Markus Wagner

Publications of Markus Wagner

• Engineering of cytomegalovirus genomes for recombinant live herpesvirus vaccines.

  Authors: Christian A Mohr, Luka Cîcîn-Saîn, Markus Wagner, Torsten Sacher, Margit Schnee, Zsolt Ruzsics, Ulrich H. Koszinowski

  International journal of medical microbiology : IJMM. 02/2008; 298(1-2):115-25.

  The advances of sequence knowledge and genetic engineering hold a great promise for a rational approach to vaccine development. Herpesviruses are important pathogens of all vertebrates. They causeThe advances of sequence knowledge and genetic engineering hold a great promise for a rational approach to vaccine development. Herpesviruses are important pathogens of all vertebrates. They cause acute and chronic infections and persist in their hosts for life. In man there are eight herpesviruses known and most of them can be linked to diseases. To date only one licensed vaccine against a human herpesvirus exists and there is no proven successful concept on rational design for herpesvirus vaccines available. Here, we use new reverse genetic systems, based on the 230-kb mouse cytomegalovirus genome to explore new methods of vaccine delivery and of virus attenuation. With regard to virus delivery, we show that the bacterial transfer of the infectious DNA in vivo is theoretically possible but not yet a practical option. With regard to a rational approach of virus attenuation, we consider a selective deletion of viral genes that modulate the immune response of the host.

• Viral interference with B7-1 costimulation: a new role for murine cytomegalovirus fc receptor-1.

  Authors: Justine D Mintern, Elizabeth J Klemm, Markus Wagner, Marie-Eve Paquet, Melanie D Napier, You-Me Kim, Ulrich H. Koszinowski, Hidde L Ploegh

  Journal of immunology (Baltimore, Md. : 1950). 01/2007; 177(12):8422-31.

  Murine CMV (MCMV), a beta-herpesvirus, infects dendritic cells (DC) and impairs their function. The underlying events are poorly described. In this study, we identify MCMV m138 as the viral geneMurine CMV (MCMV), a beta-herpesvirus, infects dendritic cells (DC) and impairs their function. The underlying events are poorly described. In this study, we identify MCMV m138 as the viral gene responsible for promoting the rapid disappearance of the costimulatory molecule B7-1 (CD80) from the cell surface of DC. This was unexpected, as m138 was previously identified as fcr-1, a putative virus-encoded FcR. m138 impaired the ability of DC to activate CD8+ T cells. Biochemical analysis and immunocytochemistry showed that m138 targets B7-1 in the secretory pathway and reroutes it to lysosomal associated membrane glycoprotein-1+ compartments. These results show a novel function for m138 in MCMV infection and identify the first viral protein to target B7-1.

• Cytomegalovirus encodes a positive regulator of antigen presentation.

  Authors: Rafaela Holtappels, Dorothea Gillert-Marien, Doris Thomas, Jürgen Podlech, Petra Deegen, Sylvia Herter, Silke A Oehrlein-Karpi, Dennis Strand, Markus Wagner, Matthias J. Reddehase

  Journal of virology. 09/2006; 80(15):7613-24.

  Murine cytomegalovirus encodes three regulators of antigen presentation to antiviral CD8 T cells. According to current paradigms, all three regulators are committed to the inhibition of theMurine cytomegalovirus encodes three regulators of antigen presentation to antiviral CD8 T cells. According to current paradigms, all three regulators are committed to the inhibition of the presentation of antigenic peptides. Whereas m152/gp40 catalyzes the retention of peptide-loaded major histocompatibility complex (MHC) class I molecules in a cis-Golgi compartment, m06/gp48 binds stably to class I molecules and directs them into the cellular cargo-sorting pathway of lysosomal degradation. Regulator m04/gp34 also binds stably to class I molecules, but unlike m152 and m06, it does not downmodulate MHC class I cell surface expression. It has entered the literature as a direct inhibitor of T-cell recognition of the MHC-peptide complex at the cell surface. In this work, we have studied the presentation of antigenic viral peptides in cells infected with a comprehensive set of mutant viruses expressing the three regulators separately as well as in all possible combinations. The results redefine m04 as a positive regulator dedicated to the facilitation of antigen presentation. When expressed alone, it did not inhibit T-cell recognition, and when expressed in the presence of m152, it restored antigen presentation by antagonizing the inhibitory function of m152. Its intrinsic positive function, however, was antagonized and even slightly overcompensated for by the negative regulator m06. In an adoptive cell transfer model, the opposing forces of the three regulators were found to govern immune surveillance in the infected host. While negative regulators, also known as immunoevasins, are common, the existence of a positive regulator is without precedent and indicates an intriguing genetic potential of this virus to influence antigen presentation.

