Vaccine 27 (2009) 6324–6329
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MDCK cells that express proteases TMPRSS2 and HAT provide a cell system to
propagate influenza viruses in the absence of trypsin and to study cleavage
of HA and its inhibition
Eva Böttchera, Catharina Freuera, Torsten Steinmetzerb, Hans-Dieter Klenka, Wolfgang Gartena,∗
aInstitut für Virologie, Philipps-Universität Marburg, Hans-Meerwein-Strasse 2, 35043 Marburg, Germany
bInstitut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marbacher Weg 6, 35037 Marburg, Germany
a r t i c l e i n f o
Received 19 November 2008
Received in revised form 6 March 2009
Accepted 16 March 2009
a b s t r a c t
Cleavage of the influenza virus hemagglutinin (HA) by host cell proteases is essential for virus infectivity
and, therefore, relevant proteases may present promising new drug targets. We recently demonstrated
that serine proteases TMPRSS2 and HAT from human airways activate influenza virus HA with monobasic
cleavage site in vitro. In the present study we generated MDCK cells with inducible expression of either
TMPRSS2 or HAT. MDCK-TMPRSS2 and MDCK-HAT cells supported growth of human and avian influenza
viruses of different subtypes in the absence of exogenous trypsin. Further, we used these cell lines to
investigate the efficacy of protease inhibitors to prevent proteolytic activation of HA by TMPRSS2 and
HAT. Multicycle viral replication in both cell lines was markedly suppressed in the presence of serine
protease inhibitors and we found that particularly in MDCK-HAT cells proteolytic activation of progeny
and MDCK-TMPRSS2 cells are useful experimental systems to study cleavage of HA by host cell protease
and its inhibition and in addition represent applicable cell lines to propagate influenza viruses in the
absence of trypsin.
© 2009 Elsevier Ltd. All rights reserved.
The influenza virus surface glycoprotein hemagglutinin (HA)
mediates both binding to cell surface receptors and fusion of viral
and endosomal membranes following endocytosis of the virus in
order to release the viral nucleocapsid into the cytoplasm of the
target cell. HA is synthesized as a precursor protein HA0 and has
to be cleaved by a host cell protease into the subunits HA1 and
HA2 to gain its fusogenic capacity [15,16,28]. Therefore, cleavage
of HA is an important determinant of viral pathogenicity. Highly
pathogenic avian influenza A viruses of subtypes H5 and H7 pos-
intracellularly by ubiquitously expressed endoproteases furin or
trast, HA of low pathogenic avian and mammalian viruses usually
contains a single arginine at the cleavage site. Cleavage of HA with
such a monobasic cleavage site is restricted to the respiratory tract
in mammals and to the respiratory and intestinal tracts in birds
and is thought to occur extracellularly by a trypsin-like protease
∗Corresponding author. Tel.: +49 6421 2865145; fax: +49 6421 2868962.
E-mail address: firstname.lastname@example.org (W. Garten).
We previously demonstrated that the human serine proteases
HAT (human airway trypsin-like protease) and TMPRSS2 (trans-
membrane protease, serine S1 family member 2) cleave HA with
monobasic cleavage site and support multicycle viral replication
in vitro . TMPRSS2 and HAT belong to the family of type II
transmembrane serine proteases  and are expressed in the
human airway epithelium. HAT is expressed mainly in the tra-
chea and bronchi  and was shown to enhance mucin gene
expression and to stimulate bronchial fibroblast proliferation in
protease-activated receptor-2 (PAR-2) dependent pathways [4,20].
Furthermore, HAT was shown to cleave fibrinogen and to inactivate
the urokinase receptor [2,33]. TMPRSS2 is expressed predomi-
nantly in human prostate, but has also been detected in human
airway epithelial cells [5,17]. TMPRSS2 is assumed to be involved
in prostate cancer progression. It was shown to be overexpressed
in androgen-dependent prostate cancer . Furthermore, fusion
of the androgen-responsive promoter elements of the TMPRSS2
gene with ETS transcripiton factor genes was found in a majority
of patients with prostate cancer . TMPRSS2 is expressed as a
full-length protein of 70kDa, which contains a 32kDa serine pro-
tease domain . Activation of the protease requires its cleavage,
that occurs by an autocatalytic cleavage mechanism and leads to
shedding of the protease domain in prostate tissues . In the
airway epithelium TMPRSS2 was demonstrated to be involved in
0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved.
E. Böttcher et al. / Vaccine 27 (2009) 6324–6329
vich, Tatyana Matrosovich and Michaela Beyerle (Institute of
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