Article

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.

0 0
 · 
0 Bookmarks
 · 
64 Views
  • [show abstract] [hide abstract]
    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;
  • [show abstract] [hide abstract]
    ABSTRACT: Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin protein in order to fuse with the infected host cell. Recent progress in the understanding of human proteases responsible for influenza hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2, TMPRSS4 and human airway trypsin-like protease, however none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study we identify and characterize matriptase as an influenza-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1, but not from H2 and H3 viruses in a broad pH range. We showed that siRNA knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza replication in these cells. Lastly, we provide a selective slow tight-binding inhibitor of matriptase that significantly reduces viral replication (1.5-log) of H1N1 influenza including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form; upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells; within endosomes by its membrane-bound form where viral fusion takes place.
    Journal of Virology 01/2013; · 5.08 Impact Factor
  • [show abstract] [hide abstract]
    ABSTRACT: TMPRSS2 is a multidomain type II transmembrane serine protease that cleaves the surface glycoprotein hemagglutinin (HA) of influenza viruses with monobasic cleavage site, which is a prerequisite for virus fusion and propagation. Furthermore, it activates the fusion protein F of the human metapneumovirus and the spike protein S of the SARS coronavirus. Increased TMPRSS2 expression was also described in several tumor entities. Therefore, TMPRSS2 emerged as a potential target for drug design. The catalytic domain of TMPRSS2 was expressed in E. coli and used for an inhibitor screen with previously synthesized inhibitors of various trypsin-like serine proteases. Two inhibitor types were identified, which inhibit TMPRSS2 in the nanomolar range. The first series comprises substrate analogue inhibitors containing a 4-amidinobenzylamide moiety in P1 position, whereby some of these analogues possess inhibition constants around 20 nM. An improved potency was found for second type derived from sulfonylated 3-amindinophenylalanylamide derivates. The most potent derivative of this series inhibits TMPRSS2 with a Ki value of 0.9 nM and showed an efficient blockage of influenza virus propagation in human airway epithelial cells. Based on the inhibitor studies a series of new fluorogenic substrates containing a d-arginine residue in P3 position was synthesized, some of them were efficiently cleaved by TMPRSS2.
    Biochemical Journal 03/2013; · 4.65 Impact Factor