Stephanie Bertram

Publications (20)119.02 Total impact

• Article: TMPRSS2 activates the human coronavirus 229E for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium.

  Stephanie Bertram, Ronald Dijkman, Matthias Habjan, Adeline Heurich, Stefanie Gierer, Ilona Glowacka, Kathrin Welsch, Michael Winkler, Heike Schneider, Heike Hofmann-Winkler, Volker Thiel, Stefan Pöhlmann
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  ABSTRACT: Infection with the human coronavirus 229E (HCoV-229E) is associated with the common cold and may result in pneumonia in immunocompromised patients. The viral spike (S) protein is incorporated into the viral envelope and mediates infectious entry of HCoV-229E into host cells, a process that depends on the activation of the S-protein by host cell proteases. However, the proteases responsible for HCoV-229E activation are incompletely defined. Here, we show that the type II transmembrane serine proteases TMPRSS2 and HAT cleave the HCoV-229E S-protein (229E-S) and augment 229E-S-driven cell-cell fusion, suggesting that TMPRSS2 and HAT can activate 229E-S. Indeed, engineered expression of TMPRSS2 and HAT rendered 229E-S-driven virus-cell fusion insensitive to an inhibitor of cathepsin L, a protease previously shown to facilitate HCoV-229E infection. Inhibition of endogenous cathepsin L or TMPRSS2 demonstrated that both proteases can activate 229E-S for entry into cells naturally susceptible to infection. In addition, evidence was obtained that activation by TMPRSS2 rescues 229E-S-dependent cell entry from inhibition by IFITM proteins. Finally, immunohistochemistry revealed that TMPRSS2 is coexpressed with CD13, the HCoV-229E receptor, in human airway epithelial (HAE) cells, and that CD13(+), TMPRSS2(+) cells are preferentially targeted by HCoV-229E, suggesting that TMPRSS2 can activate HCoV-229E in infected humans. In sum, our results indicate that HCoV-229E can employ redundant proteolytic pathways to ensure its activation in host cells. In addition, our observations and previous work underline that diverse human respiratory viruses are activated by TMPRSS2, which may constitute a target for antiviral intervention.
  Journal of Virology 03/2013; · 5.40 Impact Factor
• Article: The spike-protein of the emerging betacoronavirus EMC uses a novel coronavirus receptor for entry, can be activated by TMPRSS2 and is targeted by neutralizing antibodies.

  Stefanie Gierer, Stephanie Bertram, Franziska Kaup, Florian Wrensch, Adeline Heurich, Annika Krämer-Kühl, Kathrin Welsch, Michael Winkler, Benjamin Meyer, Christian Drosten, Ulf Dittmer, Thomas von Hahn, Graham Simmons, Heike Hofmann, Stefan Pöhlmann
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  ABSTRACT: The novel human coronavirus EMC (hCoV-EMC), which recently emerged in Saudi Arabia, is highly pathogenic and could pose a significant threat to public health. The elucidation of hCoV-EMC interactions with host cells is critical to our understanding of the viral pathogenesis and to the identification of targets for antiviral intervention. Here, we investigated the viral and cellular determinants governing hCoV-EMC entry into host cells. We found that the spike-protein of hCoV-EMC (EMC-S) is incorporated into lentiviral particles and mediates transduction of human cell lines derived from different organs, including lung, kidney and colon, as well as primary human macrophages. Expression of known coronavirus receptors, ACE2, CD13 and CEACAM1, did not facilitate EMC-S-driven transduction, suggesting that hCoV-EMC uses a novel receptor for entry. Directed protease expression and inhibition analyses revealed that TMPRSS2 and endosomal cathepsins activate EMC-S for virus-cell fusion and constitute potential targets for antiviral intervention. Finally, EMC-S-driven transduction was abrogated by serum from an hCoV-EMC infected patient, indicating that EMC-S-specific neutralizing antibodies can be generated in patients. Collectively, our results indicate that hCoV-EMC uses a novel receptor for protease-activated entry into human cells and might be capable of extrapulmonary spread. In addition, they define TMPRSS2 and cathepsins B and L as potential targets for intervention and suggest that neutralizing antibodies contribute to the control of hCoV-EMC infection.
  Journal of Virology 03/2013; · 5.40 Impact Factor
• Article: The role of the alternative coreceptor GPR15 in SIV tropism for human cells.

