Figure 1.
The release of VACV cores and initiation of viral gene expression is delayed in DCs compared to BS-C-1 cells.
(A) MDDCs or BS-C-1 cells were spinoculated with MV-GFP (MOI 10) at 4°C then incubated at 37°C for 0, 30 or 60 min to allow virus entry. Cells were washed, fixed in 4% PFA and permeabilised with 0.1% Triton X-100 for 10 min at room temperature. MV was detected by confocal microscopy by GFP fluorescence (green) and virus cores were detected using an anti-GFP Ab and GAR-633 (red). Scale bars represent 10 µm. (B) MDDCs or (C) BS-C-1 cells were spinoculated with MV-GFP (MOI 3) or EV-GFP (MOI 1) at 4°C, washed and then incubated at 37°C for up to 4 h to allow virus entry. At various time points, cells were lysed and the expression of immediate early viral transcripts for two genes, E3L and B2R, was analysed by qPCR. Viral gene expression was normalised to GAPDH.
Figure 2.
VACV entry in MDDCs is dependent on cellular factors consistent with an endocytic uptake mechanism.
The effect of depletion of ATP (A–C) or intracellular calcium (D) and sequestration of actin (E) on the entry of MV (white bars) and EV (shaded bars) was assayed by FACS. MDDCs were pre-treated with inhibitors antimycin A (AntiA), EGTA/AM, cytochalasin D (CytD) and latrunculin A (Lat A) at the concentrations indicated for 60 min at 37°C prior to spinoculation with MV-GFP (MOI 20) or EV-GFP (MOI 0.5–5) at 4°C. Cells were shifted to 37°C for 30 min to allow virus entry then washed and the remaining surface-bound virus stripped by trypsinisation. The percentage of GFP-positive cells was analysed by flow cytometry and representative data is shown for AntiA in (A) dot plot and (B) histogram form with uninfected (shaded), untreated, infected control (black line) and 20 µM AntiA treatment (red line) overlaid. The EV MOI in this experiment was 1.2. (C–E) Data from three independent experiments was normalised as a percentage of the untreated control. * p<0.05. ** p<0.01. *** p<0.001.
Figure 3.
VACV does not bind to C-type lectin receptors (CLRs) expressed on MDDCs.
MDDCs were pre-treated with the CLR inhibitors mannan, a neutralising anti-DC-SIGN mAb (AZN-D1), D-mannose (D-man) and EGTA or media alone at 4°C for 30 min then spinoculated at 4°C with MV-GFP (MOI 50) or EV-GFP (MOI 5–10). Cells were then washed and fixed in 4% PFA for flow cytometry or lysed for qPCR. (A) The percentage of GFP-positive cells was analysed by flow cytometry and normalised as a percentage of the untreated control. (B) Virally-encoded GFP DNA copy numbers were quantitated by qPCR and normalised to GAPDH and expressed as a percentage of the untreated control. (C) Confocal microscopy images of MV or EV bound to MDDCs at 4°C in the absence or presence of mannan. (D) As a positive control, pre-treated MDDCs were incubated with biotinylated HIV-1 gp120 at 4°C which was detected with streptavidin-PE and measured by flow cytometry (white bars). Alternatively, cells were infected with HIV-1 (MOI 10) in the presence of inhibitors for 72 h then integrated viral transcripts were measured by qPCR (shaded bars). (E, F) Colocalisation of MV or EV on the surface of MDDCs with DC-SIGN and MR was assessed by confocal microscopy. DC-SIGN and MR mAbs were detected with GAM-546. Images are maximum projections of z-series and the scale bars represent 5 µm. Inserts are enlargements of the boxed areas in the main images.
Figure 4.
VACV entry in MDDCs does not require dynamin.
(A) MDDCs were pretreated with dynamin II inhibitors Bis-T-23 (100 µM) and Dynasore (Dyngo7a; 200 µM) or media alone at 37°C for 30 min then incubated with 5 µg/mL transferrin-Alexafluor 647 (Tf-647) for 10 min at 37°C. Cells were washed, and then any remaining surface-bound transferrin was stripped by a low pH wash (pH 2.8). Uptake of transferrin was measured by flow cytometry and the mean fluorescence intensity (MFI) was expressed as a percentage of the untreated control MFI. (B) Alternatively, MDDCs were pre-treated with the dynamin II inhibitors then infected with MV-GFP or EV-GFP and analysed, as in Fig. 2. (C) MDDCs were spinoculated with MV-GFP or EV-GFP at 4°C then incubated in pre-warmed media with 200 µg/mL Tf-647 at 37°C for 1-45 min. Cells were then washed and trypsinised to remove residual surface bound virus and fixed in 4% PFA for confocal microscopy. Representative maximum projections of z-series taken at 15 min are shown. Scale bars represent 5 µm.
