Report

Endosomal Proteolysis of the Ebola Virus Glycoprotein Is Necessary for Infection

See allHide authors and affiliations

Science  10 Jun 2005:
Vol. 308, Issue 5728, pp. 1643-1645
DOI: 10.1126/science.1110656
  • 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 GP118K. VSV-GPΔMuc was incubated with enzyme for 1 hour at pH 5.5 and 37°C. Mock-treated VSV particles containing GP118K (open arrowhead) and CatL-treated VSV particles containing GP118K (18K) (solid arrowhead) were used in (C) and (D). (C) VSV particles containing GP118K 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 GP118K 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, GP118K only, or increasing amounts of GP118K (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 GP118K. 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 GP118K 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; Mr, relative molecular weight in kilodaltons (K).

  • Fig. 3.

    Endosomal cysteineproteaseinhibitors 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.

Additional Files


  • Abstract
    Full Text
    Endosomal Proteolysis of the Ebola Virus Glycoprotein Is Necessary for Infection
    Kartik Chandran, Nancy J. Sullivan, Ute Felbor, Sean P. Whelan, James M. Cunningham

    Supporting Online Material

    This file is in Adobe Acrobat PDF format. If you have not installed and configured the Adobe Acrobat Reader on your system, please see Help with Printing for instructions.

    This supplement contains:

    Materials and Methods
    SOM Text
    Figs. S1 to S5
    Table
    References

    Download supplement

Stay Connected to Science

Navigate This Article