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

Institut für Virologie, Philipps-Universität Marburg, Hans-Meerwein-Strasse 2, 35043 Marburg, Germany.
Vaccine (Impact Factor: 3.49). 10/2009; 27(45):6324-9. DOI: 10.1016/j.vaccine.2009.03.029
Source: PubMed

ABSTRACT 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 viruses was very susceptible to inhibitor treatment. Taken together, our data demonstrate that MDCK-HAT 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.

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    • "Knockdown of TMPRSS2 expression using an antisense peptide-conjugated morpholino oligomer (PPMO) strongly suppressed influenza virus replication in human airway epithelial cells without affecting cell viability (B€ ottcher-Friebertsh€ auser et al., 2011). Substrate analogue peptide mimetic inhibitors of HAT containing a 4-amidinobenzylamide moiety as the P1 residue have been demonstrated to efficiently suppress influenza virus replication in HAT-expressing cells (B€ ottcher et al., 2009; B€ ottcher-Friebertsh€ auser et al., 2010; Sielaff et al., 2011). Moreover, a peptide mimetic inhibitor of TMPRSS2 was shown to drastically reduce virus titres and to delay influenza virus propagation by 24–48 h in airway epithelial cells in vitro (B€ ottcher-Friebertsh€ auser et al., 2012). "
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    ABSTRACT: Influenza is an acute infection of the respiratory tract, which affects each year millions of people. Influenza virus infection is initiated by the surface glycoprotein hemagglutinin (HA) through receptor binding and fusion of viral and endosomal membranes. HA is synthesized as a precursor protein and requires cleavage by host cell proteases to gain its fusion capacity. Although cleavage of HA is crucial for virus infectivity, little was known about relevant proteases in the human airways for a long time. Recent progress in the identification and characterization of HA-activating host cell proteases has been considerable however and supports the idea of targeting HA cleavage as a novel approach for influenza treatment. Interestingly, certain bacteria have been demonstrated to support HA activation either by secreting proteases that cleave HA or due to activation of cellular proteases and thereby may contribute to virus spread and enhanced pathogenicity. In this review, we give an overview on activation of influenza viruses by proteases from host cells and bacteria with the main focus on recent progress on HA cleavage by proteases HAT and TMPRSS2 in the human airway epithelium. In addition, we outline investigations of HA-activating proteases as potential drug targets for influenza treatment.
    Pathogens and Disease 06/2013; 69(2). DOI:10.1111/2049-632X.12053 · 2.55 Impact Factor
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    • "Importantly , TTSPs are known to activate entry of some respiratory viruses including both seasonal and pathogenic human influenzae as well as human metapneumoviruses ( Bottcher et al . , 2006 ; Chaipan et al . , 2009 ; Shirogane et al . , 2008 ) . A recent report has implicated TMPRSS11a in the proteolysis of SARS - CoV S proteins ( Kam et al . , 2009 ) ."
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    ABSTRACT: Viruses have evolved complex ways to penetrate host barriers and cause disease. One of the most important barriers the virus has to cross is the cellular membrane. Enveloped viruses accomplish this task by viral glycoprotein-mediated binding to host cells and fusion of virus and host cell membranes. For the coronaviruses, viral spike (S) proteins execute these cell entry functions. In my dissertation research I focused on understanding the coronavirus spike proteins as well as other cofactors required for S-mediated entry into cells. The S proteins are set apart from other viral and cellular membrane fusion proteins by their extensively palmitoylated membrane-associated tails. In our experiments, substitution of alanines for the cysteines that are subject to palmitoylation had effects on both S incorporation into virions and S- mediated membrane fusions. In specifically dissecting the effects of endodomain mutations on the fusion process, we used antiviral peptides that bind only to folding intermediates in the S-mediated fusion process, and found that mutants lacking three palmitoylated cysteines remained in transitional folding states nearly ten times longer than native S proteins. This slower refolding was also reflected in the paucity of post-fusion six-helix bundle configurations amongst the mutant S proteins. Viruses with fewer palmitoylated S protein cysteines entered cells slowly and had reduced specific infectivities. These findings indicate that lipid adducts anchoring S proteins into virus membranes are necessary for the rapid, productive S protein refolding events that culminate in membrane fusions. The membrane fusion process also requires an S protein conformational flexibility that is facilitated by proteolytic cleavages. The severe acute respiratory syndrome (SARS) coronavirus S proteins rely on host cell proteases for fusion activation. I identified the human lung transmembrane serine protease, TMPRSS2, as an important factor for SARS coronavirus entry. TMPRSS2 co-localized on cell surfaces with the virus receptor ACE2, and enhanced the cell entry of both SARS S - pseudotyped HIV and authentic SARS-CoV. Enhanced entry correlated with TMPRSS2-mediated proteolysis of both S and ACE2. These findings indicate that a cell-surface complex comprising a primary receptor and a separate endoprotease operate as portals for activation of SARS coronavirus cell entry.
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    ABSTRACT: Influenza viruses are major human and animal pathogens. In man, they are responsible for annual epidemics and less frequent but more severe pandemics. Avian influenza viruses cause devastating outbreaks in poultry, and influenza virus infections in pigs and horses also lead to high economic losses. Vaccination is an effective instrument to control the disease burden of human and, to some extent, also of animal influenza. Inactivated human vaccines have been used for more than 60 years. Furthermore, cold-adapted, live attenuated vaccines have been licensed in some countries. Attenuated viruses with reduced pathogenicity can also be obtained when the cleavage site of the hemagglutinin is mutated. Such protease activation mutants have not only been generated for the production of inactivated vaccines against highly pathogenic avian influenza viruses, but they have also the potential to be used as live vaccines. Two types of protease activation mutants have been investigated for use as live vaccines. In the first group, the polybasic cleavage site of the hemagglutinin, a prime determinant of pathogenicity, was cut short to a single arginine. These viruses require additional mutations in other genes for full attenuation. In the second group, polybasic or monobasic cleavage sites are replaced by an elastase cleavage site. These viruses are fully attenuated, yet have retained their immunogenicity.
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