Figure 5.
VACV entry in MDDCs is dependent on cholesterol.
(A) Cells were treated with cholesterol inhibitors methyl-β-cyclodextrin (mβCD) or filipin III (Fil) for 60 min at 37°C then infected and analysed as in Fig. 2. (B) Alternatively, virus binding at 4°C was analysed immediately after spinoculation of mβCD-treated cells with MV-GFP or (C) mβCD was added at the times indicated from 60 min prior to, up to 60 min post MV binding and entry. (D) Following treatment with mβCD for 60 min, cells were washed and incubated for a further 60 min at 37°C in the absence or presence of 0.1 mM soluble cholesterol to replenish cellular cholesterol, followed by MV binding and entry as in (A). ** p<0.01. *** p<0.001.
Figure 6.
VACV is taken up by macropinocytosis in MDDCs.
The effect of (A) macropinocytosis inhibitors rottlerin, DMA and EIPA or (C) kinase inhibitors wortmannin (wort) or GF109203X on the entry of MV-GFP and EV-GFP was assayed by FACS. MDDCs were pretreated with inhibitors at the concentrations indicated for 30 min at 37°C then infected and assayed as in Fig. 2. * p<0.05, ** p<0.01, *** p<0.001. (B) The kinetics of rottlerin inhibition were investigated. Cells were treated with 10 µM rottlerin at the times indicated relative to infection with MV-GFP or EV-GFP, then assayed as in Fig. 2. (D) MV and EV colocalise with dextran during uptake by MDDCs. Cells were spinoculated with MV-GFP or EV-GFP at 4°C then incubated in pre-warmed media with 0.5 mg/mL dextran-Texas Red at 37°C for up to 30 min. Cells were then washed and trypsinised to remove residual surface bound virus and fixed in 4% PFA for confocal microscopy. Representative maximum projections of z-series taken at 15 min are shown. Scale bars represent 5 µm. Inserts are enlargements of the boxed areas in the main images.
Figure 7.
VACV does not colocalise with endolysosomal markers and is not dependent on low pH to enter the cytoplasm.
MDDCs were spinoculated with MV-GFP (MOI 10) or EV-GFP (MOI 2–5) at 4°C. After washing to remove any unbound virions, cells were incubated at 37°C to allow virus entry for 0-120 min. Cells were then fixed and permeabilised with methanol:acetone (1∶1 v/v) for 2 min at -20°C and stained for (A) early endosomes using EEA1 mAb or (B) lysosomes with Lamp2 mAb, and GAM-546. Maximum projections of z-series for EEA1 at 30 min and Lamp2 at 60 min are shown and are representative of three different donors. Scale bars represent 5 µm. (C) MDDCs were spinoculated MV-GFP (MOI 3) or EV-GFP (MOI 1), washed and incubated at 37°C in the presence or absence of 250 nM bafilomycin A (BafA) for up to 3 h. At various time points, cells were lysed and the expression of the immediate early viral gene E3L was analysed by qPCR. Viral gene expression was normalised to GAPDH.
Figure 8.
Blocking components of the macropinocytosis pathway blocks viral gene transcription.
MDDCs were pre-treated with the LatA (10 µM), mβCD (2.5 mM), EGTA/AM (250 mM), rottlerin (10 µM) and wortmannin (200 nM) for 30 min prior to spinoculation with (A) MV-GFP (MOI 3) or (B) EV-GFP (MOI 1) at 4°C. Cells were washed and incubated at 37°C to allow virus entry for 2 h then lysed for analysis of immediate early viral transcripts E3L (shaded bars) and B2R (white bars) by qPCR. The expression of viral transcripts was normalised to expression of GAPDH and is expressed as a percentage of the untreated control from two independent donors with standard error bars.