  Miriam Kiene, Andrea Marzi, Andreas Urbanczyk, Stephanie Bertram, Tanja Fisch, Inga Nehlmeier, Kerstin Gnirß, Christina B Karsten, David Palesch, Jan Münch, Francesca Chiodi, Stefan Pöhlmann, Imke Steffen
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  ABSTRACT: Many SIV isolates can employ the orphan receptor GPR15 as coreceptor for efficient entry into transfected cell lines, but the role of endogenously expressed GPR15 in SIV cell tropism is largely unclear. Here, we show that several human B and T cell lines express GPR15 on the cell surface, including the T/B cell hybrid cell line CEMx174, and that GPR15 expression is essential for SIV infection of CEMx174 cells. In addition, GPR15 expression was detected on subsets of primary human CD4(+), CD8(+) and CD19(+) peripheral blood mononuclear cells (PBMCs), respectively. However, GPR15(+) PBMCs were not efficiently infected by HIV and SIV, including cells from individuals homozygous for the defective Δ32 ccr5 allele. These results suggest that GPR15 is coexpressed with CD4 on PBMCs but that infection of CD4(+), GPR15(+) cells is not responsible for the well documented ability of SIV to infect CCR5(-) blood cells.
  Virology 08/2012; 433(1):73-84. · 3.35 Impact Factor
• Article: Cathepsins B and L activate Ebola but not Marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of TMPRSS2 expression.

  Kerstin Gnirss, Annika Kühl, Christina Karsten, Ilona Glowacka, Stephanie Bertram, Franziska Kaup, Heike Hofmann, Stefan Pöhlmann
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  ABSTRACT: Ebola (EBOV) and Marburg virus (MARV) cause severe hemorrhagic fever. The host cell proteases cathepsin B and L activate the Zaire ebolavirus glycoprotein (GP) for cellular entry and constitute potential targets for antiviral intervention. However, it is unclear if different EBOV species and MARV equally depend on cathepsin B/L activity for infection of cell lines and macrophages, important viral target cells. Here, we show that cathepsin B/L inhibitors markedly reduce 293T cell infection driven by the GPs of all EBOV species, independent of the type II transmembrane serine protease TMPRSS2, which cleaved but failed to activate EBOV-GPs. Similarly, a cathepsin B/L inhibitor blocked macrophage infection mediated by different EBOV-GPs. In contrast, MARV-GP-driven entry exhibited little dependence on cathepsin B/L activity. Still, MARV-GP-mediated entry was efficiently blocked by leupeptin. These results suggest that cathepsins B/L promote entry of EBOV while MARV might employ so far unidentified proteases for GP activation.
  Virology 03/2012; 424(1):3-10. · 3.35 Impact Factor
• Source

  Available from: PubMed Central

  Article: Influenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts.

  Stephanie Bertram, Adeline Heurich, Hayley Lavender, Stefanie Gierer, Simon Danisch, Paula Perin, Jared M Lucas, Peter S Nelson, Stefan Pöhlmann, Elizabeth J Soilleux
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  ABSTRACT: The type II transmembrane serine proteases TMPRSS2 and HAT activate influenza viruses and the SARS-coronavirus (TMPRSS2) in cell culture and may play an important role in viral spread and pathogenesis in the infected host. However, it is at present largely unclear to what extent these proteases are expressed in viral target cells in human tissues. Here, we show that both HAT and TMPRSS2 are coexpressed with 2,6-linked sialic acids, the major receptor determinant of human influenza viruses, throughout the human respiratory tract. Similarly, coexpression of ACE2, the SARS-coronavirus receptor, and TMPRSS2 was frequently found in the upper and lower aerodigestive tract, with the exception of the vocal folds, epiglottis and trachea. Finally, activation of influenza virus was conserved between human, avian and porcine TMPRSS2, suggesting that this protease might activate influenza virus in reservoir-, intermediate- and human hosts. In sum, our results show that TMPRSS2 and HAT are expressed by important influenza and SARS-coronavirus target cells and could thus support viral spread in the human host.
  PLoS ONE 01/2012; 7(4):e35876. · 4.09 Impact Factor
• Article: Influenza A Virus Does Not Encode a Tetherin Antagonist with Vpu-Like Activity and Induces IFN-Dependent Tetherin Expression in Infected Cells.

