Activation of the Nipah virus fusion protein in MDCK cells is mediated by cathepsin B within the endosome-recycling compartment.
ABSTRACT Proteolytic activation of the fusion protein of the highly pathogenic Nipah virus (NiV F) is a prerequisite for the production of infectious particles and for virus spread via cell-to-cell fusion. Unlike other paramyxoviral fusion proteins, functional NiV F activation requires endocytosis and pH-dependent cleavage at a monobasic cleavage site by endosomal proteases. Using prototype Vero cells, cathepsin L was previously identified to be a cleavage enzyme. Compared to Vero cells, MDCK cells showed substantially higher F cleavage rates in both NiV-infected and NiV F-transfected cells. Surprisingly, this could not be explained either by an increased F endocytosis rate or by elevated cathepsin L activities. On the contrary, MDCK cells did not display any detectable cathepsin L activity. Though we could confirm cathepsin L to be responsible for F activation in Vero cells, inhibitor studies revealed that in MDCK cells, cathepsin B was required for F-protein cleavage and productive replication of pathogenic NiV. Supporting the idea of an efficient F cleavage in early and recycling endosomes of MDCK cells, endocytosed F proteins and cathepsin B colocalized markedly with the endosomal marker proteins early endosomal antigen 1 (EEA-1), Rab4, and Rab11, while NiV F trafficking through late endosomal compartments was not needed for F activation. In summary, this study shows for the first time that endosomal cathepsin B can play a functional role in the activation of highly pathogenic NiV.
- SourceAvailable from: John R White[Show abstract] [Hide abstract]
ABSTRACT: Background Hendra virus (HeV) is a pleomorphic virus belonging to the Paramyxovirus family. Our long-term aim is to understand the process of assembly of HeV virions. As a first step, we sought to determine the most appropriate cell culture system with which to study this process, and then to use this model to define the morphology of the virus and identify the site of assembly by imaging key virus encoded proteins in infected cells.MethodsA range of primary cells and immortalised cell lines were infected with HeV, fixed at various time points post-infection, labelled for HeV proteins and imaged by confocal, super-resolution and transmission electron microscopy.ResultsSignificant differences were noted in viral protein distribution depending on the infected cell type. At 8 hpi HeV G protein was detected in the endoplasmic reticulum and M protein was seen predominantly in the nucleus in all cells tested. At 18 hpi, HeV-infected Vero cells showed M and G proteins throughout the cell and in transmission electron microscope (TEM) sections, in pleomorphic virus-like structures. In HeV infected MDBK, A549 and HeLa cells, HeV M protein was seen predominantly in the nucleus with G protein at the membrane. In HeV-infected primary bovine and porcine aortic endothelial cells and two bat-derived cell lines, HeV M protein was not seen at such high levels in the nucleus at any time point tested (8,12, 18, 24, 48 hpi) but was observed predominantly at the cell surface in a punctate pattern co-localised with G protein. These HeV M and G positive structures were confirmed as round HeV virions by TEM and super-resolution (SR) microscopy. SR imaging demonstrated for the first time sub-virion imaging of paramyxovirus proteins and the respective localisation of HeV G, M and N proteins within virions.Conclusion These findings provide novel insights into the structure of HeV and show that for HeV imaging studies the choice of tissue culture cells may affect the experimental results. The results also indicate that HeV should be considered a predominantly round virus with a mean diameter of approximately 280 nm by TEM and 310 nm by SR imaging.Virology Journal 11/2014; 11(1):200. · 2.09 Impact Factor
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ABSTRACT: Several cellular disorders have been related to the overexpression of the cysteine protease cathepsin B (CatB), such as rheumatic arthritis, muscular dystrophy, osteoporosis, Alzheimer's disease, and tumor metastasis. Therefore, inhibiting CatB may be a way to control unregulated cellular functions and prevent tissue malformations. The inhibitory action of 1,2,4-thiadiazole (TDZ) derivatives has been associated in the literature with their ability to form disulfide bridges with the catalytic cysteine of CatB. In this work, we present molecular modeling and docking studies of a series of eight 1,2,4-thiadiazole compounds. Substitutions at two positions (3 and 5) on the 1,2,4-thiadiazole ring were analyzed, and the docking scores were correlated to experimental data. A correlation was found with the sequence of scores of four related compounds with different substituents at position 5. No correlation was observed for changes at position 3. In addition, quantum chemistry calculations were performed on smaller molecular models to study the mechanism of inhibition of TDZ at the active site of CatB. All possible protonation states of the ligand and the active site residues were assessed. The tautomeric form in which the proton is located on N2 was identified as the species that has the structural and energetic characteristics that would allow the ring opening of 1,2,4-thiadiazole.Journal of Molecular Modeling 06/2014; 20(6):2254. · 1.87 Impact Factor
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ABSTRACT: Newly synthesized envelope glycoproteins of neuroinvasive viruses can be sorted in a polarized manner to the somatodendritic and/or axonal domains of neurons. Although critical for transneuronal spread of viruses, the molecular determinants and interregulation of this process are largely unknown. We studied the polarized sorting of the attachment (NiV-G) and fusion (NiV-F) glycoproteins of Nipah virus (NiV), a paramyxovirus that causes fatal human encephalitis, in rat hippocampal neurons. When expressed individually, NiV-G exhibited a non-polarized distribution, whereas NiV-F was specifically sorted to the somatodendritic domain. Polarized sorting of NiV-F was dependent on interaction of tyrosine-based signals in its cytosolic tail with the clathrin adaptor complex AP-1. Co-expression of NiV-G with NiV-F abolished somatodendritic sorting of NiV-F due to incorporation of NiV-G•NiV-F complexes into axonal transport carriers. We propose that faster biosynthetic transport of unassembled NiV-F allows for its proteolytic activation in the somatodendritic domain prior to association with NiV-G and axonal delivery of NiV-G•NiV-F complexes. Our study reveals how interactions of viral glycoproteins with the host's transport machinery and between themselves regulate their polarized sorting in neurons.PLoS Pathogens 05/2014; 10(5):e1004107. · 8.06 Impact Factor
Activation of the Nipah Virus Fusion Protein in MDCK Cells Is
Mediated by Cathepsin B within the Endosome-Recycling
Sandra Diederich,a* Lucie Sauerhering,aMichael Weis,aHermann Altmeppen,a* Norbert Schaschke,bThomas Reinheckel,c
Stephanie Erbar,aand Andrea Maisnera
Institute of Virology, Philipps University of Marburg, Marburg, Germanya; Faculty of Chemistry, University of Bielefeld, Bielefeld, Germanyb; and Institute of Molecular
Medicine and Cell Research and BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germanyc
imals. Due to the lack of established antiviral therapeutics and
vaccines, NiV is classified as a biosafety level 4 (BSL-4) agent.
