Hartmut Hengel

Publications of Hartmut Hengel

• The Human Cytomegalovirus-Specific UL1 Gene Encodes a Late-Phase Glycoprotein Incorporated in the Virion Envelope.

  Authors: Medya Shikhagaie, Eva Mercé-Maldonado, Elena Isern, Aura Muntasell, M Mar Albà, Miguel López-Botet, Hartmut Hengel, Ana Angulo

  Journal of virology. 02/2012; 86(8):4091-101.

  We have investigated the previously uncharacterized human cytomegalovirus (HCMV) UL1 open reading frame (ORF), a member of the rapidly evolving HCMV RL11 family. UL1 is HCMV specific; the absence ofWe have investigated the previously uncharacterized human cytomegalovirus (HCMV) UL1 open reading frame (ORF), a member of the rapidly evolving HCMV RL11 family. UL1 is HCMV specific; the absence of UL1 in chimpanzee cytomegalovirus (CCMV) and sequence analysis studies suggest that UL1 may have originated by the duplication of an ancestor gene from the RL11-TRL cluster (TRL11, TRL12, and TRL13). Sequence similarity searches against human immunoglobulin (Ig)-containing proteins revealed that HCMV pUL1 shows significant similarity to the cellular carcinoembryonic antigen-related (CEA) protein family N-terminal Ig domain, which is responsible for CEA ligand recognition. Northern blot analysis revealed that UL1 is transcribed during the late phase of the viral replication cycle in both fibroblast-adapted and endotheliotropic strains of HCMV. We characterized the protein encoded by hemagglutinin (HA)-tagged UL1 in the AD169-derived HB5 background. UL1 is expressed as a 224-amino-acid type I transmembrane glycoprotein which becomes detectable at 48 h postinfection. In infected human fibroblasts, pUL1 colocalized at the cytoplasmic site of virion assembly and secondary envelopment together with TGN-46, a marker for the trans-Golgi network, and viral structural proteins, including the envelope glycoprotein gB and the tegument phosphoprotein pp28. Furthermore, analyses of highly purified AD169 UL1-HA epitope-tagged virions revealed that pUL1 is a novel constituent of the HCMV envelope. Importantly, the deletion of UL1 in HCMV TB40/E resulted in reduced growth in a cell type-specific manner, suggesting that pUL1 may be implicated in regulating HCMV cell tropism.

• Vaccination against infection in patients with multiple sclerosis.

  Authors: Micha Loebermann, Alexander Winkelmann, Hans-Peter Hartung, Hartmut Hengel, Emil C Reisinger, Uwe K Zettl

  Nature reviews. Neurology. 01/2012; 8(3):143-51.

  Bacterial and viral infections have been shown to induce relapses and accelerate the progression of multiple sclerosis (MS). Vaccination to prevent communicable disease in such patients is,Bacterial and viral infections have been shown to induce relapses and accelerate the progression of multiple sclerosis (MS). Vaccination to prevent communicable disease in such patients is, therefore, of key importance. Reports of potentially detrimental effects of immunization on the course of MS, however, have prompted patients and physicians to adopt a cautious attitude towards the use of vaccines. The risks associated with a number of vaccines have been investigated in patients with MS. Vaccines against some diseases, such as tetanus and hepatitis B, are not associated with an elevated risk of MS exacerbation, whereas vaccines against other diseases, such as yellow fever, are contraindicated in patients with MS. Many patients with MS receive immunosuppressive or immunomodulatory therapy, which could make them more susceptible to infectious diseases and might also affect their ability to respond to immunization. Here, we review the indications for and possible adverse effects of vaccines in patients with MS, and address issues of vaccination in the context of immunomodulatory therapy for MS.

• The cytomegaloviral protein pUL138 acts as potentiator of tumor necrosis factor (TNF) receptor 1 surface density to enhance ULb'-encoded modulation of TNF-α signaling.

  Authors: Vu Thuy Khanh Le, Mirko Trilling, Hartmut Hengel

  Journal of virology. 12/2011; 85(24):13260-70.

