Progeny vaccinia and human cytomegalovirus particles utilize early endosomal cisternae for their envelopes

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.