During the first outbreak beginning in 1998 in Malaysia, NiV was
transmitted from Pteropus fruit bats to pigs and then to humans
(12, 13). Since then, NiV has reemerged in Bangladesh and in
India, causing encephalitis with mortality rates up to 80% (10).
NiV transmission occurs via the respiratory route, and virus rep-
lication in vivo is mostly observed in epithelial and endothelial
cells. The systemic infection of endothelia is accompanied by vas-
culitis and is a hallmark of NiV infection in all species (60, 79). In
nervous system (CNS) resulting in severe damage of the micro-
vasculature is thought to be the basis for the development of en-
cephalitis (27, 40).
Successful NiV entry into host cells is accomplished by the
concerted action of the two viral envelope glycoproteins. After
branes, leading to virus entry. After productive NiV replication,
the infected cell and can trigger cell-to-cell fusion with receptor-
bearing neighboring cells, resulting in the formation of multinu-
cleated syncytia (68). To gain fusion competence, the NiV F pro-
tein, which is synthesized in host cells as inactive precursor F0,
must be cleaved by cellular proteases to generate a fusion-active,
disulfide-linked F1-F2heterodimer (48). Unlike cleavage of most
transport of precursor NiV F0to the cell surface and subsequent
ipah virus (NiV) is a zoonotic paramyxovirus that causes se-
vere encephalitic and respiratory diseases in humans and an-
its cytoplasmic tail (525YSRL) (18, 77). Within the endolysosomal
proteases at its monobasic cleavage site (arginine 109) (16, 48).
After cleavage and release of the hydrophobic peptide at the N
terminus of F1, the fusion-active F1-F2form is recycled to the cell
surface, where it can induce syncytium formation or is incorpo-
rated into budding virus particles. The ubiquitously expressed
cysteine protease cathepsin L has conclusively been proven to be
the NiV F-activating protease in Vero cells (53). In agreement
with the precise NiV F activation at the monobasic cleavage site,
cathepsin L had been shown to specifically process host cell pro-
teins at dibasic and monobasic cleavage sites in endosomal com-
to be involved in NiV F activation. This study however, provides
strong evidence that F cleavage in Madin-Darby canine kidney
(MDCK) cells depends on cathepsin B, another pH-dependent
monobasic cleavage sites (2). Using cathepsin L- and B-specific
Received 21 October 2011 Accepted 6 January 2012
Published ahead of print 25 January 2012
Address correspondence to Andrea Maisner, firstname.lastname@example.org.
*Present address: Sandra Diederich, Canadian Food Inspection Agency, National
Centre for Foreign Animal Diseases, Winnipeg, Manitoba, Canada; Hermann
Altmeppen, Institute of Neuropathology, University Medical Center Hamburg-
Eppendorf, Hamburg, Germany.
S.D. and L.S. contributed equally to this article.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.org0022-538X/12/$12.00 Journal of Virologyp. 3736–3745
sin L activity was not detectable, whereas cathepsin B was highly
inhibitor NS134P. We furthermore showed that block of trans-
port from early to late endosomes by nocodazole did not affect
fusion activity, suggesting that F cleavage does not require traf-
ficking through late endosomal compartments but rather occurs
within early-recycling endosomal compartments of MDCK cells.
Supporting this view, endocytosed F proteins and cathepsin B
substantially colocalized with early endosomal antigen 1 (EEA-
1)-, Rab4-, and Rab11-positive endosomes. Together, these data
can function as a NiV-activating protease within the endosomal
MATERIALS AND METHODS
Cell culture. MDCK cells were maintained in Eagle’s minimal essential
medium (MEM; Gibco) supplemented with 10% fetal calf serum (FCS),
100 units/ml penicillin, and 0.1 mg/ml streptomycin. Vero cells (African
green monkey kidney), HeLa cells, 293 cells, Huh7 cells, porcine aortic
endothelial cells (PAECs) (20), and mouse embryo fibroblasts (MEFs)
derived from wild-type (wt), cathepsin L-knockout (L?/?), cathepsin
B-knockout (B?/?), or double-knockout (L?/?/B?/?) mice (65) were
grown in Dulbecco’s modified MEM (DMEM; Gibco) containing 10%
FCS, penicillin, and streptomycin. Primary porcine brain microvascular
endothelial cells (PBMECs) were cultured in medium 199 (Gibco) sup-
plemented with 20% FCS and antibiotics.
burg. The NiV strain used in this study was isolated from human and
propagated as described earlier (48). For NiV infection studies, confluent
monolayers of Vero, MDCK cells, or MEFs were infected with NiV at a
multiplicity of infection (MOI) of 0.2, 0.5, or 2. After incubation for 1
h at 37°C, input virus was removed by extensive washings and cells were
cultured in the absence or presence of inhibitors with DMEM without
FCS for 24 h at 37°C.
