Figure 1.
CMV internalization into large uncoated vesicles in MDDCs is an active process that requires actin cytoskeleton polymerization.
A) Transmission electron microscopy (TEM) picture is shown of day 6 immature MDDCs incubated with HCMV (VHL/E; MOI=10) for two hours then extensively washed with PBS. A scale bar is indicated (magnification x7,500). N=nucleus. B) Close-ups of different pictures are represented. Black arrows indicate HCMV virions. A scale bar is indicated for each picture. Cells were prepared as described in 1A. C) Kinetic analysis of HCMV internalization in MDDCs. The data represent the quantification of the mean number of particles per cell from the TEM pictures (n≥20 independent cells) of viral particles (VHL/E; MOI=10) immobilized at the plasma membrane (OUT, white bars) or internalized into vacuoles (IN, black bars) as function of time (30 minutes, two, six and 24 hours incubations). Cells were extensively washed with PBS after incubation with HCMV. These results are representative of two distinct experiments. D) MDDCs were pre-incubated with drugs (cytochalasin, cytD: 50-5-0.5 µM) or vehicle (DMSO: 1/100) prior to be infected with HCMV (MOI=2) for two hours. Cells were extensively washed then subcultured for 48 hours. The cells were coated onto poly-L-lysine-coated slides, fixed and permeabilized with acetone and were stained with mAbs against anti-IE and –E antigens (Argene Biosoft, France). Four distinct fields were digitalized and analyzed with ImageJ software to determine the percentage of I.E.A./E.A.+ MDDCs. n=6 independent experiments with eight different donors in total.
Figure 2.
Internalized HCMV virions partially co localize with EEA1 and not with LAMP2.
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.
Figure 3.
Uncoating of internalized particles results in naked capsids in cytoplasm of infected MDDCs.
Transmission electron microscopy (TEM) picture of a Day 6 immature MDDC incubated with HCMV (VHL/E; MOI=10) for six hours then extensively washed with PBS. Naked capsids in the cytoplasm are marked by arrowheads. A scale bar is indicated in the lower left part of the micrograph (magnification x200,000). A white dashed line indicates the nuclear envelope. N= nucleus; C= cytoplasm.
Figure 4.
HCMV internalization into MDDCs is impaired by macropinocytosis inhibitors.
A) MDDCs were pre-incubated with drugs blocking macropinocytosis (Gö6983∶ 13, 7.3, 3.75 nM and amiloride, Ami: 100, 20 µM) or clathrin-mediated endocytosis (chlorpromazine, CPZ: 30, 6, 1.2 µM) or with vehicle (DMSO, 1/100) prior to be infected with HCMV (MOI=2) for two hours. Cells were extensively washed then subcultured for 48 hours. The cells were then prepared and analyzed as described in the legend for Figure 1D. For Gö6983, n=2 independent experiments with 4 different donors in total, for Ami and CPZ n=5 independent experiments with 7 different donors in total. ns: not significant (p=0,0733) B) TEM picture of (500µM) amiloride-treated MDDCs incubated with HCMV (VHL/E; MOI=10). Black arrows indicate HCMV virions. C) Quantification of infectious HCMV particles by TEM immobilized at the plasma membrane (OUT, white bars) or internalized into vacuoles (IN, black bars) of (500 µM) amiloride-treated MDDCs incubated for two hours with VHL/E (MOI=10) (n=6 cells per conditions). The results are displayed as the median values of the percentage (±SD) of plasma membrane-associated and internalized HCMV particles.
Figure 5.
Cholesterol depletion is detrimental to the HCMV entry into MDDCs.
A) Cells were pre-incubated with filipin (7.66, 1.5, 0.3 µM), nystatin (21.2, 4.3, 0.85 µM) or methyl-β-cyclodextrin (MβCD; 5, 1, 0.2 mM) or with vehicle (DMSO, 1/100) and were processed as described in the legend for Figure 1D. For nystatin, n= 2 independent experiments with 2 different donors in total; for Filipin and MβCD n=4 independent experiments with 6 different donors in total. ns: not significant (p=0,0535).
Figure 6.
Endosomal pH neutralization does not inhibit HCMV internalization or MDDC infection.
A) Cells were pre-incubated with NH4Cl-containing buffer (50, 5, 0.5 mM) or bafilomycin A1 (320, 32, 3.2 nM) and compared to the vehicle (DMSO; 1/100). The cells were processed as described in the legend for Figure 1D. For NH4Cl, n= 3 independent experiments with 3 different donors in total, for bafilomycin A1 n= 6 independent experiments with 8 different donors in total. ns: not significant (p=0,0939). B) TEM picture of (50 nM) bafilomycin A1 treated MDDCs incubated with HCMV (VHL/E; MOI=10). Black arrows indicate HCMV virions. C) Quantification of infectious HCMV particles by TEM immobilized at the plasma membrane (OUT, white bars) or internalized into vacuoles (IN, black bars) of (50 nM) BafA1-treated MDDCs incubated for two hours with VHL/E (MOI=10) (n=5-6 cells per condition). The results are displayed as the median values of the percentage (±SD) of plasma membrane-associated and internalized HCMV particles. These results are representative of two distinct experiments.
Figure 7.
MDDCs can mediate HCMV trans-infection through both plasma membrane-associated virions and the release of internalized virions.
MDDCs, MDMs or monocytes from the same blood donor were obtained as described in the Materials and Methods section. Cells were pretreated with 40 nM BafA1 (black bars) or the vehicle (DMSO) (white bars) prior to incubation with the VHL/E HCMV strain for two hours (MOI=2). Cells were then extensively washed with a low-pH buffer (glycine 0.2M, pH=2.8) or with PBS alone as indicated above each panel of the figure and were subcultured in close contact with HFFs. After 48 hours, fibroblasts were processed as previously described [14] to evaluate the infection rate due to trans-infection by HCMV-loaded cells (absolute number of IE/E antigen-positive fibroblasts among 105 total cells). n= 4 independent experiments with four different donors in total.