  Michael Winkler, Stephanie Bertram, Kerstin Gnirß, Inga Nehlmeier, Ali Gawanbacht, Frank Kirchhoff, Christina Ehrhardt, Stephan Ludwig, Miriam Kiene, Anna-Sophie Moldenhauer, Ulrike Goedecke, Christina B Karsten, Annika Kühl, Stefan Pöhlmann
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  ABSTRACT: The interferon-induced host cell factor tetherin inhibits release of human immunodeficiency virus (HIV) from the plasma membrane of infected cells and is counteracted by the HIV-1 protein Vpu. Influenza A virus (FLUAV) also buds from the plasma membrane and is not inhibited by tetherin. Here, we investigated if FLUAV encodes a functional equivalent of Vpu for tetherin antagonism. We found that expression of the FLUAV protein NS1, which antagonizes the interferon (IFN) response, did not block the tetherin-mediated restriction of HIV release, which was rescued by Vpu. Similarly, tetherin-mediated inhibition of HIV release was not rescued by FLUAV infection. In contrast, FLUAV infection induced tetherin expression on target cells in an IFN-dependent manner. These results suggest that FLUAV escapes the antiviral effects of tetherin without encoding a tetherin antagonist with Vpu-like activity.
  PLoS ONE 01/2012; 7(8):e43337. · 4.09 Impact Factor
• Source

  Available from: Marcel A. Müller

  Article: Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease.

  Stephanie Bertram, Ilona Glowacka, Marcel A Müller, Hayley Lavender, Kerstin Gnirss, Inga Nehlmeier, Daniela Niemeyer, Yuxian He, Graham Simmons, Christian Drosten, Elizabeth J Soilleux, Olaf Jahn, Imke Steffen, Stefan Pöhlmann
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  ABSTRACT: The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) poses a constant threat to human health. The viral spike protein (SARS-S) mediates host cell entry and is a potential target for antiviral intervention. Activation of SARS-S by host cell proteases is essential for SARS-CoV infectivity but remains incompletely understood. Here, we analyzed the role of the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), in SARS-S activation. We found that HAT activates SARS-S in the context of surrogate systems and authentic SARS-CoV infection and is coexpressed with the viral receptor angiotensin-converting enzyme 2 (ACE2) in bronchial epithelial cells and pneumocytes. HAT cleaved SARS-S at R667, as determined by mutagenesis and mass spectrometry, and activated SARS-S for cell-cell fusion in cis and trans, while the related pulmonary protease TMPRSS2 cleaved SARS-S at multiple sites and activated SARS-S only in trans. However, TMPRSS2 but not HAT expression rendered SARS-S-driven virus-cell fusion independent of cathepsin activity, indicating that HAT and TMPRSS2 activate SARS-S differentially. Collectively, our results show that HAT cleaves and activates SARS-S and might support viral spread in patients.
  Journal of Virology 12/2011; 85(24):13363-72. · 5.40 Impact Factor
• Article: The Ebola virus glycoprotein and HIV-1 Vpu employ different strategies to counteract the antiviral factor tetherin.

  Annika Kühl, Carina Banning, Andrea Marzi, Jörg Votteler, Imke Steffen, Stephanie Bertram, Ilona Glowacka, Andreas Konrad, Michael Stürzl, Ju-Tao Guo, Ulrich Schubert, Heinz Feldmann, Georg Behrens, Michael Schindler, Stefan Pöhlmann
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  ABSTRACT: The antiviral protein tetherin/BST2/CD317/HM1.24 restricts cellular egress of human immunodeficiency virus (HIV) and of particles mimicking the Ebola virus (EBOV), a hemorrhagic fever virus. The HIV-1 viral protein U (Vpu) and the EBOV-glycoprotein (EBOV-GP) both inhibit tetherin. Here, we compared tetherin counteraction by EBOV-GP and Vpu. We found that EBOV-GP but not Vpu counteracted tetherin from different primate species, indicating that EBOV-GP and Vpu target tetherin differentially. Tetherin interacted with the GP2 subunit of EBOV-GP, which might encode the determinants for tetherin counteraction. Vpu reduced cell surface expression of tetherin while EBOV-GP did not, suggesting that both proteins employ different mechanisms to counteract tetherin. Finally, Marburg virus (MARV)-GP also inhibited tetherin and downregulated tetherin in a cell type-dependent fashion, indicating that tetherin antagonism depends on the cellular source of tetherin. Collectively, our results indicate that EBOV-GP counteracts tetherin by a novel mechanism and that tetherin inhibition is conserved between EBOV-GP and MARV-GP.
  The Journal of Infectious Diseases 11/2011; 204 Suppl 3:S850-60. · 6.41 Impact Factor
• Article: Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion.

