Internalized HCMV virions partially co localize with EEA1 and not with LAMP2.

<p>Confocal imaging was performed on immobilized immature MDDCs incubated for two hours on ice with the VHL/E strain (MOI=2) and subsequently cultured at 37°C for three hours (A) and 24 hours (B). Cells were then fixed/permeabilized and immunostained as indicated above each column with anti-EEA1 or -LAMP2 antibodies (green) and with an anti-HCMV gB (red). Images were obtained on a 510 LSM Meta (Zeiss, Germany). Single confocal planes are presented. In merged panels, green and red staining intensities were directly analyzed along a virtual section indicated by a thin white line with the ImageJ RGB profiler plugin. The results are displayed in graphs on the right (X-axis=distance in pixels; Y-axis=fluorescence intensity). White arrowheads indicate small CMV aggregates or isolated particles. C) Between 7 and 10 x 10⁷ HCMV-infected MDDCs (VHL/E; MOI=10) were subjected to three consecutive subcellular fractionation steps on sucrose and Percoll gradients to harvest early and late endosome-enriched and lysosome-enriched fractions. Here early endosomes (EE) and late endosomes (LE) enriched fractions were first pooled (EE+LE) and the EE+LE and the lysosomes (Ly) enriched fractions were concentrated before being used in a western blot analysis using anti-EEA1, LAMP-2, HCMV gB and MCP antibodies. Recombinant HCMV gB (Biomérieux, France) was used as a positive control. The molecular weight of the gB is 160 kDa and 153 kDa for the MCP. PN means post nuclear fraction.