Virus titers in the supernatant were determined by the 50% tissue
culture infective dose (TCID50) method on Vero cells (57).
of the NiV genome (GenBank accession number AF212302) was cloned
into the pczCFG5 vector as described earlier (45). To allow detection of
the F protein with commercially available antibodies, a tagged version of
the F protein was established by inserting the amino acids YPYDVPDYA
terminus (45). Plasmids encoding cyan fluorescent protein (CFP)- or
green fluorescent protein (GFP)-tagged Rab genes (Rab4-CFP, Rab11-
CFP, Rab7-GFP) (14) were a kind gift of R. Jacob (Marburg, Germany).
Vero and MDCK cells were transfected by using the cationic lipid
transfection reagent Lipofectamine 2000 (Invitrogen).
Inhibitors. Cathepsin L activity was inhibited by incubation with 20
?M CatLIII (Calbiochem), and cathepsin B activity was inhibited by ad-
dition of 10 or 20 ?M NS134P (62). Both cathepsins were blocked by 20
?M the membrane-permeant pan-cysteine cathepsin inhibitor E64d
(Sigma). Nocodazole (5 ?M; Sigma) was used to analyze the effect of
dimethyl sulfoxide (DMSO).
postinfection (p.i.) by scraping the cells into phosphate-buffered saline
(PBS) containing 1% sodium dodecyl sulfate (SDS). Proteins were sepa-
rated on a 12% reducing SDS-gel and transferred onto nitrocellulose.
Membranes were then incubated with an antibody directed against the
rabbit immunoglobulins (Amersham) and horseradish peroxidase-
conjugated streptavidin (Amersham). Proteins were visualized with an
enhanced chemiluminescent system (SuperSignalWest Pico; Pierce).
Band intensities were quantified using Tina (version 2.0) software. To
ence of inhibitors. At 24 h posttransfection (p.t.), cells were lysed in ra-
dioimmunoprecipitation assay (RIPA) buffer (1% Triton X-100, 1% so-
dium deoxycholate, 0.1% SDS, 0.15 M NaCl, 10 mM EDTA, 10 mM
iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 50 units/ml apro-
tinin, and 20 mM Tris-HCl, pH 8.5), followed by centrifugation for 45
min at 100,000 ? g, and F proteins were immunoprecipitated using a
sion of protein A–Sepharose CL-4B (Sigma), immune complexes were
washed with RIPA buffer and suspended in reducing sample buffer for
cellulose and probed with a monoclonal anti-HA antibody (HA.11; Co-
vance) and IRDye 800 infrared dye antimouse antibody (LI-COR). Pro-
system. The amount of F1protein as a percentage of total F protein (F1
plus F0) was calculated to yield percent cleavage.
Metabolic labeling. For pulse-chase analysis, MDCK or Vero cells
transiently expressing NiV F proteins were incubated at 24 h p.t. for 40
min with medium lacking cysteine and methionine, followed by incuba-
tion with medium containing [35S]cysteine and -methionine (Promix;
Perkin Elmer) at a final concentration of 100 ?Ci/ml for 10 min (pulse).
Then, labeling medium was replaced by serum-free nonradioactive me-
dium and cells were incubated at 37°C for 2 or 3 h with or without the
sively washed with PBS, followed by cell lysis in RIPA buffer. F proteins
were then immunoprecipitated as described above and separated on a
12% polyacrylamide gel under reducing conditions. Dried gels were sub-
jected to autoradiography and analyzed with a BAS1000 Bio-Image ana-
different cell lines were quantified as described earlier (47). Briefly, Vero
and MDCK cells transiently expressing NiV F were surface labeled with
shifted to 37°C for 0, 5, or 15 min to allow endocytosis to occur. After
rapid cooling to 4°C, biotin still bound to the cell surface was cleaved by
incubation with 50 mM 2-mercaptoethanesulfonic acid (MESNA;
Sigma). To determine the total amount of biotinylated protein, one sam-
ple was neither incubated at 37°C nor reduced with MESNA (control).
After cell lysis, F proteins were immunoprecipitated as described above,
separated on an SDS-gel under nonreducing conditions, and transferred
to nitrocellulose. Biotinylated proteins were detected with horseradish
peroxidase-conjugated streptavidin (Amersham), visualized with the en-
hanced chemiluminescence system, and quantified densitometrically.
ratio of intracellular biotinylated protein after 5, 10, and 15 min of inter-
nalization and total F protein (control) divided by 5, 10, and 15, respec-
Cathepsin activity assays. Confluent cell monolayers were lysed with
lysates were centrifuged at 4°C for 7 min at 14,000 ? g and then stored at
4°C. Enzyme activity of 10 ?g of total protein was measured using
InnoZyme cathepsin L and B activity kits (Calbiochem) following the
instructions of the supplier. Fluorescence was measured with a Perkin
Elmer LS55 luminescence spectrometer at excitation and emission wave-
lengths set at 370 nm and 450 nm, respectively. For each sample, enzy-
cence units (RFU).
glycoprotein-mediated cell-to-cell fusion, syncytium formation was
quantified as described earlier (46). Vero and MDCK cells were cotrans-
fected with NiV F- and G-encoding plasmids. Inhibitors at a concentra-
Proteolytic Activation of Nipah Virus by Cathepsins
April 2012 Volume 86 Number 7 jvi.asm.org 3737
tion of 20 ?M were added at 2.5 h p.t., and cells were fixed and stained
with 1:10-diluted Giemsa staining solution 18 h later. To quantify fusion,
five randomly chosen microscopic fields for each sample were photo-
nuclei in syncytia were counted and averaged. The relation of the total
number of nuclei and the number of nuclei in syncytia per microscopic
field indicated the percent cell fusion.
