The growth and envelopment processes of three representative herpesviruses, equine abortion, pseudorabies, and herpes simplex, were examined in baby hamster kidney (BHK 21/13) cells by bioassay (plaque-forming units) and electron microscopy. The envelopment process was identical for all three viruses. After assembly in the nucleus, the nucleocapsid acquired an envelope by budding from the inner nuclear membrane. This membrane was reduplicated as the enveloped particle was released so that the budding process did not result in disruption of the continuity of the nuclear membrane. That portion of the nuclear membrane which comprised the viral envelope was appreciably thicker than the remainder of the membrane and exhibited numerous projections on its surface. Once enveloped, the viral particles were seen in vesicles and vacuoles in the cell cytoplasm. These appeared to open at the cytoplasmic membrane, releasing the virus from the cell. There was no detectable difference in the size or appearance of enveloped particles in intra- or extracellular locations.

Abstract. Several previous studies have demonstrated that mammary epithelial cells from pregnant mice retain their differentiated characteristics and their secretory potential in culture only when maintained on stromal collagen gels floated in the culture medium. The cellular basis for these culture requirements was investigated by the monitoring of milk protein synthesis and polarized secretion from the mouse mammary epithelial cell line, COMMA-1-D. Experiments were directed towards gaining an understanding of the possible roles of cell-extracellular matrix interactions and the requirements for meeting polarity needs of the epithelium.When cells are cultured on floating collagen gels they assemble a basal lamina-like structure composed of laminin, collagen (IV), and heparan sulfate proteoglycan at the interface of the cells with the stromal collagen. To assess the role of these components, an exogenous basement membrane containing these molecules was generated using the mouse endodermal cell line, PFHR-9. This matrix was isolated as a thin sheet attached to the culture dish, and mammary cells were then plated onto it. It was found that cultures on attached PFHR-9 matrices expressed slightly higher levels of fl-casein than did cells on plastic tissue culture dishes, and also accumulated a large number of fat droplets. However, the level of 13-casein was approximately fourfold lower than that in cultures on floating collagen gels. Moreover, the 13-casein made in cells on attached matrices was not secreted but was instead rapidly degraded intracellularly. If, however, the PFHR-9 matrices with attached cells were floated in the culture medium, ~casein expression became equivalent to that in cells cultured on floating stromal collagen gels, and the casein was also secreted into the medium. The possibility that floatation of the cultures was necessary to allow access to the basolateral surface of cells was tested by culturing cells on nitrocellulose filters in Millicell (MiUipore Corp., Bedford, MA) chambers. These chambers permit the monolayers to interact with the medium and its complement of hormones and growth factors through the basal cell surface. Significantly, under these conditions ctl-, 0t2-, and 13-casein synthesis was equivalent to that in cells on floating gels and matrices, and, additionally, the caseins were actively secreted. Similar results were obtained independently of whether or not the filters were coated with matrices,In conclusion, providing the cells with a culture environment that permits access to the basolateral surface and caters to the polarity requirements of the cell allows optimal cellular differentiation and secretion. Moreover, the simple interaction of any of the basement membrane components, laminin, collagen IV, or heparan sulfate proteoglycan, with the basal epithelial surface, is not in itself sufficient to promote maintenance of differentiation.Finally, while secretion of 0tl-, ~t2-, and 13-casein from Millicell chambers was polarized to the apical (top) chamber, transferrin wa...

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