  Human cytomegalovirus is a ubiquitous herpesvirus that establishes lifelong latent infection. Changes in immune homeostasis induce the reactivation of lytic infection, which is mostly inapparent inHuman cytomegalovirus is a ubiquitous herpesvirus that establishes lifelong latent infection. Changes in immune homeostasis induce the reactivation of lytic infection, which is mostly inapparent in healthy individuals but often causes overt disease in immunocompromised hosts. Based on discrepant tumor necrosis factor receptor 1 surface disposition between human cytomegalovirus AD169 variants differing in the ULb' region, we identified the latency-associated gene product pUL138, which also is expressed during productive infection, as a selective potentiator of tumor necrosis factor receptor 1, one of the key receptors of innate immunity. Ectopically expressed pUL138 coprecipitated with tumor necrosis factor receptor 1, extended the protein half-life, and enhanced its signaling responses, thus leading to tumor necrosis factor receptor 1 hyperresponsiveness. Conversely, the targeted deletion of UL138 from the human cytomegaloviral genome strongly reduced tumor necrosis factor receptor 1 surface densities of infected cells. Remarkably, the comparison of UL138 deficiency to ULb' deficiency revealed the presence of further positive modulators of tumor necrosis factor alpha signal transduction encoded within the human cytomegalovirus ULb' region, identifying this region as a hub for multilayered tumor necrosis factor alpha signaling regulation.

• Enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus.

  Authors: Nadine Honke, Namir Shaabani, Giuseppe Cadeddu, Ursula R Sorg, Dong-Er Zhang, Mirko Trilling, Karin Klingel, Martina Sauter, Reinhard Kandolf, Nicole Gailus [......] Stephan E Baldus, Melanie Grusdat, Max Löhning, Hartmut Hengel, Klaus Pfeffer, Masato Tanaka, Dieter Häussinger, Mike Recher, Philipp A Lang, Karl S Lang

  Nature immunology. 11/2011; 13(1):51-7.

  The innate immune system limits viral replication via type I interferon and also induces the presentation of viral antigens to cells of the adaptive immune response. Using infection of mice withThe innate immune system limits viral replication via type I interferon and also induces the presentation of viral antigens to cells of the adaptive immune response. Using infection of mice with vesicular stomatitis virus, we analyzed how the innate immune system inhibits viral propagation but still allows the presentation of antigen to cells of the adaptive immune response. We found that expression of the gene encoding the inhibitory protein Usp18 in metallophilic macrophages led to lower type I interferon responsiveness, thereby allowing locally restricted replication of virus. This was essential for the induction of adaptive antiviral immune responses and, therefore, for preventing the fatal outcome of infection. In conclusion, we found that enforced viral replication in marginal zone macrophages was an immunological mechanism that ensured the production of sufficient antigen for effective activation of the adaptive immune response.

• Modulation of NKp30- and NKp46-mediated natural killer cell responses by poxviral hemagglutinin.

  Authors: Mostafa Jarahian, Manuela Fiedler, André Cohnen, Dominik Djandji, Günter J Hämmerling, Cornelius Gati, Adelheid Cerwenka, Peter C Turner, Richard W Moyer, Carsten Watzl, Hartmut Hengel, Frank Momburg

  PLoS pathogens. 08/2011; 7(8):e1002195.

  Natural killer (NK) cells are an important element in the immune defense against the orthopox family members vaccinia virus (VV) and ectromelia virus (ECTV). NK cells are regulated through inhibitoryNatural killer (NK) cells are an important element in the immune defense against the orthopox family members vaccinia virus (VV) and ectromelia virus (ECTV). NK cells are regulated through inhibitory and activating signaling receptors, the latter involving NKG2D and the natural cytotoxicity receptors (NCR), NKp46, NKp44 and NKp30. Here we report that VV infection results in an upregulation of ligand structures for NKp30 and NKp46 on infected cells, whereas the binding of NKp44 and NKG2D was not significantly affected. Likewise, infection with ectromelia virus (ECTV), the mousepox agent, enhanced binding of NKp30 and, to a lesser extent, NKp46. The hemagglutinin (HA) molecules from VV and ECTV, which are known virulence factors, were identified as novel ligands for NKp30 and NKp46. Using NK cells with selectively silenced NCR expression and NCR-CD3ζ reporter cells, we observed that HA present on the surface of VV-infected cells, or in the form of recombinant soluble protein, was able to block NKp30-triggered activation, whereas it stimulated the activation through NKp46. The net effect of this complex influence on NK cell activity resulted in a decreased NK lysis susceptibility of infected cells at late time points of VV infection when HA was expression was pronounced. We conclude that poxviral HA represents a conserved ligand of NCR, exerting a novel immune escape mechanism through its blocking effect on NKp30-mediated activation at a late stage of infection.

• Absence of cross-presenting cells in the salivary gland and viral immune evasion confine cytomegalovirus immune control to effector CD4 T cells.

