JOURNAL OF VIROLOGY, Mar. 2010, p. 2972–2982
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 6
Biochemical and Structural Characterization of Cathepsin L-Processed
Ebola Virus Glycoprotein: Implications for Viral
Entry and Immunogenicity?
Chantelle L. Hood,† Jonathan Abraham,‡ Jeffrey C. Boyington, Kwanyee Leung,
Peter D. Kwong, and Gary J. Nabel*
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
Room 4502, Building 40, MSC-3005, 40 Convent Drive, Bethesda, Maryland 20892-3005
Received 10 October 2009/Accepted 22 December 2009
Ebola virus (EBOV) cellular attachment and entry is initiated by the envelope glycoprotein (GP) on the
virion surface. Entry of this virus is pH dependent and associated with the cleavage of GP by proteases,
including cathepsin L (CatL) and/or CatB, in the endosome or cell membrane. Here, we characterize the
product of CatL cleavage of Zaire EBOV GP (ZEBOV-GP) and evaluate its relevance to entry. A stabilized
recombinant form of the EBOV GP trimer was generated using a trimerization domain linked to a cleavable
histidine tag. This trimer was purified to homogeneity and cleaved with CatL. Characterization of the trimeric
product by N-terminal sequencing and mass spectrometry revealed three cleavage fragments, with masses of
23, 19, and 4 kDa. Structure-assisted modeling of the cathepsin L-cleaved ZEBOV-GP revealed that cleavage
removes a glycosylated glycan cap and mucin-like domain (MUC domain) and exposes the conserved core
residues implicated in receptor binding. The CatL-cleaved ZEBOV-GP intermediate bound with high affinity
to a neutralizing antibody, KZ52, and also elicited neutralizing antibodies, supporting the notion that the
processed intermediate is required for viral entry. Together, these data suggest that CatL cleavage of EBOV
GP exposes its receptor-binding domain, thereby facilitating access to a putative cellular receptor in steps that
lead to membrane fusion.
Ebola virus (EBOV) is a member of the Filoviridae family
and causes severe hemorrhagic fever in humans and nonhu-
man primates, with case fatality rates of up to 90%. Virus entry
and attachment is mediated by a single envelope glycoprotein
(GP) as a class I fusion protein, which is proteolytically pro-
cessed during maturation into two subunits, GP1 and GP2. The
GP1 N terminus contains a putative receptor-binding domain
(RBD) (2, 9, 11, 12), and the GP2 C terminus contains a fusion
peptide, two heptad-repeat regions, and a transmembrane do-
main. GP1 and GP2 are linked by a disulfide bond (Cys53-
Cys609) and form trimers of heterodimers on the surface of
virions. EBOV GP is also extensively glycosylated, especially
within a region of GP1 termed the mucin-like domain (MUC
domain), which contains multiple N- and O-linked glycans. We
and others have previously shown the MUC domain of GP1 to
be cytotoxic and to induce cell rounding (17, 21), and deletion
of this region increases pseudovirus infectivity compared to
that of full-length GP (11). The MUC domain, however, is also
known to enhance cell binding through the human macro-
phage C-type lectin specific for galactose and N-acetylglu-
cosamine (hMGL) (18), suggesting that glycans in this domain
may be involved in the initial cellular attachment. Several other
studies have identified factors that enhance cell binding and/or
infectivity, including folate receptor ? (4), ? integrins (19), C-type
lectins DC-SIGN and L-SIGN (1), and Tyro3 family members
(16). However, the critical cellular receptor(s) thought to interact
directly with the GP1 RBD have yet to be identified.