Colocalization studies. For colocalization studies of NiV F with early
endosomes or Rab proteins, antibody uptake assays were performed as
described previously (18). Briefly, at 24 h p.t., NiV F-expressing MDCK
cells were incubated with a polyclonal guinea pig anti-NiV serum (1:
1,000; kindly provided by H. Feldmann) for 1 h at 4°C. After stringent
occur. Then, surface-bound primary antibodies were blocked with a
peroxidase-conjugated secondary antibody (1:50; Dako). Following fixa-
tion with 4% paraformaldehyde (PFA) for 20 min and permeabilization
with 0.2% Triton X-100–PBS, internalized primary antibodies were
probed with Alexa Fluor (AF) 568-conjugated secondary antibodies (1:
200; Molecular Probes). For costaining of early endosomes, an antibody
against EEA-1 (1:100; BD Biosciences) was added after fixation and per-
AF 488-conjugated secondary antibodies (1:250; Molecular Probes). To
analyze colocalization of endocytosed NiV F with marker proteins of the
recycling and late endosomal compartments, pczCFG5-NiV-F was
cotransfected with plasmids encoding either Rab4-CFP, Rab11-CFP, or
Rab7-GFP. At 18 h p.t., antibody uptake assays were performed as de-
For colocalization studies of endogenous cathepsin B with marker
proteins of endosomal compartments, MDCK cells were transfected with
Rab-encoding plasmids. Cells were fixed and permeabilized at 18 h p.t.
After incubation with an anti-cathepsin B antibody from goat (1:100;
Neuromics), primary antibodies were probed by consecutive incubation
with AF 568-conjugated secondary antibodies (1:200). Costaining of
EEA-1 was performed as described above. Representative images were
recorded with a confocal laser scanning microscope (Leica SP5).
To visualize active cathepsin B, Rab11-CFP-transfected MDCK cells
were seeded on 8-well glass-bottomed ? slides (Ibidi). At 16 h p.t., cells
were washed with PBS and incubated with the cell-permeant cathepsin
B-specific Magic Red fluorogenic substrate MR-(RR)2(Immunochemis-
try Technologies, LLC). Optimal substrate concentration and incubation
times were titrated in comparison to those for 293 cells, which have low
cathepsin B activity. Finally, the fluorogenic substrate was used at a con-
centration 10-fold lower than that recommended by the manufacturer,
PBS, red fluorescence generated as a result of intracellular cathepsin
cells by confocal microscopy (Leica SP5).
Immunostaining of NiV-infected cells. For the visualization of NiV-
induced syncytia in MEFs at 48 h p.i., infected cells were inactivated and
fixed with 4% PFA for 48 h and then permeabilized with methanol-
acetone and stained with a polyclonal guinea pig anti-NiV serum (1:
1,000). Primary antibodies were detected with rhodamine-conjugated
secondary antibodies (1:200), and nuclei were counterstained with 4=,6=-
For the analysis of NiV-induced cell-to-cell fusion in the presence of
nocodazole, cells were infected in the absence or presence of 5 ?M no-
codazole (added at 8 h p.i.). At 24 h p.i., infected cells were fixed, perme-
abilized, and incubated with primary anti-NiV antibodies as described
ary antibodies (1:250) and nuclei were counterstained with DAPI. Stain-
ing of microtubules was performed using mouse anti-?-tubulin (1:1,000;
Sigma) and AF 568-conjugated anti-mouse IgG. Images were recorded
using a Zeiss Axiovert 200 M microscope.
NiV F cleavage efficiency varies between different cell types. As
for all paramyxoviruses, NiV production from infected host cells
and virus spread via cell-to-cell fusion depend among other fac-
the NiV proteolytic activation pathway differs from that for other
rus (MV). F proteins of MV are activated by the ubiquitous Golgi
apparatus protease furin during transport to the cell surface, re-
sulting in almost complete MV F cleavage (?95%) (16, 38). In
contrast, the inactive precursor NiV F0is transported to the cell
surface and must then undergo clathrin-mediated endocytosis to
get in contact with its activating protease in the endolysosomal
compartment. After pH-dependent cleavage, fusion-active F1-F2
efficiencies differ significantly depending on the cell line (1, 7, 24,
48, 53). We found F processing rates to vary between 20 and 70%
in cells from different species and tissues or in cell lines stably
transfected with NiV glycoproteins. Interestingly, MDCK cells,
well-established epithelial cells used to study virus replication in
rates than other cell types, including Vero cells, which are gener-
to be the NiV F-activating protease (53). A representative experi-
of 0.2 to 0.5. At 24 h p.i., infected cells were lysed and inactivated,
and Western blot analysis under reducing conditions was per-
formed using a specific antiserum directed against the NiV F cy-
toplasmic tail. For quantification of the cleavage efficiency, the
percentage of the F1subunit was determined. Vero cells displayed
an F cleavage of about 55%, whereas MDCK cells showed a pro-
the absence of virus infection, F cleavage in lysates of cells trans-
fected with a NiV F-encoding plasmid was analyzed at 24 h after
transfection. The Western blot analysis displayed in Fig. 1B re-
vealed similar differences in cleavage efficiencies. Again, MDCK
FIG 1 NiV F cleavage efficiency in Vero and MDCK cells. (A) Vero and
MDCK cells were infected with NiV at an MOI of 0.2 or 0.5. At 24 h p.i., cells
were lysed. Samples were subjected to SDS-PAGE under reducing conditions,
transferred to nitrocellulose, and probed with NiV F-specific antibodies and
peroxidase-conjugated secondary antibodies. Proteins were visualized by en-
hanced chemiluminescence and quantified densitometrically. (B and C) Cells
were transfected with a NiV F-encoding plasmid (pczCFG5-NiV-F). At 24 h
p.t., F proteins were immunoprecipitated from cell lysates and subjected to
Western blot analysis using an Odyssey infrared imaging system. (C) The
amounts of F0and F1proteins were quantified from four independent exper-
F0) was calculated to yield the percent cleavage (% F1).