  Authors: Senta M Walton, Sanja Mandaric, Nicole Torti, Albert Zimmermann, Hartmut Hengel, Annette Oxenius

  PLoS pathogens. 08/2011; 7(8):e1002214.

  Horizontal transmission of cytomegaloviruses (CMV) occurs via prolonged excretion from mucosal surfaces. We used murine CMV (MCMV) infection to investigate the mechanisms of immune control inHorizontal transmission of cytomegaloviruses (CMV) occurs via prolonged excretion from mucosal surfaces. We used murine CMV (MCMV) infection to investigate the mechanisms of immune control in secretory organs. CD4 T cells were crucial to cease MCMV replication in the salivary gland (SG) via direct secretion of IFNγ that initiated antiviral signaling on non-hematopoietic cells. In contrast, CD4 T cell helper functions for CD8 T cells or B cells were dispensable. Despite SG-resident MCMV-specific CD8 T cells being able to produce IFNγ, the absence of MHC class I molecules on infected acinar glandular epithelial cells due to viral immune evasion, and the paucity of cross-presenting antigen presenting cells (APCs) prevented their local activation. Thus, local activation of MCMV-specific T cells is confined to the CD4 subset due to exclusive presentation of MCMV-derived antigens by MHC class II molecules on bystander APCs, resulting in IFNγ secretion interfering with viral replication in cells of non-hematopoietic origin.

• Identification of DNA-damage DNA-binding protein 1 as a conditional essential factor for cytomegalovirus replication in interferon-γ-stimulated cells.

  Authors: Mirko Trilling, Vu Thuy Khanh Le, Manuela Fiedler, Albert Zimmermann, Elke Bleifuss, Hartmut Hengel

  PLoS pathogens. 06/2011; 7(6):e1002069.

  The mouse cytomegaloviral (MCMV) protein pM27 represents an indispensable factor for viral fitness in vivo selectively, antagonizing signal transducer and activator of transcription 2The mouse cytomegaloviral (MCMV) protein pM27 represents an indispensable factor for viral fitness in vivo selectively, antagonizing signal transducer and activator of transcription 2 (STAT2)-mediated interferon signal transduction. We wished to explore by which molecular mechanism pM27 accomplishes this effect. We demonstrate that pM27 is essential and sufficient to curtail the protein half-life of STAT2 molecules. Pharmacologic inhibition of the proteasome restored STAT2 amounts, leading to poly-ubiquitin-conjugated STAT2 forms. PM27 was found in complexes with an essential host ubiquitin ligase complex adaptor protein, DNA-damage DNA-binding protein (DDB) 1. Truncation mutants of pM27 showed a strict correlation between DDB1 interaction and their ability to degrade STAT2. SiRNA-mediated knock-down of DDB1 restored STAT2 in the presence of pM27 and strongly impaired viral replication in interferon conditioned cells, thus phenocopying the growth attenuation of M27-deficient virus. In a constructive process, pM27 recruits DDB1 to exploit ubiquitin ligase complexes catalyzing the obstruction of the STAT2-dependent antiviral state of cells to permit viral replication.

• c-Src is required for complex formation between the hepatitis C virus-encoded proteins NS5A and NS5B: a prerequisite for replication.

  Authors: Andreas Pfannkuche, Katrin Büther, Juliane Karthe, Marion Poenisch, Ralf Bartenschlager, Mirko Trilling, Hartmut Hengel, Dieter Willbold, Dieter Häussinger, Johannes Georg Bode

  Hepatology (Baltimore, Md.). 04/2011; 53(4):1127-36.

  Hepatitis C virus (HCV) is a leading cause of chronic liver disease worldwide and establishes a persistent infection in more than 60% of infected individuals. This high frequency of persistentHepatitis C virus (HCV) is a leading cause of chronic liver disease worldwide and establishes a persistent infection in more than 60% of infected individuals. This high frequency of persistent infection indicates that HCV has evolved efficient strategies to interfere with the adaptive and innate immune response and to occupy and use host cell infrastructure. The present study provides evidence that c-Src, a member of the Src family kinases that participates in many signal transduction pathways, represents an essential host factor exploited for viral replication. c-Src directly interacts with the viral RNA-dependent RNA polymerase (NS5B) via its SH3 domain and with the nonstructural phosphoprotein NS5A via its SH2 domain. Both interactions are required to maintain the protein-protein interaction of NS5A and NS5B, which has been previously demonstrated to be essential for viral replication. Accordingly, HCV genome replication and production of the viral proteins was strongly reduced upon small interfering RNA-mediated knockdown of c-Src or in the presence of the tyrosine kinase inhibitor herbimycin A. This effect could not be rescued by supplementation of the two other ubiquitously expressed Src family kinases Fyn or Yes. CONCLUSION: Our data suggest that c-Src participates in the formation of an NS5A/NS5B protein complex that is required for efficient replication of HCV.