Following virus uptake into host cells, which is presumed to
occur via receptor-mediated endocytosis (13), the virion is
transported to acidified endosomes where GP is exposed to a
low pH and enzymatic processing. EBOV entry is pH depen-
dent (19); however, unlike influenza virus, for which a low pH
alone induces the conformational changes that lead to mem-
brane fusion (20), recent studies indicate that proteolysis by
endosomal cathepsin L (CatL) and CatB (active only at pH 5
to 6) is a dependent step for EBOV entry (5, 14). Although the
intermediate EBOV GP generated by CatL cleavage is known
to have increased binding and infectivity to target cells (7),
little else is known about the cleavage product, specifically
where the proteolytic sites are within GP and whether the
cleaved product is immunogenic. Recently, Dube and col-
leagues have proposed a model for CatL cleavage based on
thermolysin cleavage (6). However, thermolysin is nonphysi-
ological in this setting and is a member of the metalloenzyme-
protease family, whereas CatL is a member of the cysteine-
protease family and essential for EBOV entry. In this study, we
have characterized the physiological CatL cleavage of the
Zaire EBOV GP (ZEBOV-GP) trimer and explored the effect
of cleavage on the immunological properties of the GP trimer.
To generate this intermediate, we expressed and purified a
recombinant form of the Ebola GP trimer ectodomain that had
* Corresponding author. Mailing address: Vaccine Research Center,
National Institute of Allergy and Infectious Diseases, National Insti-
tutes of Health, Room 4502, Building 40, MSC-3005, 40 Convent
Drive, Bethesda, MD 20892-3005. Phone: (301) 496-1852. Fax: (301)
480-0274. E-mail: firstname.lastname@example.org.
† Present address: St. Vincent’s Centre for Applied Medical Re-
search (AMR), Lowy Packer Building—St. Vincent’s Research Pre-
cinct, 405 Liverpool Street, Darlinghurst, NSW, Australia 2010.
‡ Present address: Laboratory of Molecular Medicine, Children’s
Hospital, Harvard Medical School, Boston, MA.
?Published ahead of print on 6 January 2010.
been stabilized with a trimerization motif derived from T4
fibritin (foldon) and purified to homogeneity. The recombi-
nant protein was cleaved with CatL, and the stable cleavage
intermediate was characterized biochemically and immunolog-
ically. We identified several sites of CatL cleavage within the
ZEBOV-GP ectodomain which are different than those ob-
served with thermolysin. The cleaved intermediate product
retained binding to the EBOV-neutralizing antibody KZ52 and
elicited EBOV-neutralizing antibodies in vaccinated mice. Our
data, in conjunction with the recently determined structure of
the ZEBOV-GP ectodomain (10), shed light on the critical
role of CatL processing in GP structure and function.
MATERIALS AND METHODS
Cell lines and plasmids. Recombinant baculovirus vectors containing
ZEBOV-GP (1976 Mayinga; GenBank accession no. AAC54887) or Sudan
EBOV GP (SEBOV-GP, 1976 Boniface; GenBank accession no. AAB37096)
were generated by cloning the appropriate EBOV GP into a baculovirus back-
bone vector (BD Pharmingen, San Diego, CA). A foldon trimerization sequence
from bacteriophage T4 fibritin was cloned in place of the transmembrane region
at the C terminus, followed by a thrombin cleavage site and His tag. The purified
protein contained the following additional C-terminal residues: GSGYIPEAPR
DGQAYVRKDGEWVLLSTFLGGSLVPRGSPHHHHHH, with the foldon trim-
erization domain shown in italics, the thrombin cleavage site underlined, and the
His tag in boldface. Hi5 insect cells were maintained in Express Five serum-free
media (Invitrogen, Carlsbad, CA). Pseudotyped EBOV GP plasmids included
Zaire GP (GenBank accession no. AAC54887), Sudan GP (GenBank accession
no. AAB37096), Ivory Coast GP (GenBank accession no. ACI28632), Reston GP
(GenBank accession no. AAC54889), and from the newest EBOV strain, Bun-
dibugyo GP (BEBOV-GP; GenBank accession no. ACI28624), which were syn-
thesized using human-preferred codons as described previously (8) by GeneArt
Baculovirus production. GPs were produced by cotransfection of baculovirus
transfer vector with BaculoGold-linearized baculovirus DNA (BD Pharmingen)
into Spodoptera frugiperda (Sf9) cells (Invitrogen) using a BaculoGold transfec-
tion buffer set (BD Pharmingen) and subsequently were amplified in the same
cells according to the manufacturer’s instructions.
