Figure 4.

Incoming HSV-1 capsids colocalize with MTs. (a) Conventional immunofluorescence microscopy. Vero cells infected at an MOI of 50 in the presence of CH were fixed in methanol at 2 h PI, and double labeled with anti-VP5 (NC-1; FITC anti–rabbit) and antitubulin antibodies (IA2; rhodamine anti–mouse). The FITC and the rhodamine signals were documented simultaneously using the FITC filter set. Almost all capsids (arrows) not localized to the nucleus (N) colocalize with microtubules. (b) Confocal immunofluorescence microscopy. Vero cells infected at an MOI of 50 in the presence of CH were fixed in methanol 2 h PI, and double labeled with anti-capsid (HC, white) and anti-tubulin antibodies (IA2, gray). Almost all capsids not localized to the nucleus (N) colocalize with microtubules. Note that in some cells, the viral capsids accumulate at the microtubule-organizing center (MTOC). (c–e) Conventional EM. At 1 h postinfection, Vero cells infected at an MOI of 500 in the presence of CH were fixed with 2% glutaraldehyde in PBS. Epon sections were cut parallel to the substrate. Numerous incoming viral capsids are localized to microtubules (MT) as identified by their typical morphology, localization, and their 24-nm diameter. (f–h) Immunoelectron microscopy. Vero cells infected at an MOI of 500 in the presence of CH were extracted at 1 h (f) or 4 h PI (g and h) with 0.5% TX-100 in MT buffer before fixation. They were labeled with rabbit anti– tubulin followed by protein A–9 nm gold, and Epon sections were cut parallel to the substrate. The cytoskeleton, nucleus, and nuclear pore complexes (NPC), as well as cytoplasmic capsids, were preserved excellently, whereas all membranous organelles were extracted. The microtubules (MT) are easily identified, since they are heavily decorated with antibody and protein A–gold. In rather thick sections (f), the microtubules can be traced over long distances, and many capsids (filled arrows) are localized on them. These capsids still contain their electron-dense DNA core. At later time points (g and h), most of the capsids have lost their electron-dense core (h, open arrow) and are mostly located in close proximity to the nucleus. Very often the capsids are bound directly to the outer ring of the nuclear pore complex (g, NPC). Virions bound to the plasma membrane (h, curved arrowhead) have lost the trilaminar appearance of their membrane, but apparently a lot of glycoprotein and tegument remains attached to them, causing a distinct morphology easily distinguished from cytoplasmic capsids (g). Bar, 100 nm.