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: 3.77). 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Influenza viruses are major human pathogens responsible for respiratory diseases affecting millions of people worldwide and characterized by high morbidity and significant mortality. Influenza infections can be controlled by vaccination and antiviral drugs. However, vaccines need annual updating and give limited protection. Only two classes of drugs are currently approved for the treatment of influenza: M2 ion channel blockers and neuraminidase inhibitors. However, they are often associated with limited efficacy and adverse side effects. In addition, the currently available drugs suffer from rapid and extensive emergence of drug resistance. All this highlights the urgent need for developing new antiviral strategies with novel mechanisms of action and with reduced drug resistance potential. Several new classes of antiviral agents targeting viral replication mechanisms or cellular proteins/processes are under development. This review gives an overview of novel strategies targeting the virus and/or the host cell for counteracting influenza virus infection.
    Cellular and Molecular Life Sciences CMLS 04/2014; · 5.62 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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.
    PLoS ONE 01/2014; 9(5):e97695. · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cleavage of the hemagglutinin (HA) by host proteases is essential for the infectivity of influenza viruses. We have analysed here the role of the serine protease TMPRSS2 activating HA in the human respiratory tract on pathogenesis in a mouse model.Replication of the human H7N9 isolate A/Anhui/1/13 and of human H1N1 and H3N2 viruses was compared in TMPRSS2-knockout (TMPRSS2(-/-)) and wildtype (WT) mice. Knockout of TMPRSS2 expression inhibited H7N9 influenza virus replication in explants of murine trachea, bronchi and lung. H1N1 virus replication was also strongly suppressed in airway explants of TMPRSS2(-/-) mice, while H3N2 virus replication was only marginally affected. H7N9 and H1N1 viruses were apathogenic in TMPRSS2(-/-) mice, whereas WT mice developed severe disease with mortality rates of 100% and 20%, respectively. In contrast, all H3N2 infected TMPRSS2(-/-) and WT mice succumbed to lethal infection. Cleavage analysis showed that H7 and H1 are efficiently activated by TMPRSS2, whereas H3 is less susceptible to the protease.Our data demonstrate that TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 and H1N1 influenza virus in mice. In contrast, replication of H3N2 virus appears to depend on another, not yet identified protease, supporting the concept that human influenza viruses differ in protease specificity. Cleavage of the hemagglutinin (HA) by host proteases is essential for the infectivity of influenza virus, but little is known about its relevance for pathogenesis in mammals. Here, we show that knockout mice that do not express the HA-activating protease TMPRSS2 are resistant to pulmonary disease with lethal outcome when infected with influenza A viruses of subtypes H7N9 and H1N1, whereas they are not protected from lethal H3N2 virus infection. These findings demonstrate that human influenza viruses differ in protease specificity, and that expression of the appropriate protease in respiratory tissues is essential for pneumotropism and pathogenicity. Our observations also demonstrate that HA-activating proteases and in particular TMPRSS2 are promising targets for influenza therapy.
    Journal of Virology 02/2014; · 5.08 Impact Factor


Available from