• Transposon-assisted cloning and traceless mutagenesis of adenoviruses: Development of a novel vector based on species D.

  Authors: Zsolt Ruzsics, Markus Wagner, Andrea Osterlehner, Jonathan Cook, Ulrich Koszinowski, Hans-Gerhard Burgert

  Journal of virology. 09/2006; 80(16):8100-13.

  Until recently, adenovirus (Ad)-mediated gene therapy was almost exclusively based on human Ad type 5 (Ad5). Preexisting immunity and the limited, coxsackievirus and adenovirus receptor-dependentUntil recently, adenovirus (Ad)-mediated gene therapy was almost exclusively based on human Ad type 5 (Ad5). Preexisting immunity and the limited, coxsackievirus and adenovirus receptor-dependent tropism of Ad5 stimulated attempts to exploit the natural diversity in tropism of the other 50 known human Ad serotypes. Aiming in particular at immunotherapy and vaccination, we have screened representative serotypes from different Ad species for their ability to infect dendritic cells. Ad19a, an Ad from species D, was selected for development as a new vector for vaccination and cancer gene therapy. To clone and manipulate its genome, we have developed a novel methodology, coined "exposon mutagenesis," that allows the rapid and precise introduction of virtually any genetic alteration (deletions, point mutations, or insertions) into recombinant Ad bacterial artificial chromosomes. The versatility of the system was exemplified by deleting the E3 region of Ad19a, by specifically knocking out expression of a species-specific E3 gene, E3/49K, and by reinserting E3/49K into an E3 null Ad19a mutant. The technology requires only limited sequence information and is applicable to other Ad species. Therefore, it should be extremely valuable for the analysis of gene functions from any Ad species. In addition, a basic, replication-defective E1- and E3-deleted Ad19a vector expressing GFP (Ad19aGFP) was generated. This new vector based on species D Ads exhibits a very promising tropism for lymphoid and muscle cells and shows great potential as an alternative vector for transduction of cell types that are resistant to or only poorly transduced by conventional Ad5-based vectors.

• Manipulating cytomegalovirus genomes by BAC mutagenesis: Strategies and applications

  Authors: Wolfram Brune, Markus Wagner, Martin Messerle

  01/2006: pages 61-69;

  ISBN: 1-904455-02-6

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• A cytomegaloviral protein reveals a dual role for STAT2 in IFN-{gamma} signaling and antiviral responses.

  Authors: Albert Zimmermann, Mirko Trilling, Markus Wagner, Manuel Wilborn, Ivan Bubic, Stipan Jonjic, Ulrich Koszinowski, Hartmut Hengel

  The Journal of experimental medicine. 06/2005; 201(10):1543-53.

  A mouse cytomegalovirus (MCMV) gene conferring interferon (IFN) resistance was identified. This gene, M27, encodes a 79-kD protein that selectively binds and down-regulates for signal transducer andA mouse cytomegalovirus (MCMV) gene conferring interferon (IFN) resistance was identified. This gene, M27, encodes a 79-kD protein that selectively binds and down-regulates for signal transducer and activator of transcription (STAT)-2, but it has no effect on STAT1 activation and signaling. The absence of pM27 conferred MCMV susceptibility to type I IFNs (alpha/beta), but it had a much more dramatic effect on type II IFNs (gamma) in vitro and in vivo. A comparative analysis of M27(+) and M27(-) MCMV revealed that the antiviral efficiency of IFN-gamma was partially dependent on the synergistic action of type I IFNs that required STAT2. Moreover, STAT2 was directly activated by IFN-gamma. This effect required IFN receptor expression and was independent of type I IFNs. IFN-gamma induced increasing levels of tyrosine-phosphorylated STAT2 in M27(-) MCMV-infected cells that were essential for the antiviral potency of IFN-gamma. pM27 represents a new strategy for simultaneous evasions from types I and II IFNs, and it documents an unknown biological significance for STAT2 in antiviral IFN-gamma responses.

• The human cytomegalovirus UL78 gene is highly conserved among clinical isolates, but is dispensable for replication in fibroblasts and a renal artery organ-culture system.