Diederich et al.
jvi.asm.orgJournal of Virology
cells showed larger amounts of F1cleavage products than Vero
cells (Fig. 1C).
NiV F endocytosis rates do not significantly differ in Vero
and MDCK cells. Due to the fact that F endocytosis is an indis-
ferent internalization rates could be the reason for the various
efficiencies in F processing. We therefore analyzed NiV F uptake
by a quantitative biotin-based endocytosis assay (77). For that
purpose, F-expressing cells were surface labeled with NHS-SS-
biotin and then incubated for 0, 5, or 15 min at 37°C to allow
endocytosis to occur. Surface-remained biotin was then removed
of surface-biotinylated F, one sample was neither incubated at
37°C nor reduced with MESNA (control). After immunoprecipi-
tation, F proteins were separated by SDS-PAGE under nonreduc-
ing conditions and transferred to nitrocellulose, and biotinylated
proteins were detected with peroxidase-conjugated streptavidin.
As shown in Fig. 2, the F internalization rates in Vero and MDCK
endocytosis kinetics are unlikely the reason for the substantial
variations in F processing. Supporting the idea that minor varia-
protein, no correlations between F cleavage and internalization
PAECs; data not shown).
Cathepsin L and B activities differ principally between Vero
and MDCK cells. Differences in the endocytosis rates did not ex-
plain the more pronounced F processing rates in MDCK cells
compared to Vero cells. We thus hypothesized that variations in
ity of cathepsin L, the endolysosomal protease shown to mediate
L activity assays with lysates from Vero and MDCK cells using
commercial InnoZyme cathepsin assays. Confirming previous
data, cathepsin L activity in Vero cells was very high (52), but to
our surprise, we did not detect any cathepsin L activity in MDCK
cells (Fig. 3A). The efficient F processing, despite the lack of any
detectable cathepsin L activity, strongly hints to a functional role
L, the only other cysteine protease known to be widely expressed
in the endolysosomal compartment and shown to process sub-
strates at basic cleavage sites is cathepsin B (2). Since cathepsins B
in several viral infections (11, 19, 36, 64), we wanted to evaluate
that cathepsin B is the most abundant cysteine protease in most
cell types (32, 73), we detected high cathepsin B activities in
MDCK cells (Fig. 3B). Though cathepsin expression patterns and
activities are differently regulated across various tissues and cells
(26, 31, 35, 67), there is no cell type described so far completely
lacking either cathepsin L or cathepsin B. We thus assume that
low to detect. The idea of a potential role for cathepsin B in NiV F
cleavage was further supported by the finding that several cell
types aside from MDCK cells which readily support NiV F cleav-
the described high cathepsin B and low cathepsin L expression in
endothelial cells (30), both endothelial cell types tested, primary
cathepsin L and B activities.
F cleavage, cell-to-cell fusion, and production of infectious
sin B in NiV F activation, we performed inhibitor studies using
cathepsin L (CatLIII) and cathepsin B (NS134P) inhibitors (62).
To analyze the effect of the inhibitors on cleavage of transiently
expressed NiV F protein, MDCK and Vero cells were metaboli-
cally labeled at 24 h after transfection with [35S]cysteine and
cells were further incubated at 37°C for 2 h in the presence of 20
FIG 2 Endocytosis rate of NiV F in Vero and MDCK cells. At 24 h p.t., NiV
F-expressing cells were surface labeled with cleavable NHS-SS-biotin at 4°C
and then shifted to 37°C for the times indicated to allow endocytosis to occur.
Subsequently, cell surface proteins were reduced with MESNA at 4°C. To de-
termine the total amount of surface-biotinylated proteins, samples were com-
pared to cells that were neither incubated at 37°C nor treated with MESNA
(control). Following cell lysis, F proteins were immunoprecipitated and sam-
ples were separated on a 12% SDS-gel under nonreducing conditions and
transferred to nitrocellulose. Biotinylated proteins were then detected with
represents 50% of the total amount of biotinylated F proteins. After quantifi-
cation of three independent experiments, the internalization rate was calcu-
lated and is indicated as percent per min.
FIG 3 Cathepsin L and B activities. Cathepsin L (A) and cathepsin B (B)
enzyme activities in Vero and MDCK cell lysates are plotted as relative fluo-
rescence units (RFU). (C) Cathepsin L and B activities in different human
(293, HeLa, Huh7) and porcine (PBMECs, PAECs) cell types are shown.
Proteolytic Activation of Nipah Virus by Cathepsins
April 2012 Volume 86 Number 7 jvi.asm.org 3739
?M CatLIII, 10 ?M NS134P, or both inhibitors. F proteins were
then immunoprecipitated with an F-specific antiserum and sepa-
rated by SDS-PAGE under reducing conditions. In agreement
blot analysis (Fig. 1), cleavage efficiency at 2 h after labeling was
higher in control MDCK cells than Vero cells (41% and 32%,
respectively). Supporting our idea of different protease usage in
the two cell types, F cleavage in Vero cells was drastically reduced
to 4% and 3% in the presence of the cathepsin L inhibitor (Fig. 4,
Vero cells with CatLIII) and with the addition of cathepsin L and
B inhibitors (Vero cells with CatLIII and NS134P), respectively.
Inhibition of cathepsin B with NS134P barely affected F process-
ing. In contrast to Vero cells, CatLIII had no influence on F pro-
cessing in MDCK cells, while cathepsin B inhibition alone (Fig. 4,
MDCK cells with NS134P) or the combination of both inhibitors
efficiently reduced F cleavage to 3% and 0.5%, respectively. In
agreement, syncytium formation upon F and G coexpression,
which directly depends on functional F cleavage, was effectively
inhibited by CatLII in Vero cells and by NS134P in MDCK cells.
