FIG 5.

Makona GP mutations R574H and I584L reduce the dependency on cathepsin-B activity for VSV-MAK-GP infection of Vero E6 cells. (A) Immunofluorescence labeling of virally infected cells pretreated with the cathepsin-B inhibitor CA-074. The top row shows representative images of cells pretreated with 20 μM CA-074 and infected with the indicated virus. The second row shows cells pretreated with 80 μM CA-074 and infected with the indicated virus. Cells are labeled with 4′,6′-diamidino-2-phenylindole (blue) and antibodies against VSV matrix protein (green) and EBOV GP (red). Images were captured using a 10× objective. (B) Western blots measuring VSV matrix (M) protein accumulation in infected cell lysates at the indicated CA-074 concentrations. Relative levels of M protein accumulation (compared to the level with no treatment) are below each lane. Heat shock protein 90 (Hsp90) was used to normalize M protein values. (C) Reduction of virus growth titers by CA-074 inhibitor treatment. Values represent the mean and range of two independent experiments. (D) Representative experiment measuring growth titers in the absence and presence of E64D. Infection was performed at an MOI of 4, and growth was analyzed at 16 hpi.