A Differential Role for Macropinocytosis in Mediating Entry of the Two Forms of Vaccinia Virus into Dendritic Cells
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.