Syncytium formation was blocked as efficiently by the single in-
hibitors selective for cathepsin L or cathepsin B as by the combi-
nation of the two inhibitors or the pan-cysteine cathepsin inhibi-
tor E64d (Fig. 5). These findings clearly support the idea that
cathepsin B is the F-activating protease in MDCK cells, whereas
cathepsin L represents the key protease in Vero cells.
We further analyzed the effects of the inhibitors on NiV infec-
tion. Since cleaved and fusion-active F proteins are essentially re-
sin inhibitors and titrated the amount of infectious virus released
into the cell supernatant at 24 h p.i. Confirming previous data
(17), virus titers in Vero cell supernatants were reduced by more
than 3 log steps in the presence of CatLIII (Table 1). As expected,
cathepsin B inhibition had no effect. In contrast to Vero cells,
infectious virus production in NiV-infected MDCK cells was not
affected by CatLIII but was drastically reduced by the cathepsin B
inhibitor NS134P (Table 1). These findings again demonstrate
that protease usage differs in the two cell types. Cathepsin L rep-
resents the main activating protease in Vero cells, whereas NiV F
cleavage in MDCK cells predominantly depends on cathepsin B.
NiV replication is completely blocked only in cathepsin L
and B double-deficient MEFs. To further evaluate the impact of
B?/?, or L?/?/B?/?mice were infected with NiV at an MOI of 2.
6A). Syncytium formation was observed in wt, B?/?, and L?/?
FIG 4 Effect of specific cathepsin L and B inhibitors on NiV F cleavage. Vero
metabolically labeled with35S-labeled Promix for 10 min and then incubated
for 2 h in the absence (?) or presence (?) of 20 ?M CatLIII, 10 ?M NS134P,
or both inhibitors. F proteins were immunoprecipitated from cell lysates and
analyzed by autoradiography after separation on a 12% SDS-gel under reduc-
ing conditions. The amount of F1protein (% F1) as a percentage of total F
protein (F1plus F0) was calculated to yield percent cleavage.
FIG 5 Effect of cathepsin inhibitors on NiV glycoprotein-mediated fusion activity. Vero and MDCK cells were cotransfected with NiV G- and F-encoding
plasmids. Cathepsin inhibitors at a final concentration of 20 ?M were added at 2.5 h p.t. Eighteen hours later, syncytium formation was visualized by Giemsa
staining. Quantification (percent cell fusion) was performed as described in Materials and Methods. Magnification, ?100.
TABLE 1 Effect of cathepsin inhibitors on production of
Virus titer (TCID50/ml)
4 ? 105
2.5 ? 102
4.5 ? 105
?2 ? 102
1.2 ? 104
1.5 ? 104
7 ? 102
?2 ? 102
CatLIII (20 ?M)
NS134P (10 ?M)
CatLIII ? NS134P
aVero and MDCK cells were infected with NiV at MOIs of 0.2 and 0.5, respectively.
After 1 h virus adsorption at 37°C, cells were washed to remove input virus and then
further incubated with the indicated inhibitors. At 24 h p.i., virus titers in the
supernatant were determined by the TCID50method.
Diederich et al.
jvi.asm.orgJournal of Virology
or cathepsin B alone did not completely abolish functional F
and B (L?/?/B?/?). To quantify virus production, virus titers in
the MEF supernatants were determined (Fig. 6B). In agreement
with the reduced syncytium formation, production of infectious
viruses was slightly impaired by knockout of cathepsin B (B?/?),
whereas knockout of cathepsin L (L?/?) led to a more enhanced
reduction. A complete block of production of infectious viruses
was observed only in L?/?/B?/?MEFs. Even if the knockout of
data support the idea that cathepsin B can functionally activate
NiV in the absence of cathepsin L.
Microtubule-dependent transport to late endosomes is not
needed for F processing and NiV-mediated cell-to-cell fusion.
Efficient processing of NiV F by cathepsin B in MDCK cells re-
quires a colocalization of cathepsin B and endocytosed F in the
same endosomal compartments of MDCK cells. To evaluate this
idea, we first determined which endosomes are reached by NiV F
tibody uptake assay with F-expressing MDCK cells and analyzed
F was labeled with a NiV-specific antiserum at 4°C, and then cells
were shifted to 37°C for 10 min to allow endocytosis to occur.
Subsequently, surface-bound primary antibodies were blocked
with a peroxidase-conjugated antibody. After fixation and per-
meabilization, internalized F proteins were probed with AF 568-
conjugated secondary antibodies. Early endosomes were stained
with an EEA-1-specific and an AF 488-conjugated secondary an-
endosomes after 10 min of internalization (Fig. 7A). To further
monitor endosomal F trafficking, NiV F was coexpressed with
CFP (marker for recycling endosomes ), and Rab7-GFP
(marker for late endosomes ). After surface labeling of the F
proteins, endocytosis was allowed to proceed for 30 min. Then,
surface-bound antibodies were blocked and internalized F pro-
teins were detected as described above. Figure 7B clearly shows a
prominent colocalization of endocytosed NiV F with Rab4 and
Rab11, indicating that F proteins are mainly present in early and
endosomal compartments (Fig. 7B).
FIG 6 NiV infection in cathepsin-knockout MEFs. MEFs derived from wild-type (wt), cathepsin L-knockout (L?/?), cathepsin B-knockout (B?/?), or
and rhodamine-conjugated secondary antibodies, and nuclei were counterstained with DAPI. ?, average. (B) To quantify virus release, supernatants were
collected at 48 h p.i., and virus titers were determined by the TCID50method. One representative example from three independent experiments is shown. Virus
titers in wild-type MEFs were set at 100%.
(A) At 24 h p.t., an antibody uptake assay with NiV F-expressing MDCK cells
at 4°C and then incubated for 10 min at 37°C. Following endocytosis, surface-
bound primary antibodies were blocked by incubation with a peroxidase-
ized F proteins were detected with AF 568-conjugated secondary antibodies.