  Graham Simmons, Stephanie Bertram, Ilona Glowacka, Imke Steffen, Chawaree Chaipan, Juliet Agudelo, Kai Lu, Andrew J Rennekamp, Heike Hofmann, Paul Bates, Stefan Pöhlmann
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  ABSTRACT: Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation.
  Virology 03/2011; 413(2):265-74. · 3.35 Impact Factor
• Source

  Available from: Marcel A. Müller

  Article: Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response.

  Ilona Glowacka, Stephanie Bertram, Marcel A Müller, Paul Allen, Elizabeth Soilleux, Susanne Pfefferle, Imke Steffen, Theodros Solomon Tsegaye, Yuxian He, Kerstin Gnirss, Daniela Niemeyer, Heike Schneider, Christian Drosten, Stefan Pöhlmann
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  ABSTRACT: The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) can be proteolytically activated by cathepsins B and L upon viral uptake into target cell endosomes. In contrast, it is largely unknown whether host cell proteases located in the secretory pathway of infected cells and/or on the surface of target cells can cleave SARS S. We along with others could previously show that the type II transmembrane protease TMPRSS2 activates the influenza virus hemagglutinin and the human metapneumovirus F protein by cleavage. Here, we assessed whether SARS S is proteolytically processed by TMPRSS2. Western blot analysis revealed that SARS S was cleaved into several fragments upon coexpression of TMPRSS2 (cis-cleavage) and upon contact between SARS S-expressing cells and TMPRSS2-positive cells (trans-cleavage). cis-cleavage resulted in release of SARS S fragments into the cellular supernatant and in inhibition of antibody-mediated neutralization, most likely because SARS S fragments function as antibody decoys. trans-cleavage activated SARS S on effector cells for fusion with target cells and allowed efficient SARS S-driven viral entry into targets treated with a lysosomotropic agent or a cathepsin inhibitor. Finally, ACE2, the cellular receptor for SARS-CoV, and TMPRSS2 were found to be coexpressed by type II pneumocytes, which represent important viral target cells, suggesting that SARS S is cleaved by TMPRSS2 in the lung of SARS-CoV-infected individuals. In summary, we show that TMPRSS2 might promote viral spread and pathogenesis by diminishing viral recognition by neutralizing antibodies and by activating SARS S for cell-cell and virus-cell fusion.
  Journal of Virology 02/2011; 85(9):4122-34. · 5.40 Impact Factor
• Article: TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells.

  Stephanie Bertram, Ilona Glowacka, Paulina Blazejewska, Elizabeth Soilleux, Paul Allen, Simon Danisch, Imke Steffen, So-Young Choi, Youngwoo Park, Heike Schneider, Klaus Schughart, Stefan Pöhlmann
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  ABSTRACT: Proteolysis of influenza virus hemagglutinin by host cell proteases is essential for viral infectivity, but the proteases responsible are not well defined. Recently, we showed that engineered expression of the type II transmembrane serine proteases (TTSPs) TMPRSS2 and TMPRSS4 allows hemagglutinin (HA) cleavage. Here we analyzed whether TMPRSS2 and TMPRSS4 are expressed in influenza virus target cells and support viral spread in the absence of exogenously added protease (trypsin). We found that transient expression of TMPRSS2 and TMPRSS4 resulted in HA cleavage and trypsin-independent viral spread. Endogenous expression of TMPRSS2 and TMPRSS4 in cell lines correlated with the ability to support the spread of influenza virus in the absence of trypsin, indicating that these proteases might activate influenza virus in naturally permissive cells. Indeed, RNA interference (RNAi)-mediated knockdown of both TMPRSS2 and TMPRSS4 in Caco-2 cells, which released fully infectious virus without trypsin treatment, markedly reduced the spread of influenza virus, demonstrating that these proteases were responsible for efficient proteolytic activation of HA in this cell line. Finally, TMPRSS2 was found to be coexpressed with the major receptor determinant of human influenza viruses, 2,6-linked sialic acids, in human alveolar epithelium, indicating that viral target cells in the human respiratory tract express TMPRSS2. Collectively, our results point toward an important role for TMPRSS2 and possibly TMPRSS4 in influenza virus replication and highlight the former protease as a potential therapeutic target.
  Journal of Virology 10/2010; 84(19):10016-25. · 5.40 Impact Factor
• Article: Novel insights into proteolytic cleavage of influenza virus hemagglutinin.