• Inhibition of mouse TAP by immune evasion molecules encoded by non-murine herpesviruses.

  Authors: Marieke C Verweij, Maaike E Ressing, Wilco Knetsch, Edwin Quinten, Anne Halenius, Nikki van Bel, Hartmut Hengel, Jan Wouter Drijfhout, Thorbald van Hall, Emmanuel J H J Wiertz

  Molecular immunology. 03/2011; 48(6-7):835-45.

  Herpesviruses escape elimination by cytotoxic T lymphocytes through specific interference with the antigen-presenting function of MHC class I (MHC I) molecules. The transporter associated withHerpesviruses escape elimination by cytotoxic T lymphocytes through specific interference with the antigen-presenting function of MHC class I (MHC I) molecules. The transporter associated with antigen processing (TAP) forms a bottleneck in the MHC I antigen presentation pathway. The fact that multiple viruses, especially herpesviruses, encode molecules blocking TAP function is a case in point. The action of these viral immuno evasins is usually potent and very specific, making these proteins valuable tools for studying the cell biology of antigen presentation, including alternative antigen processing pathways. Yet, no dedicated TAP inhibitor has been described for any of the mouse herpesviruses. To permit the use of immuno evasins derived from non-mouse herpesviruses in mouse models, we assessed the cross-species activity of four TAP inhibitors and one tapasin inhibitor in the context of three different mouse haplotypes, H-2(b), H-2(d), and H-2(k). Two of the four TAP inhibitors, the bovine herpesvirus 1-encoded UL49.5 and the human cytomegalovirus (HCMV)-encoded US6 protein, potently inhibited mouse TAP. ICP47 and BNLF2a, encoded by herpes simplexvirus 1 and Epstein-Barr virus, respectively, failed to inhibit TAP in all mouse cells tested. Previous work, however, demonstrated that US6 did not cross the mouse species barrier. We now show that substitution of the cysteine residue at position 108 was responsible for this lack of activity. The HCMV-encoded tapasin inhibitor US3 efficiently downregulated H-2(d) molecules on 3T3 cells, but not in other cell lines tested. Finally, we show that synthetic peptides comprising the functional domain of US6 can be exploited as a versatile TAP inhibitor. In conclusion, a complete overview is presented of the applicability of herpesvirus-encoded TAP and tapasin inhibitors in mouse cells of different genetic background.

• Human cytomegalovirus disrupts the major histocompatibility complex class I peptide-loading complex and inhibits tapasin gene transcription.

  Authors: Anne Halenius, Sebastian Hauka, Lars Dölken, Jan Stindt, Henrike Reinhard, Constanze Wiek, Helmut Hanenberg, Ulrich H Koszinowski, Frank Momburg, Hartmut Hengel

  Journal of virology. 01/2011; 85(7):3473-85.

  Major histocompatibility complex class I (MHC I) molecules present antigenic peptides for CD8(+) T-cell recognition. Prior to cell surface expression, proper MHC I loading is conducted by theMajor histocompatibility complex class I (MHC I) molecules present antigenic peptides for CD8(+) T-cell recognition. Prior to cell surface expression, proper MHC I loading is conducted by the peptide-loading complex (PLC), composed of the MHC I heavy chain (HC) and β(2)-microglobulin (β(2)m), the peptide transporter TAP, and several chaperones, including tapasin. Tapasin connects peptide-receptive MHC I molecules to the PLC, thereby facilitating loading of high-affinity peptides onto MHC I. To cope with CD8(+) T-cell responses, human cytomegalovirus (HCMV) encodes several posttranslational strategies inhibiting peptide transport and MHC I biogenesis which have been studied extensively in transfected cells. Here we analyzed assembly of the PLC in naturally HCMV-infected fibroblasts throughout the protracted replication cycle. MHC I incorporation into the PLC was absent early in HCMV infection. Subsequently, tapasin neosynthesis became strongly reduced, while tapasin steady-state levels diminished only slowly in infected cells, revealing a blocked synthesis rather than degradation. Tapasin mRNA levels were continuously downregulated during infection, while tapasin transcripts remained stable and long-lived. Taking advantage of a novel method by which de novo transcribed RNA is selectively labeled and analyzed, an immediate decline of tapasin transcription was seen, followed by downregulation of TAP2 and TAP1 gene expression. However, upon forced expression of tapasin in HCMV-infected cells, repair of MHC I incorporation into the PLC was relatively inefficient, suggesting an additional level of HCMV interference. The data presented here document a two-pronged coordinated attack on tapasin function by HCMV.

