The Membrane Fusion Step of Vaccinia Virus Entry Is Cooperatively Mediated by Multiple Viral Proteins and Host Cell Components
Figure 2
Membrane fusion and core entry.
(A) Equivalent numbers of purified MVs were untreated or loaded with R18 for 20 min at room temperature. Unbound R18 was removed by pelleting and washing the virus. Control and R18-labeled virions were resuspended and serial dilutions made to assay virus infectivity (PFU/ml) by plaque assay. The results of five independent experiments with error bars are plotted. (B) Purified R18-loaded MV particles were bound to HeLa cells at 4°C for 60 min. The cells were then incubated at 4°C, 20°C, or 37°C for 40 min while R18 fluorescence was monitored and quantified as arbitrary fluorescent units. (C) R18-loaded MVs (recombinant WRvFire) were incubated with HeLa cells at 4°C to permit binding. Washed cells were then placed in a cuvette containing pre-warmed media at 37°C and fluorescence was monitored over time (black line; left y-axis). In parallel, unlabeled MVs were bound to cells in the cold and then shifted to 37°C. Cell lysates were prepared at indicated times and assayed for LUC activity (gray line; right y-axis). (D) An equivalent number of purified R18-loaded MVs were bound to HeLa cells in the cold for 60 min. Virus-bound cells were then placed at 37°C in a pre-warmed cuvette containing media adjusted to either pH 7.4 or 5.0 while R18 fluorescence was monitored. After 3 min, cell media was adjusted back to neutral \ and R18 fluorescence monitoring continued.