  Stephanie Bertram, Ilona Glowacka, Imke Steffen, Annika Kühl, Stefan Pöhlmann
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  ABSTRACT: The influenza virus hemagglutinin (HA) mediates the first essential step in the viral life cycle, virus entry into target cells. Influenza virus HA is synthesised as a precursor protein in infected cells and requires cleavage by host cell proteases to transit into an active form. Cleavage is essential for influenza virus infectivity and the HA-processing proteases are attractive targets for therapeutic intervention. It is well established that cleavage by ubiquitously expressed subtilisin-like proteases is a hallmark of highly pathogenic avian influenza viruses (HPAIV). In contrast, the nature of the proteases responsible for cleavage of HA of human influenza viruses and low pathogenic avian influenza viruses (LPAIV) is not well understood. Recent studies suggest that cleavage of HA of human influenza viruses might be a cell-associated event and might be facilitated by the type II transmembrane serine proteases (TTSPs) TMPRSS2, TMPRSS4 and human airway trypsin-like protease (HAT). Here, we will introduce the different concepts established for proteolytic activation of influenza virus HA, with a particular focus on the role of TTSPs, and we will discuss their implications for viral tropism, pathogenicity and antiviral intervention.
  Reviews in Medical Virology 09/2010; 20(5):298-310. · 7.20 Impact Factor
• Article: A single asparagine-linked glycosylation site of the severe acute respiratory syndrome coronavirus spike glycoprotein facilitates inhibition by mannose-binding lectin through multiple mechanisms.

  Yanchen Zhou, Kai Lu, Susanne Pfefferle, Stephanie Bertram, Ilona Glowacka, Christian Drosten, Stefan Pöhlmann, Graham Simmons
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  ABSTRACT: Mannose-binding lectin (MBL) is a serum protein that plays an important role in host defenses as an opsonin and through activation of the complement system. The objective of this study was to assess the interactions between MBL and severe acute respiratory syndrome-coronavirus (SARS-CoV) spike (S) glycoprotein (SARS-S). MBL was found to selectively bind to retroviral particles pseudotyped with SARS-S. Unlike several other viral envelopes to which MBL can bind, both recombinant and plasma-derived human MBL directly inhibited SARS-S-mediated viral infection. Moreover, the interaction between MBL and SARS-S blocked viral binding to the C-type lectin, DC-SIGN. Mutagenesis indicated that a single N-linked glycosylation site, N330, was critical for the specific interactions between MBL and SARS-S. Despite the proximity of N330 to the receptor-binding motif of SARS-S, MBL did not affect interactions with the ACE2 receptor or cathepsin L-mediated activation of SARS-S-driven membrane fusion. Thus, binding of MBL to SARS-S may interfere with other early pre- or postreceptor-binding events necessary for efficient viral entry.
  Journal of Virology 09/2010; 84(17):8753-64. · 5.40 Impact Factor
• Article: Calcium-modulating cyclophilin ligand does not restrict retrovirus release.

  Annika Kühl, Jan Münch, Daniel Sauter, Stephanie Bertram, Ilona Glowacka, Imke Steffen, Anke Specht, Heike Hofmann, Heike Schneider, Georg Behrens, Stefan Pöhlmann
  Nature medicine 02/2010; 16(2):155-6; author reply 157. · 27.14 Impact Factor
• Article: Novel insights into proteolytic cleavage of influenza virus hemagglutinin

  Stephanie Bertram, Ilona Glowacka, Imke Steffen, Annika Ku¨ hl, Stefan Po¨hlmann
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  ABSTRACT: The influenza virus hemagglutinin (HA) mediates the first essential step in the viral life cycle, virus entry into target cells. Influenza virus HA is synthesised as a precursor protein in infected cells and requires cleavage by host cell proteases to transit into an active form. Cleavage is essential for influenza virus infectivity and the HA-processing proteases are attractive targets for therapeutic intervention. It is well established that cleavage by ubiquitously expressed subtilisin-like proteases is a hallmark of highly pathogenic avian influenza viruses (HPAIV). In contrast, the nature of the proteases responsible for cleavage of HA of human influenza viruses and low pathogenic avian influenza viruses (LPAIV) is not well understood. Recent studies suggest that cleavage of HA of human influenza viruses might be a cell-associated event and might be facilitated by the type II transmembrane serine proteases (TTSPs) TMPRSS2, TMPRSS4 and human airway trypsin-like protease (HAT). Here, we will introduce the different concepts established for proteolytic activation of influenza virus HA, with a particular focus on the role of TTSPs, and we will discuss their implications for viral tropism, pathogenicity and antiviral intervention. Copyright # 2010 John Wiley & Sons, Ltd.
  Reviews in Medical Virology. 01/2010;
• Source