• Exploitation of herpesviral transactivation allows quantitative reporter gene-based assessment of virus entry and neutralization.

  Authors: Henrike Reinhard, Vu Thuy Khanh Le, Mats Ohlin, Hartmut Hengel, Mirko Trilling

  PloS one. 01/2011; 6(1):e14532.

  Herpesviral entry is a highly elaborated process requiring many proteins to act in precise conjunction. Neutralizing antibodies interfere with this process to abrogate viral infection. Based onHerpesviral entry is a highly elaborated process requiring many proteins to act in precise conjunction. Neutralizing antibodies interfere with this process to abrogate viral infection. Based on promoter transactivation of a reporter gene we established a novel method to quantify herpesvirus entry and neutralization by antibodies. Following infection with mouse and human cytomegalovirus and Herpes simplex virus 1 we observed promoter transactivation resulting in substantial luciferase expression (>1000-fold). No induction was elicited by UV-inactivated viruses. The response was MOI-dependent and immunoblots confirmed a correlation between luciferase induction and pp72-IE1 expression. Monoclonal antibodies, immune sera and purified immunoglobulin preparations decreased virus-dependent luciferase induction dose-dependently, qualifying this approach as surrogate virus neutralization test. Besides the reduced hands-on time, this assay allows analysis of herpesvirus entry in semi-permissive and non-adherent cells, which were previously non-assessable but play significant roles in herpesvirus pathology.

• NKp46 and DNAM-1 NK-cell receptors drive the response to human cytomegalovirus-infected myeloid dendritic cells overcoming viral immune evasion strategies.

  Authors: Giuliana Magri, Aura Muntasell, Neus Romo, Andrea Sáez-Borderías, Daniela Pende, Daniel E Geraghty, Hartmut Hengel, Ana Angulo, Alessandro Moretta, Miguel López-Botet

  Blood. 10/2010; 117(3):848-56.

  Information on natural killer (NK)-cell receptor-ligand interactions involved in the response to human cytomegalovirus (HCMV) is limited and essentially based on the study of infected fibroblasts.Information on natural killer (NK)-cell receptor-ligand interactions involved in the response to human cytomegalovirus (HCMV) is limited and essentially based on the study of infected fibroblasts. Experimental conditions were set up to characterize the NK response to HCMV-infected myeloid dendritic cells (DCs). Monocyte-derived DCs (moDCs) infected by the TB40/E HCMV strain down-regulated the expression of human leukocyte antigen class I molecules and specifically activated autologous NK-cell populations. NKG2D ligands appeared virtually undetectable in infected moDCs, reflecting the efficiency of immune evasion mechanisms, and explained the lack of antagonistic effects of NKG2D-specific monoclonal antibody. By contrast, DNAM-1 and DNAM-1 ligands (DNAM-1L)-specific monoclonal antibodies inhibited the NK response at 48 hours after infection, although the impact of HCMV-dependent down-regulation of DNAM-1L in infected moDCs was perceived at later stages. moDCs constitutively expressed ligands for NKp46 and NKp30 natural cytotoxicity receptors, which were partially reduced on HCMV infection; yet, only NKp46 appeared involved in the NK response. In contrast to previous reports in fibroblasts, human leukocyte antigen-E expression was not preserved in HCMV-infected moDCs, which triggered CD94/NKG2A(+) NK-cell activation. The results provide an insight on key receptor-ligand interactions involved in the NK-cell response against HCMV-infected moDCs, stressing the importance of the dynamics of viral immune evasion mechanisms.

• Virology. A vaccine monkey wrench?

  Authors: Hartmut Hengel, Ulrich H Koszinowski

  Science (New York, N.Y.). 04/2010; 328(5974):51-2.

• Nonstructural 3/4A protease of hepatitis C virus activates epithelial growth factor-induced signal transduction by cleavage of the T-cell protein tyrosine phosphatase.

  Authors: Erwin Daniel Brenndörfer, Juliane Karthe, Lars Frelin, Patricia Cebula, Andreas Erhardt, Jan Schulte Am Esch, Hartmut Hengel, Ralf Bartenschlager, Matti Sällberg, Dieter Häussinger, Johannes Georg Bode

  Hepatology (Baltimore, Md.). 07/2009; 49(6):1810-20.

