herpes viruses) have evolved to utilize early endocytic compartments to achieve their egress from host cells. We also found that in cells infected by vaccinia virus the delivery of the endocytic tracer to the Golgi apparatus is greatly enhanced over that in controls or cells infected with human cytomegalovirus. The cytopathic effects of vaccinia virus therefore include perturbation of membrane traffic between endocytic and exocytic compartments.
我们通过电子显微镜研究了人巨细胞病毒子代颗粒和痘苗病毒颗粒的包膜形成过程。作为宿主细胞，我们使用了原代人包皮成纤维细胞（两种病毒均可在其中复制）以及AtT20细胞（痘苗病毒可在其中复制，但人巨细胞病毒不能）。正如我们在此处所示，原代人包皮成纤维细胞含有与AtT20细胞以及培养中的许多其他细胞类似的管状早期内体。我们的目的是确定包裹成熟子代病毒颗粒的池是否与管状内体相关。当感染细胞在感染后适当时间（痘苗病毒感染后5 - 8小时以及人巨细胞病毒感染后72小时或96小时）与液相内吞示踪剂辣根过氧化物酶（HRP）一起孵育时，我们发现细胞质中许多部分或完全包膜化的痘苗病毒和人巨细胞病毒颗粒，其池状包膜腔内有HRP反应产物。对单张和连续切片的检查表明，子代病毒粒子的池状包膜源自管状内体。用HRP进行的脉冲追踪实验表明，源自管状内体的包膜病毒颗粒不会被导向晚期内体和自噬消化，而是从细胞中释放出来。用布雷菲德菌素A进行的实验表明，在内体池对病毒颗粒的包膜形成过程在没有高尔基体的情况下至少持续2小时。我们的数据表明，痘苗病毒和人巨细胞病毒（可能还有其他痘病毒和疱疹病毒）已经进化到利用早期内吞区室来实现从宿主细胞中释放。我们还发现，在感染痘苗病毒的细胞中，内吞示踪剂向高尔基体的转运比对照细胞或感染人巨细胞病毒的细胞大大增强。因此，痘苗病毒的细胞病变效应包括内吞和外排区室之间膜运输的扰动。
