Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection

Abstract

Ebola virus (EboV) causes rapidly fatal hemorrhagic fever in humans and there is currently no effective treatment. We found that the infection of African green monkey kidney (Vero) cells by vesicular stomatitis viruses bearing the EboV glycoprotein (GP) requires the activity of endosomal cysteine proteases. Using selective protease inhibitors and protease-deficient cell lines, we identified an essential role for cathepsin B (CatB) and an accessory role for cathepsin L (CatL) in EboV GP-dependent entry. Biochemical studies demonstrate that CatB and CatL mediate entry by carrying out proteolysis of the EboV GP subunit GP1 and support a multistep mechanism that explains the relative contributions of these enzymes to infection. CatB and CatB/CatL inhibitors diminish the multiplication of infectious EboV-Zaire in cultured cells and may merit investigation as anti-EboV drugs.

Fig. 1

Endosomal cysteine proteases CatB and CatL are host factors for VSV-GPΔMuc infection. (A and B) (Top) Effect of CatB-selective inhibitor CA074 (A) and CatL/CatB inhibitor FYdmk (B) on infectivities of VSV-G and VSV-GPΔMuc in Vero cells. (Bottom) CatB (A) and CatL (B) enzymatic activities in CA074- and FYdmk-treated Vero cells. Infectivities [infectious units (iu)/ml] are relative to the infectivity of the same virus in dimethyl sulfoxide (DMSO)–treated Vero cells (set to 100%) (25). (C) Wild-type (WT) and CatB-deficient (CatB^−/− CatL^+/+) MEFs were not transfected (none) or transfected with plasmid DNAs encoding β-galactosidase (β-gal), CatB (CatB), or CatL (CatL). After 24 hours, cells were exposed to VSV-GPΔMuc (~1 iu per cell), and the percentage of infected cells was determined 24 hours later by flow cytometry (25). (D) Capacity of VSV-GPΔMuc to infect CatB/CatL-deficient (CatB^−/−CatL^−/−) MEFs was determined as in (C). Infectivities from two replicates are shown in (A) and (B) and are representative of four independent experiments. Error bars, SD from at least three replicates.

Fig. 2

Endosomal cysteine proteases act directly on EboV GPΔMuc to mediate infection of Vero cells. (A and B) Purified CatB (A) and CatL (B) cleave GPΔMuc to GP1_18K. VSV-GPΔMuc was incubated with enzyme for 1 hour at pH 5.5 and 37°C. Mock-treated VSV particles containing GP1 (open arrowhead) and CatL-treated VSV particles containing GP1_18K (18K) (solid arrowhead) were used in (C) and (D). (C) VSV particles containing GP1_18K are highly infectious and fully dependent on cellular CatB activity. Cells were not treated (solid bars) or pretreated with E-64d (300 μM) (open bars) to inactivate CatB. Approximate CatB activity (B%) in these cells is indicated above the bars. (D) VSV particles containing GP1_18K bypass a block to GP1 cleavage within cells. Cells were treated with inhibitors to obtain the approximate levels of cellular CatB (B%) and CatL (L%) activity shown (also see Fig. 1 and table S1). Open bars, 300 μM E-64d; solid black bars, 10 μM FYdmk; solid gray bars, 40 μM CA074; striped bars, 1 μM FYdmk. Cells were then infected with VSV-GPΔMuc containing GP1 only, GP1_18K only, or increasing amounts of GP1_18K (wedge) (generated by incubation with increasing concentrations of CatL for 1 hour at pH 5.5 and 37°C). (E) Purified CatB efficiently digests CatL-derived GP1_18K. VSV-GPΔMuc was incubated with the indicated enzymes for 1 hour at pH 5.5 and 37°C {CatB, 40 μg/ml; CatL, 20 μg/ml; CatB and CatL together (CatB + CatL); or CatL followed by CatB [CatL→CatB (30 min each)]}. (F) Digestion of CatL-derived GP1_18K by CatB inactivates VSV-GPΔMuc. Infectivities of VSV particles from (E) are shown. Averages of two replicates are shown in (C) and (D) and are representative of three independent experiments. Error bars, SD from three replicates; M_r, relative molecular weight in kilodaltons (K).

Fig. 3

Endosomal cysteine protease inhibitors diminish EboV-Zaire multiplication in Vero cells. (A) Yields of infectious EboV-Zaire released from cells treated with DMSO (no drug), 300 μM E-64d (to inactivate endosomal cysteine proteases), or 80 μM CA074 (to selectively inactivate CatB) for 4 hours are shown. Growth medium containing inhibitors was removed from cells at the indicated time (arrowhead) and replaced with fresh medium lacking inhibitors. pfu/ml, plaque-forming units per milliliter; h p.i., hours post infection. Averages from two replicates are shown. (B) GP expression in EboV-infected cells from (A). β-actin was used as a loading control.