Early endosomes were stained with an anti-EEA-1 antibody and AF 488-
conjugated secondary antibodies. (B) MDCK cells were cotransfected with
pczCFG5-NiV-F and Rab4-CFP-, Rab11-CFP-, or Rab7-GFP-encoding plas-
serum. Then, endocytosis was allowed to proceed for 30 min at 37°C. Inter-
nalized F proteins were detected as described above. Images were recorded
with a confocal laser scanning microscope (SP5; Leica). Insets represent ex-
pansions of the boxed regions of the merged images.
Proteolytic Activation of Nipah Virus by Cathepsins
April 2012 Volume 86 Number 7jvi.asm.org 3741
tein transport from early to late endosomes was disrupted by the
monitored by staining untreated MDCK cells (control) and cells
treated with 5 ?M nocodazole for 2 h with an anti-?-tubulin
antibody. Figure 8A demonstrates that addition of nocodazole
was effective in destroying the MT skeleton. To determine if the
block of transport to late endosomes influences F processing,
MDCK cells expressing NiV F were metabolically labeled for 10
trol) or in the presence of 5 ?M nocodazole. After cell lysis, F
proteins were immunoprecipitated, separated under reducing
conditions, and then subjected to autoradiography. As shown in
Fig. 8B, F cleavage was not significantly affected by nocodazole,
indicating that F processing did not depend on the transport to
late endosomes. To finally see if the cleaved F protein is biologi-
cally active and can induce cell-to-cell fusion in the absence of
functional transport from early to late endosomes, MDCK cells
were infected with NiV. At 8 h p.i., medium was replaced by me-
dium without (control) or with nocodazole. Sixteen hours later,
reveals that syncytium formation was not blocked by nocodazole
treatment, demonstrating that disruption of MT blocking vesicle
transport from early to late endosomes had no impact on the
generation and surface expression of fusion-active F proteins in
Cathepsin B predominantly localizes in the F cleavage com-
MDCK cells occurs in early or recycling endosomes and are pro-
posing that F cleavage is mediated by cathepsin B, we finally
wanted to investigate the intracellular distribution of cathepsin B
in MDCK cells. We thus performed colocalization studies of en-
in Fig. 9A (EEA-1), cathepsin B was detected in early endosomes.
Colocalization with Rab4- and Rab11-positive recycling endo-
somes was even more pronounced (Fig. 9B). To confirm that en-
dosomal vesicles contain active cathepsin B, Rab11-expressing
cells were incubated with a cathepsin B substrate linked to Magic
Red. MR-(RR)2represents an indicator dye that fluoresces only
when the peptide substrate is cleaved by cathepsin B and thus
sin B. In live MDCK cells, intensive punctate Magic Red staining
which partly overlapped with Rab11-positive recycling endo-
FIG 8 Influence of nocodazole on NiV F processing and NiV infection. (A)
mouse anti-?-tubulin antibodies and AF 568-labeled anti-mouse IgG. Nuclei
were visualized by DAPI staining. (B) NiV F-expressing MDCK cells were
metabolically labeled with35S-labeled Promix for 10 min and then incubated
for 3 h in the absence (control) or presence of 5 ?M nocodazole (? nocoda-
zole). F proteins were immunoprecipitated from cell lysates and analyzed by
autoradiography after separation on a 12% SDS-gel under reducing condi-
tions. (C) MDCK cells were infected with NiV at an MOI of 0.5. At 8 h p.i.,
medium was replaced by fresh medium (control) or by medium containing 5
?M nocodazole (? nocodazole). At 24 h p.i., cells were fixed and permeabil-
ized. Virus-induced syncytium formation was detected by incubation with a
polyclonal anti-NiV serum and AF 488-conjugated secondary antibodies.
MDCK cells were fixed and permeabilized. Early endosomes were visualized
with an EEA-1-specific primary antibody and AF 488-conjugated secondary
antibodies. Endogenous cathepsin B was detected with anti-cathepsin B anti-
bodies and AF 568-conjugated secondary antibodies. (B) MDCK cells were
transfected with Rab4-CFP- or Rab11-CFP-encoding plasmids. At 18 h p.t.,
cells were fixed and endogenous cathepsin B was stained as described above.
Images were recorded with a confocal laser scanning microscope (SP5; Leica).
visualize catalytically active cathepsin B, Rab11-CFP-expressing cells were in-
cells were directly examined by confocal microscopy.
Diederich et al.
jvi.asm.orgJournal of Virology
that efficient F processing in MDCK cells is due to cathepsin B
protease expression in the (early) recycling endosomal compart-
ments where F cleavage takes place.
Proteolytic activation of the NiV F protein depends on clathrin-
mediated endocytosis and subsequent cleavage at the monobasic
cleavage site (R109) by a pH-dependent endosomal protease (18,
age enzyme using Vero cells as a model cell line (53). Here, we
show that in contrast to Vero cells, F activation in MDCK cells
activity, the different protease usage in these cells strongly argues
for a redundant or alternative functional role of cathepsin B in
NiV activation. The finding that functional F cleavage and NiV
infection in MDCK cells were not prevented by nocodazole and
the prominent colocalization of internalized F and cathepsin B
with EEA-1, Rab4, and Rab11 strengthen the idea that NiV acti-
vation by cathepsin B occurs within early and recycling endo-
Despite a pronounced activity of cathepsin B in Vero cells, we
on NiV F cleavage (Fig. 4) and virus release (Table 1). This is in
Vero cells. In contrast to MDCK cells, in which cathepsin B was
found to functionally process NiV F0, the readily expressed ca-
thepsin B appeared not to be involved in NiV activation in Vero
cells. As proposed by Pager et al. (53), this might be due to an
incorrect cleavage by cathepsin B in Vero cells. An alternative
sin B in Vero cells that does not allow sufficient contact with the
teolytic processing by cathepsin B at R109.
