In the final stages of the HSV-1 lifecycle, a viral nucleocapsid buds into a vesicle of TGN/endosome origin acquiring an envelope and an outer vesicular membrane. The virus-containing vesicle then traffics to the plasma membrane where it fuses, exposing an enveloped virion. Although the process of directed egress has been studied in polarized epithelial cell lines, little work has been done in non-polarized cell types, i.e. cells without extensive junctional complexes and compositionally distinct membrane regions. In the following report we describe a molecular and cellular study of HSV-1 egress as it occurs in non-polarized cells. Infected Vero cells were examined by electron, confocal, and TIRF microscopy. The results demonstrated that HSV-1 was released at specific pocket-like areas of the plasma membrane along the substrate-adherent surface and cell-cell adherent contacts. Both the membrane composition and cytoskeletal structure of these egress sites were found to be modified by infection. The membrane at release site "patches" was rich in virus-encoded glycoproteins, and accumulation of glycoproteins at exit sites began before viral transport. The use of a mutant unable to produce mature virions showed that glycoprotein patches formed normally even when virus trafficking was inhibited. Depolymerization of the cytoskeleton indicated that microtubules and actin were both important for trafficking of virions and glycoproteins to release sites. Furthermore, the actin cytoskeleton was found to be necessary for maintaining the integrity of egress sites once they formed. When actin was depolymerized, the glycoprotein concentrations dispersed across the membrane as did the surface-associated virus. Lastly, glycoprotein accumulation at release sites was found to be dependent on the cytoplasmic tail of viral glycoprotein E. When cells were infected with gE deletion mutants, patch sizes were significantly reduced, although the total amount of virus released was increased. The results of this study are interpreted to indicate that egress of HSV-1 is directed to virally induced, specialized egress sites that form along the cell membrane. These sites share similarities with retroviral virological synapses and may function in a similar manner during cell-to-cell spread.
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