Generation of CatL-cleaved EBOV GP. ZEBOV-GP with the transmembrane
segment deleted was generated using recombinant baculovirus infection of Hi5
insect cells for 48 h at 27°C with gentle shaking. The secreted protein was
concentrated using a Pall Centramate system and purified by nickel affinity
chromatography (GE Healthcare, Piscataway, NJ), including an ATP (5 mM)
and MgCl (3 mM) (Sigma) column wash to remove a contaminating protein
interaction. The His tag was removed by thrombin digestion (3 U/mg protein)
(Novagen, Madison, WI) and further purified on a Superose6 column (GE
Healthcare). Peak EBOV-GP fractions were pooled and cleaved with recombi-
nant Cathepsin L (40 ?g/mg protein) (CalBiochem, San Diego, CA) for 90 min
at 37°C in 100 mM sodium acetate and 1 mM EDTA at pH 5.5 in a reaction
volume of 10 ml, concentrated, and purified on a Superdex200 column (GE
SDS-PAGE, Western blot, and Native Blue analysis. Purified protein was
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) and visualized with InstantBlue stain (Novexin, Cambridge, United
Kingdom). Protein was analyzed in parallel by Western blot analysis using Ebola
GP-specific rabbit immune sera (1:2,000) and a goat anti-rabbit secondary anti-
body conjugated to horseradish peroxidase (HRP; 1:3,000). All antibodies were
diluted in 2.5% skim milk and 0.5% Tween in Tris-buffered saline (TBS). Puri-
fied protein was analyzed by Native Blue gel electrophoresis using 4 to 12%
Nupage Ready gels (Invitrogen). Molecular mass standards (0.4 ?g/?l) included
thyroglobulin (669 kDa), ferritin (440 kDa), and aldolase (158 kDa) (Amersham
Biosciences, Piscataway, NJ).
PNGaseF treatment. Purified CatL-cleaved ZEBOV-GP (10 ?g) was dena-
tured with 1? glycoprotein denaturing buffer (New England BioLabs [NEB],
Ipswich, MA) at 100°C for 10 min and treated with PNGaseF (150 U/?g protein;
NEB) in 1? G7 reaction buffer (NEB), supplemented with 1% NP-40, (NEB)
and incubated at 37°C for 1 h.
Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)
analysis. Protein samples were analyzed using the modified thin-layer method
described by Cadene and Chait (3). The matrix used was a saturated solution of
?-cyano-4-hydroxycinnamic acid in a 3:1:2 (vol/vol/vol) mixture of formic acid-
water-isopropanol. The protein solution was mixed 1:5 with matrix solution and
spotted on the sample plate prepared with a thin layer of ?-cyano-4-hydroxycin-
namic acid and allowed to dry. Standard solutions of 6 mM apomyoglobin and 15
mM bovine serum albumin were mixed with matrix and spotted in the same way
for use as calibrants. Spectra were acquired on a Voyager-DE Pro (Applied
Biosystems, Foster City, CA) operating in linear mode. Instrument settings were
as follows: accelerating voltage, 25,000 V; grid voltage, 93%; extraction delay
time, 1,050 ns; laser intensity, 2,200 to 2,700.
N-terminal sequencing. Proteins were adsorbed onto polyvinylidene difluoride
(PVDF) membrane using a ProSorb sample preparation cartridge (Applied
Biosystems) and washed with 0.1% trifluoroacetic acid (TFA). The PVDF disk
was removed from the cartridge and sequenced on a Procise 494 protein se-
quencer (Applied Biosystems) using the standard protocol from the manufac-
Surface plasmon resonance. Kinetic parameters of KZ52 and CatL-cleaved
ZEBOV-GP were determined with a Biacore 3000 surface plasmon resonance
spectrometer (GE Healthcare). KZ52 was immobilized on CM5 sensor chips
using standard amine coupling to surface densities of 474, 246, 241, 238, 154, and
151 response units. CatL-cleaved ZEBOV-GP and SEBOV-GP were injected
sequentially over the coupled sensor chips with increasing concentrations of
31.25, 125, 250, and 500 nM at a flow rate of 30 ?l/min for 300 s at 25°C. The
dissociation rates of the complexes were monitored for 600 s at a flow rate of 30
?l/min. The buffer used for preparation of the ZEBOV-GP samples and buffer
blanks contained 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005%
surfactant P-20, and 0.1% carboxymethyldextran. The same buffer was used as
running buffer during the binding studies. A 1:1 Langmuir binding model with
drifting baseline (BiaEvaluation 4.1) was used to fit data globally and to extract
kinetic parameters of interaction with ZEBOV-GP.