  The hepatitis C virus (HCV) is a worldwide major cause of chronic liver disease with a high tendency to establish a persistent infection. To permit persistent replication of viral genomes through theThe hepatitis C virus (HCV) is a worldwide major cause of chronic liver disease with a high tendency to establish a persistent infection. To permit persistent replication of viral genomes through the cellular translation machinery without affecting host cell viability, viruses must have developed mechanisms to control cellular cascades required for sufficient viral replication, on the one hand, and to adapt viral replication to the cellular requirements on the other hand. The present study aimed to further elucidate mechanisms by which HCV targets growth factor signaling of the host cell and their implications for viral replication. The study describes a novel mechanism by which HCV influences the activation of the epithelial growth factor receptor/Akt pathway through a nonstructural (NS)3/4A-dependent down-regulation of the ubiquitously expressed tyrosine phosphatase T cell protein tyrosine phosphatase (TC-PTP). NS3/4A is demonstrated to cleave TC-PTP protease-dependently in vitro at two cleavage sites. The in vivo relevance of this finding is supported by the fact that down-regulation of TC-PTP protein expression could also be demonstrated in HCV-infected individuals and in transgenic mice with intrahepatic expression of NS3/4A. CONCLUSION: This down-regulation of TC-PTP results in an enhancement of epithelial growth factor (EGF)-induced signal transduction and increases basal activity of Akt, which is demonstrated to be essential for the maintenance of sufficient viral replication. Hence, therapeutic targeting of NS3/4A may not only disturb viral replication by blocking the processing of the viral polyprotein but also exerts unforeseen indirect antiviral effects, further diminishing viral replication.

• An IFN-{gamma}-Induced IRF-1-Dependent Antiviral Response Inhibits Vaccinia Virus Replication in Mouse but Not Human Fibroblasts.

  Authors: Mirko Trilling, Vu Thuy Khanh Le, Albert Zimmermann, Holger Ludwig, Klaus Pfeffer, Gerd Sutter, Geoffrey L. Smith, Hartmut Hengel

  Journal of virology. 03/2009;

  Vaccinia virus (VACV) replicates in mouse and human fibroblasts with comparable kinetics and efficiency, yielding similar titers of infectious progeny. Here we demonstrate that interferon (IFN)-gammaVaccinia virus (VACV) replicates in mouse and human fibroblasts with comparable kinetics and efficiency, yielding similar titers of infectious progeny. Here we demonstrate that interferon (IFN)-gamma but not IFN-alpha or IFN-beta pretreatment of mouse fibroblasts prior to VACV infection induces a long lasting antiviral state blocking VACV replication. In contrast, high doses of IFN-gamma failed to establish an antiviral state in human fibroblasts. In mouse fibroblasts IFN-gamma impeded the viral replication cycle at the level of late gene transcription and blocked multiplication of VACV genomes. The IFN-gamma induced antiviral state invariably prevented the growth of different VACV strains but was not effective against replication of ectromelia virus (ECTV). The IFN-gamma effect required intact IFN-gamma receptor signalling prior to VACV infection through Janus kinase (Jak) 2 and signal transducer and activator of transcription (STAT) 1. The permissive state of IFN-gamma-treated human cells was unrelated to the VACV-encoded IFN decoy receptors B8 and B18 and associated with a complete disruption of STAT1 homodimer formation and DNA binding. Unlike human fibroblasts, mouse cells responded with long lasting STAT1 activation which was preserved after VACV infection. Deletion of IFN response factor (IRF)-1 gene from mouse cells rescued efficient VACV replication, demonstrating that IRF-1 target genes have a critical role in VACV control. These data have implications for the understanding of VACV pathogenesis and identify an incongruent IFN-gamma response between the human host and the mouse model.

• Human cytomegalovirus interferes with signal transducer and activator of transcription (STAT) 2 protein stability and tyrosine phosphorylation.

  Authors: Vu Thuy Khanh Le, Mirko Trilling, Manuel Wilborn, Hartmut Hengel, Albert Zimmermann

  The Journal of general virology. 11/2008; 89(Pt 10):2416-26.