  Available from: Ilona Glowacka

  Article: Incorporation of podoplanin into HIV released from HEK-293T cells, but not PBMC, is required for efficient binding to the attachment factor CLEC-2.

  Chawaree Chaipan, Imke Steffen, Theodros Solomon Tsegaye, Stephanie Bertram, Ilona Glowacka, Yukinari Kato, Jan Schmökel, Jan Münch, Graham Simmons, Rita Gerardy-Schahn, Stefan Pöhlmann
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  ABSTRACT: Platelets are associated with HIV in the blood of infected individuals and might modulate viral dissemination, particularly if the virus is directly transmitted into the bloodstream. The C-type lectin DC-SIGN and the novel HIV attachment factor CLEC-2 are expressed by platelets and facilitate HIV transmission from platelets to T-cells. Here, we studied the molecular mechanisms behind CLEC-2-mediated HIV-1 transmission. Binding studies with soluble proteins indicated that CLEC-2, in contrast to DC-SIGN, does not recognize the viral envelope protein, but a cellular factor expressed on kidney-derived 293T cells. Subsequent analyses revealed that the cellular mucin-like membranous glycoprotein podoplanin, a CLEC-2 ligand, was expressed on 293T cells and incorporated into virions released from these cells. Knock-down of podoplanin in 293T cells by shRNA showed that virion incorporation of podoplanin was required for efficient CLEC-2-dependent HIV-1 interactions with cell lines and platelets. Flow cytometry revealed no evidence for podoplanin expression on viable T-cells and peripheral blood mononuclear cells (PBMC). Podoplanin was also not detected on HIV-1 infected T-cells. However, apoptotic bystander cells in HIV-1 infected cultures reacted with anti-podoplanin antibodies, and similar results were obtained upon induction of apoptosis in a cell line and in PBMCs suggesting an unexpected link between apoptosis and podoplanin expression. Despite the absence of detectable podoplanin expression, HIV-1 produced in PBMC was transmitted to T-cells in a CLEC-2-dependent manner, indicating that T-cells might express an as yet unidentified CLEC-2 ligand. Virion incorporation of podoplanin mediates CLEC-2 interactions of HIV-1 derived from 293T cells, while incorporation of a different cellular factor seems to be responsible for CLEC-2-dependent capture of PBMC-derived viruses. Furthermore, evidence was obtained that podoplanin expression is connected to apoptosis, a finding that deserves further investigation.
  Retrovirology 01/2010; 7:47. · 6.47 Impact Factor
• Chapter: Cellular Entry of the SARS Coronavirus: Implications for Transmission, Pathogenicity and Antiviral Strategies

  Ilona Glowacka, Stephanie Bertram, Stefan Pöhlmann
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  ABSTRACT: A novel coronavirus was identified as the causative agent of the lung disease severe acute respiratory syndrome (SARS). The outbreak of SARS in 2002/2003 was associated with high morbidity and mortality and sparked international research efforts to develop antiviral strategies. Many of these efforts focussed on the viral surface protein spike (S), which facilitates the first indispensable step in the viral replication cycle, infectious entry into target cells. For infectious cellular entry to occur, the S protein must engage a cellular receptor, the carboxypeptidase angiotensin-converting enzyme 2 (ACE2). The interface between ACE2 and S protein, which has been characterized at the structural level, constitutes a key target for vaccines and inhibitors, and is believed to be an important determinant of viral pathogenesis and interspecies transmission. In this chapter, we will discuss how SARS-S mediates cellular entry and we will review the implications of this process for SARS coronavirus (SARS-CoV) transmission, disease development and antiviral intervention.
  12/2009: pages 3-22;
• Article: Differential downregulation of ACE2 by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus NL63.