  Authors: Detlef Michel, Irena Milotić, Markus Wagner, Bianca Vaida, Jens Holl, Ramona Ansorge, Thomas Mertens

  The Journal of general virology. 03/2005; 86(Pt 2):297-306.

  The human cytomegalovirus (HCMV) UL78 ORF is considered to encode a seven-transmembrane receptor. However, neither the gene nor the UL78 protein has been characterized so far. The objective of thisThe human cytomegalovirus (HCMV) UL78 ORF is considered to encode a seven-transmembrane receptor. However, neither the gene nor the UL78 protein has been characterized so far. The objective of this study was to investigate the UL78 gene and to clarify whether it is essential for replication. UL78 transcription was activated early after infection, was inhibited by cycloheximide but not by phosphonoacetic acid, and resulted in a 1.7 kb mRNA. Later in the replication cycle, a second mRNA of 4 kb evolved, comprising the UL77 and UL78 ORFs. The 5' end of the UL78 mRNA initiated 48 bp upstream of the translation start and the polyadenylated tail started 268 bp downstream of the UL78 translation stop codon within the UL79 ORF. By using bacterial artificial chromosome technology, a recombinant HCMV lacking most of the UL78 coding region was constructed. Successful reconstitution of the UL78-deficient virus proved that the gene was not essential for virus replication in fibroblasts. The deletion also did not reduce virus replication in ex vivo-cultured sections of human renal arteries. Analysis of viral proteins at different stages of the replication cycle confirmed these results. Among clinical HCMV isolates, the predicted UL78 protein was highly conserved. However, an accumulation of different single mutations could be found in the N-terminal region and at the very end of the C terminus. Due to the absence of an in vivo HCMV model, the role of UL78 in the pathogenesis of HCMV infection in humans remains unclear.

• Identification of a mouse cytomegalovirus gene selectively targeting CD86 expression on antigen-presenting cells.

  Authors: Andrea Loewendorf, Corinna Krüger, Eva Maria Borst, Markus Wagner, Ursula Just, Martin Messerle

  Journal of virology. 01/2005; 78(23):13062-71.

  We and others have shown that infection of dendritic cells with murine cytomegalovirus (MCMV) leads to severe functional impairment of these antigen-presenting cells (D. M. Andrews, C. E. Andoniou,We and others have shown that infection of dendritic cells with murine cytomegalovirus (MCMV) leads to severe functional impairment of these antigen-presenting cells (D. M. Andrews, C. E. Andoniou, F. Granucci, P. Ricciardi-Castagnoli, and M. A. Degli-Esposti, Nat. Immunol. 2:1077-1084, 2001; S. Mathys, T. Schroeder, J. Ellwart, U. H. Koszinowski, M. Messerle, and U. Just, J. Infect. Dis. 187:988-999, 2003). Phenotypically, reduced surface expression of costimulatory molecules and major histocompatibility complex molecules was detected. In order to identify the molecular basis for the observed effects, we generated MCMV mutants with large deletions of nonessential genes. The study was facilitated by the finding that a monocyte-macrophage cell line displayed similar phenotypic alterations after MCMV infection. By analyzing the expression of cell surface molecules on infected cells, we identified a mutant virus which is no longer able to downmodulate the expression of the costimulatory molecule CD86. Additional mutants with smaller deletions allowed us to pin down the responsible gene to a certain genomic region. RNA analysis led to the identification of the spliced gene m147.5, encoding a protein with 145 amino acids. Experiments with an m147.5 mutant revealed that the protein affects CD86 expression only, suggesting that additional MCMV genes are responsible for downmodulation of the other surface molecules. Identification of viral gene products interfering with functionally important proteins of antigen-presenting cells will provide the basis to dissect the complex interaction of CMV with these important cells and to evaluate the biological importance of these viral genes in vivo.

• A cyclooxygenase-2 homologue encoded by rhesus cytomegalovirus is a determinant for endothelial cell tropism.

  Authors: Cary A Rue, Michael A Jarvis, Amber J Knoche, Heather L Meyers, Victor R DeFilippis, Scott G Hansen, Markus Wagner, Klaus Früh, David G Anders, Scott W Wong, Peter A Barry, Jay A Nelson

  Journal of virology. 12/2004; 78(22):12529-36.