  We have investigated the role of signal transducer and activator of transcription (STAT) 2 during human cytomegalovirus (HCMV) replication and found that protein levels of STAT2 are downregulated.We have investigated the role of signal transducer and activator of transcription (STAT) 2 during human cytomegalovirus (HCMV) replication and found that protein levels of STAT2 are downregulated. STAT2 downregulation was observed in HCMV clinical isolates and laboratory strains with the exception of strain Towne. The HCMV-induced loss of STAT2 protein occurred despite an increased accumulation of STAT2 mRNA; it required HCMV early gene expression. The decrease in STAT2 was sensitive to proteasome inhibition, suggesting degradation of STAT2 via the ubiquitin proteasome pathway. Notably, pUL27, the HCMV homologue of the mouse CMV pM27 protein, which mediates the selective proteolysis of STAT2, did not induce STAT2 downregulation. Moreover, preceding STAT2 degradation, alpha/beta interferon (IFN)-receptor-mediated tyrosine phosphorylation of STAT2 was inhibited in HCMV-infected cells. This effect was paralleled by impaired tyrosine activation of STAT1 and STAT3. Accordingly, IFNs affected the replication efficiency of STAT2 degrading and non-degrading HCMV strains to a similar degree. In summary, HCMV abrogates IFN receptor signalling at multiple checkpoints by independent mechanisms including UL27-independent degradation of STAT2 and a preceding blockade of STAT2 phosphorylation.

• Construction of a fully retargeted herpes simplex virus 1 capable to enter cells solely via the human epidermal growth factor receptor 2 (HER2).

  Authors: Laura Menotti, Arianna Cerretani, Hartmut Hengel, Gabriella Campadelli-Fiume

  Journal of virology. 09/2008;

  A novel frontier in the treatment of tumors difficult to treat is oncolytic virotherapy, whereby a replication competent virus selectively infects and destroys tumor cells. Herpes simplex virus (HSV)A novel frontier in the treatment of tumors difficult to treat is oncolytic virotherapy, whereby a replication competent virus selectively infects and destroys tumor cells. Herpes simplex virus (HSV) represents a particularly attractive system. Effective retargeting to tumor-specific receptors has been achieved by insertion in gD of heterologous ligands. Previously, our laboratory generated a HSV retargeted to the human epidermal growth factor receptor 2 (HER2), a receptor overexpressed in about one third of mammary tumors, and in some ovary tumors. HER2 overexpression correlates with increased metastaticity and poor prognosis. Because HER2 has no natural ligand, the inserted ligand was a single chain antibody to HER2. The objective of this work was to genetically engineer a HSV that selectively targets the HER2-expressing tumor cells and has lost the ability to enter cells through the natural gD receptors, HVEM and nectin1. Detargeting from nectin1 was attempted by two different strategies, point mutations or insertion of the single chain antibody at a different site in gD relative to previously described sites of insertion. We report that point mutations at gD aa 34, 215, 222 and 223 fail to generate a nectin1-detargeted HSV. A HSV simultaneously detargeted from nectin1 and HVEM, and retargeted to HER2 was successfully engineered by moving the site of single chain antibody insertion at residue 39, i.e. in front of the nectin1-interacting surface and not laterally to it, and by deleting aa residues 6-38. The resulting recombinant R-LM113 entered cells and spread from cell to cell solely via HER2.

• The dynamics of mouse cytomegalovirus-specific CD4 T cell responses during acute and latent infection.

  Authors: Senta M Walton, Philippe Wyrsch, Michael W Munks, Albert Zimmermann, Hartmut Hengel, Ann B Hill, Annette Oxenius

  Journal of immunology (Baltimore, Md. : 1950). 08/2008; 181(2):1128-34.

  The dynamics of mouse cytomegalovirus (MCMV)-specific CD4 T cell responses and the mechanisms by which these cells contribute to viral control are not well understood, mainly due to lack ofThe dynamics of mouse cytomegalovirus (MCMV)-specific CD4 T cell responses and the mechanisms by which these cells contribute to viral control are not well understood, mainly due to lack of appropriate tools to characterize MCMV-specific CD4 T cells. We therefore generated MCMV-specific CD4 T cell hybridomas, then used an MCMV expression library and overlapping peptides to identify CD4 T cell epitopes. We used these novel tools to study the long-term kinetics and organ distribution of MCMV-specific CD4 T cells in comparison to MCMV-specific CD8 T cell responses. We demonstrate that the overall MCMV-specific CD4 T cell response stabilizes during the latent stage, which stands in contrast to subpopulations of MCMV-specific CD8 T cells and HCMV-specific CD4 T cells which accumulate over the course of CMV latency. Furthermore, MCMV-specific CD4 T cells displayed a Th1 phenotype, secreting high levels of IFN-gamma and TNF-alpha and to some extent IL-2, cytokines which are involved in protection from CMV disease.

