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.2012 Mar 1;424(1):3-10.
doi: 10.1016/j.virol.2011.11.031. Epub 2012 Jan 4.

Cathepsins B and L activate Ebola but not Marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of TMPRSS2 expression

Affiliations

Cathepsins B and L activate Ebola but not Marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of TMPRSS2 expression

Kerstin Gnirss et al. Virology..

Abstract

Ebola (EBOV) and Marburg virus (MARV) cause severe hemorrhagic fever. The host cell proteases cathepsin B and L activate the Zaire ebolavirus glycoprotein (GP) for cellular entry and constitute potential targets for antiviral intervention. However, it is unclear if different EBOV species and MARV equally depend on cathepsin B/L activity for infection of cell lines and macrophages, important viral target cells. Here, we show that cathepsin B/L inhibitors markedly reduce 293T cell infection driven by the GPs of all EBOV species, independent of the type II transmembrane serine protease TMPRSS2, which cleaved but failed to activate EBOV-GPs. Similarly, a cathepsin B/L inhibitor blocked macrophage infection mediated by different EBOV-GPs. In contrast, MARV-GP-driven entry exhibited little dependence on cathepsin B/L activity. Still, MARV-GP-mediated entry was efficiently blocked by leupeptin. These results suggest that cathepsins B/L promote entry of EBOV while MARV might employ so far unidentified proteases for GP activation.

Copyright © 2011 Elsevier Inc. All rights reserved.

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Figures

Fig. 1
Fig. 1
Inhibition of cathepsin B, L and S activity by protease inhibitors. 293T cells were incubated with the indicated inhibitors and cathepsin activity was determined 8 h later using commercially available kits. The cathepsin B, L and S activity in cells treated with protease inhibitors is shown relative to that measured upon treatment of cells with PBS, which was set as 100%. Results for cathepsin B and L activity represent the average of three independent experiments. Error bars indicate standard error of the mean. The results of a representative experiment performed in duplicates are shown for cathepsin S and were confirmed in a separate experiment. Error bars indicate standard deviation (SD). CatB, cathepsin B; CatL, cathepsin L; CatS, cathepsin S.
Fig. 2
Fig. 2
Entry driven by the glycoproteins of the four Ebola virus species depends on cathepsin activity. A. 293T target cells were preincubated with the indicated concentrations of cathepsin B and L inhibitors (MDL28170, E64c), a serine protease inhibitor (AEBSF) and an inhibitor blocking cysteine and serine proteases (leupeptin). Subsequently, cells were infected with pseudotypes bearing the indicated GPs, normalized for equal infectivity. Luciferase activity in cell lysates was measured at 72 h post infection. B. The experiment was carried out as described in (A), using two different concentrations of cathepsin L inhibitor III and cathepsin B inhibitor CA074. C. The experiment was carried out as described in (A) but cells were incubated with the cathepsin B inhibitor CA074Me. The results of representative experiments performed in triplicates are shown in A–C; error bars indicate SD. Similar results were obtained in three separate experiments.
Fig. 3
Fig. 3
Cellular entry mediated by Marburg virus glycoprotein is largely cathepsin-independent. 293T target cells were preincubated with the indicated concentrations of protease inhibitors leupeptin, AEBSF and CA074Me. Subsequently, cells were infected with pseudotypes bearing the indicated GPs, normalized for equal infectivity and luciferase activity in cell lysates was measured at 72 h post infection. The results ± SD of a representative experiment performed in triplicates are shown and were confirmed in at least two separate experiments.
Fig. 4
Fig. 4
Proteolytic processing of the Ebola virus glycoprotein by TMPRSS2 and hepsin. Lysates of 293T cells coexpressing the indicated proteases and EBOV-GPs or empty vector (pCAGGS) were analyzed for GP cleavage by Western blot using a V5-tag specific antibody. Detection of ß-actin served as a loading control. The results are representative for at least three independent experiments.
Fig. 5
Fig. 5
TMPRSS2 and hepsin do not facilitate cathepsin-independent, Ebola virus glycoprotein-dependent host cell entry. A. Pseudotypes were generated in 293T cells expressing the indicated GPs in combination with the indicated proteases. Released pseudoparticles were normalized for equal content of p24-antigen and used to infect mock-transfected 293T cells pretreated with PBS or PBS containing 1 μM CA074Me. At 8 h post infection, medium was replaced, and luciferase activity was measured in cell lysates at 72 h post infection. B. Pseudotypes bearing the indicated GPs were normalized for comparable infectivity prior to infection of PBS or CA074Me treated 293T cells expressing the indicated proteases. C. Infectivity-normalized SARS-S pseudotypes were used to infect PBS or MDL 28170 treated 293T-ACE2 target cells expressing TMPRSS2 or no protease. The results of representative experiments performed in triplicates are shown in A–C; error bars indicate SD. Similar results were obtained in at least two separate experiments. For testing of statistical significance, a two-tailed student'st-test was employed.
Fig. 6
Fig. 6
Macrophage infection driven byZaire andReston ebolavirus as well as Marburg virus glycoproteins is cathepsin-dependent. A. Macrophages were preincubated with 4 μM CA074Me before infection with pseudotypes bearing the indicated GPs, normalized for equal infectivity. Luciferase activity in cell lysates was measured at 72 h post infection. B. Macrophages were preincubated with the indicated concentrations of leupeptin prior to infection with ZEBOV-GP or MARV-GP pseudoparticles. C. The experiment was carried out as described in (B) but the cells were treated with the indicated concentrations CA074Me prior to infection. The results of representative experiments performed in triplicates are shown in A–C; error bars indicate SD. Similar results were obtained in two independent experiments.
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