Cathepsins L and B are ubiquitous cysteine proteases of the
papain superfamily not only involved in bulk protein turnover in
the lysosomal compartment but also critically engaged in proteo-
growth factors, and protein antigens (see reference 59 and refer-
cleavage, cathepsins exhibit a broad substrate and cleavage site
specificity, albeit some prefer certain amino acids to others in the
target sequence. For example, cathepsin L prefers hydrophobic
amino acids in P2 and basic residues in P1 (56). In contrast, ca-
a hydrophobic amino acid (23). Analysis of the P4-P2= NiV F
cleavage site by the software tool PEPS (Prediction of Endopepti-
dase Substrates ), which is based on known cathepsin B or
cathepsin L cleavage sites in full-length protein substrates, re-
a more than 1,000-fold likelihood of NiV F cleavage by cathepsin
B or L than by a random amino acid sequence. The major differ-
ence in the structures of cathepsins L and B is that the latter con-
tains a so-called occluding loop that covers part of the catalytic
center, thereby limiting access of substrate proteins for endopro-
teolytic cleavage. Recently, the cleavage specificities of cathepsins
B and L on naturally occurring peptides have been reassessed by
the proteomic approach PICS (proteomic identification of pro-
tease cleavage sites [5, 63]). Remarkably, the PICS cleavage site
logo at pH 6 for cathepsin B contains 6 of the 8 amino acids
present in the P4-P4= NiV F cleavage site. The P3= glycine in the
NiV F site is especially typical for the cathepsin B endoprotease
catalytic center of the protease and determines the specific cleav-
age site. In contrast, the PICS profile for cathepsin L highlights
only 4 of the 8 residues present in the P4-P4= NiV F cleavage site.
These structure-substrate considerations indicate that cathepsin
by the biochemical environment, the set of cleaving enzymes, the
time that a protein substrate resides within the same compart-
ment, and the change of endosomal pH from almost neutral to
acidic (29). Due to different pH optima, proteases are active only
at certain stages in the endolysosomal compartment (32, 42).
pH of 5.5 to 6 and the protease is unstable under neutral condi-
between pH 6 and 7 (3, 55), thereby strongly supporting our
model that cathepsin B-mediated NiV F cleavage occurs in early
somal compartments (pH 5.0 to 6.0).
Even though the substrate specificity and pH dependence of
cathepsins L and B are not fully identical, they share many sub-
strates. The idea of a partial redundancy of both enzymes was
supported by studies with knockout mice. Here, the loss of one
protease can be compensated for by the remaining protease (9,
65). Knockout mice deficient in only cathepsin B or cathepsin L
knockout mice lacking both cathepsin L and cathepsin B died
early due to CNS atrophy (21). We also observed a redundant
function of cathepsins L and B when we infected MEFs from
knockout mice. Even though syncytium formation and release of
infectious NiV in cathepsin L-knockout MEFs were drastically
reduced and the reduction was more pronounced than that in
MEFs lacking cathepsin B, productive virus replication was com-
pletely blocked only in the absence of both cathepsin L and ca-
thepsin B. This again supports our view that both cathepsins have
the ability to produce fusion-active NiV F proteins and further
in NiV activation in cells lacking sufficient cathepsin L activity.
Redundant functions of cathepsins L and B, especially if one
cathepsin is lacking, were also reported for the proteolytic pro-
cessing of other viral proteins. For example, Ebola virus (EBOV)
cathepsins B and L (11, 64). However, cathepsin L-deficient hu-
man monocyte-derived dendritic cells also supported EBOV en-
try, thus indicating that cathepsin L is dispensable for infection in
these cells (41). In Moloney murine leukemia virus (MLV) infec-
tion, virus entry is facilitated by cathepsin B, which cleaves the
surface unit SU in the early endosome (36). While inhibition of
cathepsin L had no effect on infection in cells expressing both
cathepsin B and cathepsin L, a block of cathepsin L significantly
inhibited MLV infection in cathepsin B-deficient NIH 3T3 cells
Proteolytic Activation of Nipah Virus by Cathepsins
April 2012 Volume 86 Number 7jvi.asm.org 3743
sin L can support MLV infection, with cathepsin B being the fa-
vored protease. These data are consistent with our idea of an al-
ternative usage of cathepsins L and B for NiV activation,
depending on cell type and availability of the respective protease.
Recent studies on reovirus infections also highlighted distinct
roles of cathepsins in vivo. While reoviruses are known to utilize
of the two cathepsins for cell or organ tropism of NiV infection in
vivo might be an interesting question for the future.
available, even though a variety of approaches have been investi-
ing of NiV activation by host cell proteases could be a further
starting point for developing antivirals acting against cellular tar-
processes, including cancer, osteoarthritis, and autoimmune dis-
orders, some promising results for the effectiveness of cathepsin
inhibitors in tumor invasion, in osteoporosis prevention, and as
immunomodulators have been obtained (15, 66, 69). Short-term
not yet clear which protease is predominantly used in in vivo in-
fections, antiviral strategies should include inhibitors blocking
Besides the restricted expression of cellular receptors, limited
expression of activating proteases is an important factor for virus
(8, 33, 38, 49, 54). Our results showing that NiV can use two
broadly expressed cathepsins, however, suggest that protease ex-
pression does not restrict NiV spread in vivo. The ability to utilize
either cathepsin L or cathepsin B likely allows the virus to princi-
pally replicate in any receptor-positive cell type.
burg, Germany) for providing anti-NiV serum and Rab constructs, re-
L.S. was supported by a fellowship of the Jürgen-Manchot-Stiftung.
This work was supported by grants of the German Research Foundation
(DFG) to A.M. (GK 1216/2 and SFB 593/TP B11).
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