  Ilona Glowacka, Stephanie Bertram, Petra Herzog, Susanne Pfefferle, Imke Steffen, Marcus O Muench, Graham Simmons, Heike Hofmann, Thomas Kuri, Friedemann Weber, Jutta Eichler, Christian Drosten, Stefan Pöhlmann
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  ABSTRACT: The human coronaviruses (CoVs) severe acute respiratory syndrome (SARS)-CoV and NL63 employ angiotensin-converting enzyme 2 (ACE2) for cell entry. It was shown that recombinant SARS-CoV spike protein (SARS-S) downregulates ACE2 expression and thereby promotes lung injury. Whether NL63-S exerts a similar activity is yet unknown. We found that recombinant SARS-S bound to ACE2 and induced ACE2 shedding with higher efficiency than NL63-S. Shedding most likely accounted for the previously observed ACE2 downregulation but was dispensable for viral replication. Finally, SARS-CoV but not NL63 replicated efficiently in ACE2-positive Vero cells and reduced ACE2 expression, indicating robust receptor interference in the context of SARS-CoV but not NL63 infection.
  Journal of Virology 10/2009; 84(2):1198-205. · 5.40 Impact Factor
• Article: Type II transmembrane serine proteases in cancer and viral infections.

  So-Young Choi, Stephanie Bertram, Ilona Glowacka, Young Woo Park, Stefan Pöhlmann
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  ABSTRACT: Regulated proteolysis of cellular factors is pivotal to tissue development and homeostasis, whereas uncontrolled proteolytic activity is linked to disease. Type II transmembrane serine proteases (TTSPs) are expressed at the cell surface and are thus ideally located to regulate cell-cell and cell-matrix interactions. Increasing evidence demonstrates that aberrant expression of TTSPs is a hallmark of several cancers and recent studies have defined molecular mechanisms underlying TTSP-promoted carcinogenesis. In addition, new findings suggest that influenza and other respiratory viruses could exploit TTSPs to promote their spread, making these proteases potential targets for intervention in cancer and viral infections. Here, we review the role of TTSPs in tumorigenesis and viral infection and discuss potential approaches to therapy.
  Trends in Molecular Medicine 08/2009; 15(7):303-12. · 10.35 Impact Factor
• Article: Proteolytic activation of the 1918 influenza virus hemagglutinin.

  Chawaree Chaipan, Darwyn Kobasa, Stephanie Bertram, Ilona Glowacka, Imke Steffen, Theodros Solomon Tsegaye, Makoto Takeda, Thomas H Bugge, Semi Kim, Youngwoo Park, Andrea Marzi, Stefan Pöhlmann
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  ABSTRACT: Proteolytic activation of the hemagglutinin (HA) protein is indispensable for influenza virus infectivity, and the tissue expression of the responsible cellular proteases impacts viral tropism and pathogenicity. The HA protein critically contributes to the exceptionally high pathogenicity of the 1918 influenza virus, but the mechanisms underlying cleavage activation of the 1918 HA have not been characterized. The neuraminidase (NA) protein of the 1918 influenza virus allows trypsin-independent growth in canine kidney cells (MDCK). However, it is at present unknown if the 1918 NA, like the NA of the closely related strain A/WSN/33, facilitates HA cleavage activation by recruiting the proprotease plasminogen. Moreover, it is not known which pulmonary proteases activate the 1918 HA. We provide evidence that NA-dependent, trypsin-independent cleavage activation of the 1918 HA is cell line dependent and most likely plasminogen independent since the 1918 NA failed to recruit plasminogen and neither exogenous plasminogen nor the presence of the A/WSN/33 NA promoted efficient cleavage of the 1918 HA. The transmembrane serine protease TMPRSS4 was found to be expressed in lung tissue and was shown to cleave the 1918 HA. Accordingly, coexpression of the 1918 HA with TMPRSS4 or the previously identified HA-processing protease TMPRSS2 allowed trypsin-independent infection by pseudotypes bearing the 1918 HA, indicating that these proteases might support 1918 influenza virus spread in the lung. In summary, we show that the previously reported 1918 NA-dependent spread of the 1918 influenza virus is a cell line-dependent phenomenon and is not due to plasminogen recruitment by the 1918 NA. Moreover, we provide evidence that TMPRSS2 and TMPRSS4 activate the 1918 HA by cleavage and therefore may promote viral spread in lung tissue.
  Journal of Virology 02/2009; 83(7):3200-11. · 5.40 Impact Factor