Vaccination. Female BALB/c mice (6 to 8 weeks old; Jackson Laboratories,
Bar Harbor, ME) were immunized intramuscularly with 20 ?g of purified CatL-
cleaved ZEBOV-GP or uncleaved ZEBOV-GP in 100 ?l of phosphate-buffered
saline (PBS; pH 7.4) at weeks 0, 4, and 8. The collected immune sera were
analyzed for neutralizing activity. All animal experiments were conducted in full
compliance with all relevant federal guidelines and NIH policies.
Production of GP-pseudotyped lentiviral vectors and measurement of neu-
tralizing activity of immune sera. Ebola GP-pseudotyped lentiviral vectors ex-
pressing a luciferase reporter gene were produced as described previously (8, 22).
Briefly, 293T cells were cotransfected with 7 ?g of pCMV?R8.2, 7 ?g of
pHR?CMV-Luc, and 125 ng cytomegalovirus enhancer with HTLV-1 R region
(CMV/R) ZEBOV-GP, CMV/R SEBOV-GP, CMV/R Ivory Coast EBOV GP
(ICEBOV-GP), CMV/R Reston EBOV GP (REBOV-GP), CMV/R BEBOV-
GP, or vesicular stomatitis virus GP (VSV-GP). Cells were transfected overnight
and then washed the following day, and fresh medium was added. Supernatants
were harvested 48 h later, filtered through a 0.45-?m syringe filter, aliquoted, and
stored at ?80°C. For neutralization assays, immune sera were mixed with 100 ?l
of pseudovirus at various dilutions and added to 786-O cells (ATCC, Manassas,
VA) in 96-well dishes (1.5 ? 103cells/well). Plates were washed, and fresh
medium was added 14 to 16 h later. Following infection for 48 h, cells were lysed
in mammalian cell lysis buffer (Promega, Madison, WI). A standard quantity of
cell lysate was used in a luciferase assay with luciferase assay reagent (Promega)
according to the manufacturer’s protocol. The 50% inhibitory concentrations
(IC50s) were calculated using GraphPad Prism4 (GraphPad, San Diego, CA).
Preimmune sera mixed with pseudovirus were used to calculate the neutralizing
Expression, purification, and characterization of CatL-
cleaved ZEBOV-GP. CatL cleavage of Ebola virus GP is a
critical and essential step in virus entry; however, important
properties of the intermediate EBOV GP cleavage product
are currently undefined. To characterize the intermediate
ZEBOV-GP resulting from CatL cleavage, we first purified the
full-length, mucin-containing ZEBOV-GP ectodomain ex-
pressed in baculovirus using His-affinity purification. Insect
protein glycosylation pathways are not necessarily equivalent
to mammalian pathways; any lack of complex glycosylation is
not expected to interfere with proteolytic enzyme cleavage,
although the greater accessibility to CatL could lead to false-
VOL. 84, 2010 CHARACTERIZATION OF PROCESSED EBOLA VIRUS GP2973
7. Kaletsky, R. L., G. Simmons, and P. Bates. 2007. Proteolysis of the Ebola
virus glycoproteins enhances virus binding and infectivity. J. Virol. 81:13378–
8. Kong, W.-P., C. Hood, Z.-Y. Yang, C. J. Wei, L. Xu, A. Garcia-Sastre, T. M.
Tumpey, and G. J. Nabel. 2006. Protective immunity to lethal challenge of
the 1918 pandemic influenza virus by vaccination. Proc. Natl. Acad. Sci.
