Fig 3.

TIRF micrographs showing formation of glycoprotein patch sites uncoupled from virus egress. (A) Vero cells infected with VP26-GFP HSV-1 were fixed at 4, 6, and 8 hpi. Patch glycoproteins were labeled with rhodamine-WGA. All images were taken at the same exposure. In the 4-hpi image, the edge of the adherent surface of the cell (as determined by increasing the brightness of the image) is outlined. Note that neither glycoprotein patches nor virus are visible at 4 hpi. By 6 hpi, glycoprotein patches are beginning to form (arrow) on the adherent surface, but virus still is not evident. At 8 hpi, there is pronounced glycoprotein staining on the cell surface and virions are beginning to accumulate in these areas. Images indicate that patches form before viral egress. (B) Vero cells infected with wild-type KOS HSV-1 (top) or UL25Δ mutant HSV (bottom) were fixed at 12 hpi. In a UL25Δ infection, capsids are retained in the nucleus. Egress site glycoproteins were labeled with WGA. Virus was labeled with α-VP5 major capsid protein antibody and Alexa 488-labeled secondary antibody. Infections suggest that adherent surface glycoprotein patches form independently of viral release.