Figure 1.
Virion binding, lipid mixing and core entry assays.
VACV Gauss-A4, a recombinant VACV with Gaussia LUC fused to a core protein, was used to measure the binding of virions at 4°C by assaying cell-associated LUC activity. For virus-cell membrane fusion, R18-loaded virions were bound to target cells at 4°C, shifted to 37°C, and the dequenching of R18 due to lipid mixing was measured by increased fluorescence. WRvFire, a recombinant VACV that expresses firefly LUC under an early promoter, was used to infect cells and newly synthesized LUC was measured. Direct visualization of virions fusing with the plasma membrane and quantification of viral cores in the cytosol were achieved by transmission electron microscopy. The times used for pretreatment, binding and entry are depicted at the bottom of the figure.
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.
Figure 3.
Effects of inhibitors on VACV-cell attachment, membrane fusion and core entry.
HeLa cells were left untreated or pre-treated for 30 min at 37°C with: (A) mßCD (10 mM), blebbistatin (75 µM), dynasore (100 µM) and bafilomycin A1 (50 nM); (B) latrunculin A (10 µM), cytochalasin D (10 µM), CK-548 (100 µM) and CK-636 (100 µM); (C) genestein (100 µM), Iressa (40 µM), and 32674 (40 µM). For cell binding (black bars), control and inhibitor-treated cells were incubated with equivalent numbers of VACV Gauss-A4 MVs at 4°C for 60 min. Unbound virions were removed by washing and cells lysed to measure cell-associated Gaussia LUC activity. For membrane fusion (white bars), control and inhibitor-treated cells were incubated with equivalent numbers of R18-loaded WRvFire particles at 4°C for 60 min. Washed cells were then incubated at 37°C for 40 min in the presence of the indicated inhibitor while R18 fluorescence was monitored. For core entry (gray bars), equivalent numbers of WRvFire MVs were adsorbed to control and inhibitor-treated cells at 4°C for 60 min. Cells were washed and incubated for 2 h at 37°C in the presence or absence of the indicated inhibitor. Cells were then lysed and firefly LUC activity in cell extracts measured. Data are represented as percent of the untreated cell control for each assay.
Figure 4.
Effects of inhibitors on entry determined by transmission electron microscopy.
Purified MVs (350 PFU per cell) were spinoculated onto inhibitor-treated HeLa monolayers at 4°C for 60 min. Virus-bound cells were then incubated for either 30 or 90 min at 37°C in the presence or absence of the indicated inhibitor, fixed and processed for transmission electron microscopy. Representative images from untreated cells at 30 min showing full fusion of virion and plasma membranes resulting in pore formation (A) and cores in the cytosol (B). White arrowheads point to cores; scale bars indicate magnification. For each infection, a total of 90 randomly-selected cell sections were visualized and the number of plasma membrane full fusion events (C) and viral cores in the cytosol (D) were determined at 30 and 90 min.
Figure 5.
Effects of deficiencies of individual virion membrane proteins on membrane fusion and virus infectivity.
In each panel, equivalent numbers of purified R18-loaded MVs were bound to HeLa cells at 4°C for 60 min and unbound virions were removed by washing. Virus-bound cells were then incubated at 37°C for 40 min and R18 fluorescence was monitored and plotted as arbitrary units. Parallel cultures were incubated for 48 h and the yield of virus was determined by plaque assay. Recombinant viruses were as follows: (A) ΔI5L, (B) IPTG-inducible A16, (C) IPTG-inducible G9, (D) IPTG-inducible G3, (E) IPTG-inducible A21, (F) IPTG-inducible O3, (G) IPTG-inducible H2, (H) IPTG-inducible F9, (I) IPTG-inducible A28, (J) IPTG-inducible L1, and (K) IPTG-inducible L5. For panels B-K, the plus and minus signs in the upper left signifies the virus was grown in the presence or absence of IPTG, respectively. For panel A, the plus and minus refer to wild type virus and a deletion mutant, respectively. As a negative control, 56°C heat-inactivated I5+ virions (panel A) were assayed for hemifusion and infectivity (<105 PFU/ml; data not shown).
Figure 6.
Entry of virions lacking H2 or A28 protein determined by transmission electron microscopy.
Purified MVs (350 PFU per cell or equivalent number of particles) possessing (+) or lacking (-) H2 or A28 protein as indicated were spinoculated onto pre-chilled HeLa cell monolayers at 4°C for 60 min. Virus-bound cells were then incubated for either 30, 60 or 180 min at 37°C, fixed and processed for electron microscopy. For each infection, a total of 90 randomly-selected cell sections were inspected and the number of full fusion events (A and C) and free viral cores in the cytosol (B and D) were determined as described in the legend to Figure 4.
Figure 7.
Effects of anti-L1 MAb on virus-cell membrane fusion, viral core entry and virus infectivity.
Equivalent numbers of R18-loaded virions (WRvFire) were incubated with or without 100 µg/ml of anti-L1 mouse MAb or control anti-HA mouse MAb for 30 min at room temperature. Virions were then assayed for their ability to mediate virus-cell membrane fusion by R18 dequenching (A) or core entry by LUC expression (B), at either 37°C or 4°C. Infectivity (C) was assayed by adsorbing each virus sample at 37°C to BS-C-1 monolayers for 60 min and enumerating plaque formation 48 h later. Data are represented as percent of the no MAb control at 37°C for each assay.
Figure 8.
Attempt to trans-complement entry of virions lacking A28.
(A) Equivalent numbers of purified A28+ or A28− MVs expressing firefly LUC (WRvFire) were mixed with varying amounts of purified, wild type (wt) MVs as indicated. Virions were adsorbed to HeLa cell monolayers at 4°C for 60 min. Cells were washed and placed at 37°C for 2 h to allow virus entry. Cells were then lysed and firefly LUC activity measured. MOI (multiplicity of infection; PFUs per cell) of wt VACV is indicated. (B) Equivalent numbers of purified A28+ or A28− MVs expressing firefly LUC were mixed with varying amounts of bovine serum albumin (BSA) or soluble A28 protein as indicated (ng/ml). Virions were adsorbed to HeLa cell monolayers at 4°C for 60 min. Cells were washed and placed at 37°C for 2 h to allow for virus entry. Cells were then lysed and LUC activity measured.
Figure 9.
Summary of effects of inhibitors on VACV entry.
Cell binding, virus-cell membrane fusion, and viral core entry were assessed as described in the text and as depicted in Figures 1 and 3. The inhibitors are grouped according to their best-characterized effects but may also perturb cells in other ways.