Figure 5.

Time course of HSV-1 entry. Vero cells infected at an MOI of 500 in the presence of CH were fixed at various times postinfection with 2% glutaraldehyde in PBS and embedded in Epon for EM analysis. (a) Schematic cross-section perpendicular to the substrate through an adherent Vero cell. For quantitation, 25 random electron micrographs obtained from sections cut parallel to the substrate through the basal part of the cells were taken in a systematic fashion for each time point. “Basal” is defined as being within 2 μm distance from the substrate (30 sections of ∼50–70 nm). (b) The total numbers of cytosolic capsids (full and empty), cytosolic empty capsids, and virions in endosomes were determined for each time point. The number of cytosolic capsids increased rapidly until 1 h postinfection. At later time points, less cytosolic capsids were detected, suggesting that they were ultimately disassembled. In addition, the subcellular localization of each capsid was determined and the numbers were expressed as a percentage of the total (c–g). (c) At the earliest time point of 15 min, the majority of cytosolic capsids was localized close to the plasma membrane, from where they disappeared rapidly later. (d) Several capsids could not be localized to a particular organelle. At 1 h postinfection, there was the highest amount of those capsids, which were apparently free in the cytosol. (e) A significant portion of all cytosolic capsids, namely 10% at 1 h postinfection, colocalized transiently with microtubules. (f) Capsids began to appear at the nuclear pores at 2 h postinfection, and at 4 h, 60% of all cytosolic capsid had reached the nucleus. (g) The appearance of empty cytosolic capsids coincided with the arrival at the nucleus. Only at 4 h postinfection, there was a very small portion of empty, cytosolic capsids. N, nucleus; PM, plasma membrane.