• Mouse cytomegalovirus inhibits beta interferon (IFN-beta) gene expression and controls activation pathways of the IFN-beta enhanceosome.

  Authors: Vu Thuy Khanh Le, Mirko Trilling, Albert Zimmermann, Hartmut Hengel

  The Journal of general virology. 06/2008; 89(Pt 5):1131-41.

  We have investigated beta interferon (IFN-beta) and IFN-alpha4 gene expression and activation of related transcription factors in mouse cytomegalovirus (MCMV)-infected fibroblasts. mRNA analysisWe have investigated beta interferon (IFN-beta) and IFN-alpha4 gene expression and activation of related transcription factors in mouse cytomegalovirus (MCMV)-infected fibroblasts. mRNA analysis demonstrated an initial phase of IFN gene induction upon MCMV infection, which was followed by a sustained MCMV-mediated simultaneous downregulation of IFN-beta and IFN-alpha4 gene expression. The induction of IFN transcription resulted from the activation of the components of the IFN-beta enhanceosome, i.e. IFN regulatory factor (IRF) 3, nuclear factor (NF)-kappaB, activating transcription factor (ATF)-2 and c-Jun. Activation of the transcription factors occurred rapidly and in a sequential order upon infection, but only lasted a while. As a consequence, IFN-alpha/beta gene expression became undetectable 6 h post-infection and throughout the MCMV replication cycle. This effect is based on an active interference since restimulation of IFN gene induction by further external stimuli (e.g. Sendai virus infection) was completely abolished. This inhibition required MCMV gene expression and was not observed in cells infected with UV-inactivated MCMV virions. The efficiency of inhibition is achieved by a concerted blockade of IkappaBalpha degradation and a lack of nuclear accumulation of IRF3 and ATF-2/c-Jun. Using an MCMV mutant lacking pM27, a signal transducer and activator of transcription (STAT) 2-specific inhibitor of Jak/STAT signalling, we found that the initial phase of IFN induction and the subsequent inhibition does not depend on the positive-IFN feedback loop. Our findings indicate that the MCMV-mediated downregulation of IFN transcription in fibroblasts relies on a large arsenal of inhibitory mechanisms targeting each pathway that contributes to the multiprotein enhanceosome complex.

• The human cytomegalovirus Fc receptor gp68 binds the Fc CH2-CH3 interface of immunoglobulin G.

  Authors: Elizabeth R Sprague, Henrike Reinhard, Evelyn J Cheung, Alexander H. Farley, Robin Deis Trujillo, Hartmut Hengel, Pamela J Bjorkman

  Journal of virology. 05/2008; 82(7):3490-9.

  Recognition of immunoglobulin G (IgG) by surface receptors for the Fc domain of immunoglobulin G (Fcgamma), FcgammaRs, can trigger both humoral and cellular immune responses. Two humanRecognition of immunoglobulin G (IgG) by surface receptors for the Fc domain of immunoglobulin G (Fcgamma), FcgammaRs, can trigger both humoral and cellular immune responses. Two human cytomegalovirus (HCMV)-encoded type I transmembrane receptors with Fcgamma-binding properties (vFcgammaRs), gp34 and gp68, have been identified on the surface of HCMV-infected cells and are assumed to confer protection against IgG-mediated immunity. Here we show that Fcgamma recognition by both vFcgammaRs occurs independently of N-linked glycosylation of Fcgamma, in contrast with the properties of host FcgammaRs. To gain further insight into the interaction with Fcgamma, truncation mutants of the vFcgammaR gp68 ectodomain were probed for Fcgamma binding, resulting in localization of the Fcgamma binding site on gp68 to residues 71 to 289, a region including an immunoglobulin-like domain. Gel filtration and biosensor binding experiments revealed that, unlike host FcgammaRs but similar to the herpes simplex virus type 1 (HSV-1) Fc receptor gE-gI, gp68 binds to the C(H)2-C(H)3 interdomain interface of the Fcgamma dimer with a nanomolar affinity and a 2:1 stoichiometry. Unlike gE-gI, which binds Fcgamma at the slightly basic pH of the extracellular milieu but not at the acidic pH of endosomes, the gp68/Fcgamma complex is stable at pH values from 5.6 to pH 8.1. These data indicate that the mechanistic details of Fc binding by HCMV gp68 differ from those of host FcgammaRs and from that of HSV-1 gE-gI, suggesting distinct functional and recognition properties.