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: 3.62). 10/2009; 27(45):6324-9. DOI: 10.1016/j.vaccine.2009.03.029
Source: PubMed


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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    • "In fact, single cycle experiments carried out with PR8-NS1-Gluc virus stocks produced in the presence of trypsin benefit from the lack of Gaussia luciferase activity in the viral inoculum. When multiple rounds of virus replication need to be monitored, as in the measurement of the antiviral activity of zanamivir, it is necessary to use cell lines that express activating proteases like TMPRSS2, such as CaCo-2 [30] or MDCK-TMPRSS2 cells [29]. "
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    ABSTRACT: Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1-3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels.
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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.
    Preview · Article · Jun 2013 · Pathogens and Disease
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    • "The catalytic domains of the TMPRSS were thought to be only linked to the membrane-bound N-terminal chain of the enzyme by a disulfide bridge; however, soluble forms of the HAT and TMPRSS2 were also reported suggesting possible release of the catalytic domains from the cell surface [16,18]. Upon doxycycline-induced expression of HAT and TMPRSS2 in MDCK cells [19] and using both seasonal influenza virus A/Memphis/14/96 (H1N1) and pandemic virus A/Hamburg/5/2009 (H1N1), TMPRSS2 was found to cleave HA within the cell, while, HAT does it at the cell surface, thus, supporting cleavage of both newly synthesized HA and incoming virions [17]. Both activities could be blocked by appropriate peptide mimetic protease inhibitors [17]. "
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