U. S. A. 103:15987–15991.
9. Kuhn, J. H., S. R. Radoshitzky, A. C. Guth, K. L. Warfield, W. Li, M. J.
Vincent, J. S. Towner, S. T. Nichol, S. Bavari, H. Choe, M. J. Aman, and M.
Farzan. 2006. Conserved receptor-binding domains of Lake Victoria mar-
burgvirus and Zaire ebolavirus bind a common receptor. J. Biol. Chem.
10. Lee, J. E., M. L. Fusco, A. J. Hessell, W. B. Oswald, D. R. Burton, and E. O.
Saphire. 2008. Structure of the Ebola virus glycoprotein bound to an anti-
body from a human survivor. Nature 454:177–182.
11. Manicassamy, B., J. Wang, H. Jiang, and L. Rong. 2005. Comprehensive
analysis of ebola virus GP1 in viral entry. J. Virol. 79:4793–4805.
12. Mpanju, O. M., J. S. Towner, J. E. Dover, S. T. Nichol, and C. A. Wilson.
2006. Identification of two amino acid residues on Ebola virus glycoprotein
1 critical for cell entry. Virus Res. 121:205–214.
13. Sanchez, A. 2007. Analysis of filovirus entry into Vero e6 cells, using inhib-
itors of endocytosis, endosomal acidification, structural integrity, and cathep-
sin (B and L) activity. J. Infect. Dis. 196(Suppl. 2):S251–S258.
14. Schornberg, K., S. Matsuyama, K. Kabsch, S. Delos, A. Bouton, and J.
White. 2006. Role of endosomal cathepsins in entry mediated by the Ebola
virus glycoprotein. J. Virol. 80:4174–4178.
15. Schornberg, K. L., C. J. Shoemaker, D. Dube, M. Y. Abshire, S. E. Delos,
A. H. Bouton, and J. M. White. 2009. Alpha5beta1-integrin controls ebola-
virus entry by regulating endosomal cathepsins. Proc. Natl. Acad. Sci.
U. S. A. 106:8003–8008.
16. Shimojima, M., A. Takada, H. Ebihara, G. Neumann, K. Fujioka, T. Ir-
imura, S. Jones, H. Feldmann, and Y. Kawaoka. 2006. Tyro3 family-medi-
ated cell entry of Ebola and Marburg viruses. J. Virol. 80:10109–10116.
17. Simmons, G., R. J. Wool-Lewis, F. Baribaud, R. C. Netter, and P. Bates.
2002. Ebola virus glycoproteins induce global surface protein down-modu-
lation and loss of cell adherence. J. Virol. 76:2518–2528.
18. Takada, A., K. Fujioka, M. Tsuiji, A. Morikawa, N. Higashi, H. Ebihara, D.
Kobasa, H. Feldmann, T. Irimura, and Y. Kawaoka. 2004. Human macro-
phage C-type lectin specific for galactose and N-acetylgalactosamine pro-
motes filovirus entry. J. Virol. 78:2943–2947.
19. Takada, A., C. Robison, H. Goto, A. Sanchez, K. G. Murti, M. A. Whitt, and
Y. Kawaoka. 1997. A system for functional analysis of Ebola virus glycopro-
tein. Proc. Natl. Acad. Sci. U. S. A. 94:14764–14769.
20. White, J. M., and I. A. Wilson. 1987. Anti-peptide antibodies detect steps in
a protein conformational change: low-pH activation of the influenza virus
hemagglutinin. J. Cell Biol. 105:2887–2896.
21. Yang, Z.-Y., H. J. Duckers, N. J. Sullivan, A. Sanchez, E. G. Nabel, and G. J.
Nabel. 2000. Identification of the Ebola virus glycoprotein as the main viral
determinant of vascular cell cytotoxicity and injury. Nat. Med. 6:886–889.
22. Yang, Z.-Y., B. K. Chakrabarti, L. Xu, B. Welcher, W.-P. Kong, K. Leung, A.
Panet, J. R. Mascola, and G. J. Nabel. 2004. Selective modification of
variable loops alters tropism and enhances immunogenicity of human im-
munodeficiency virus type 1 envelope. J. Virol. 78:4029–4036.
2982 HOOD ET AL.J. VIROL.