Figure 6.

Glycoproteins undergo endocytosis in various complexes. A) HFF‐Tert cells were infected with VP26‐mTurquoise/gM‐EYFP recombinant virus for 8 h. Antibodies specific to HSV‐1 gD, gB, gH and gE were added in various combinations for the last 15 min of incubation. After fixing and permeabilization isotype‐specific secondary Alexa Fluor antibodies were used and images were acquired using a confocal microscope. Blue circles point to particles containing all labeled components. Yellow arrows point to dots containing only some components, while red, green and blue arrows point to dots containing only one type of glycoprotein. Scale bar = 2 µm. B) Quantification of images obtained as for Figure 5A. At least 100 capsids from multiple cells and several fields of view were counted for each combination of glycoproteins. Graph represents the percentage of capsids positive for each glycoprotein labeling as mean from six combinations of antibodies obtained in two independent experiments ± SEM. gM was imaged as fusion to EYFP while other glycoproteins were imaged after primary antibody uptake into cells for 15 min before fixation. C) Kinetics of gD and gE transport to intracellular capsids. Cells infected with VP26‐mTurquoise/gM‐EYFP virus were incubated for 8 h and then fixed with 4% formaldehyde. At 60, 30, 15 and 5 min before fixing gD (LP2) and gE (3063) antibodies were added to cells for uptake. At least 100 capsids from multiple cells and several fields of view were counted for each condition. Data are represented as mean from two independent experiments ± SEM.