  Cyclooxygenase-2 (COX-2) is a cellular enzyme in the eicosanoid synthetic pathway that mediates the synthesis of prostaglandins from arachidonic acid. The eicosanoids function as critical regulatorsCyclooxygenase-2 (COX-2) is a cellular enzyme in the eicosanoid synthetic pathway that mediates the synthesis of prostaglandins from arachidonic acid. The eicosanoids function as critical regulators of a number of cellular processes, including the acute and chronic inflammatory response, hemostasis, and the innate immune response. Human cytomegalovirus (HCMV), which does not encode a viral COX-2 isoform, has been shown to induce cellular COX-2 expression. Importantly, although the precise role of COX-2 in CMV replication is unknown, COX-2 induction was shown to be critical for normal HCMV replication. In an earlier study, we identified an open reading frame (Rh10) within the rhesus cytomegalovirus (RhCMV) genome that encoded a putative protein (designated vCOX-2) with high homology to cellular COX-2. In the current study, we show that vCOX-2 is expressed with early-gene kinetics during RhCMV infection, resulting in production of a 70-kDa protein. Consistent with the expression of a viral COX-2 isoform, cellular COX-2 expression was not induced during RhCMV infection. Finally, analysis of growth of recombinant RhCMV with vCOX-2 deleted identified vCOX-2 as a critical determinant for replication in endothelial cells.

• Identification of proteins associated with murine cytomegalovirus virions.

  Authors: Lisa M Kattenhorn, Ryan Mills, Markus Wagner, Alexandre Lomsadze, Vsevolod Makeev, Mark Borodovsky, Hidde L Ploegh, Benedikt M Kessler

  Journal of virology. 11/2004; 78(20):11187-97.

  Proteins associated with the murine cytomegalovirus (MCMV) viral particle were identified by a combined approach of proteomic and genomic methods. Purified MCMV virions were dissociated by completeProteins associated with the murine cytomegalovirus (MCMV) viral particle were identified by a combined approach of proteomic and genomic methods. Purified MCMV virions were dissociated by complete denaturation and subjected to either separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in-gel digestion or treated directly by in-solution tryptic digestion. Peptides were separated by nanoflow liquid chromatography and analyzed by tandem mass spectrometry (LC-MS/MS). The MS/MS spectra obtained were searched against a database of MCMV open reading frames (ORFs) predicted to be protein coding by an MCMV-specific version of the gene prediction algorithm GeneMarkS. We identified 38 proteins from the capsid, tegument, glycoprotein, replication, and immunomodulatory protein families, as well as 20 genes of unknown function. Observed irregularities in coding potential suggested possible sequence errors in the 3'-proximal ends of m20 and M31. These errors were experimentally confirmed by sequencing analysis. The MS data further indicated the presence of peptides derived from the unannotated ORFs ORF(c225441-226898) (m166.5) and ORF(105932-106072). Immunoblot experiments confirmed expression of m166.5 during viral infection.

• Human cytomegalovirus UL131-128 genes are indispensable for virus growth in endothelial cells and virus transfer to leukocytes.

  Authors: Gabriele Hahn, Maria Grazia Revello, Marco Patrone, Elena Percivalle, Giulia Campanini, Antonella Sarasini, Markus Wagner, Andrea Gallina, Gabriele Milanesi, Ulrich Koszinowski, Fausto Baldanti, Giuseppe Gerna

  Journal of virology. 10/2004; 78(18):10023-33.

  Human cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects and morbidity in immunocompromised patients and a potential trigger for vascular disease. HCMVHuman cytomegalovirus (HCMV), a ubiquitous human pathogen, is the leading cause of birth defects and morbidity in immunocompromised patients and a potential trigger for vascular disease. HCMV replicates in vascular endothelial cells and drives leukocyte-mediated viral dissemination through close endothelium- leukocyte interaction. However, the genetic basis of HCMV growth in endothelial cells and transfer to leukocytes is unknown. We show here that the UL131-128 gene locus of HCMV is indispensable for both productive infection of endothelial cells and transmission to leukocytes. The experimental evidence for this is based on both the loss-of-function phenotype in knockout mutants and natural variants and the gain-of-function phenotype by trans-complementation with individual UL131, UL130, and UL128 genes. Our findings suggest that a common mechanism of virus transfer may be involved in both endothelial cell tropism and leukocyte transfer and shed light on a crucial step in the pathogenesis of HCMV infection.

• Comprehensive mutational analysis of a herpesvirus gene in the viral genome context reveals a region essential for virus replication.

  Authors: Anja Bubeck, Markus Wagner, Zsolt Ruzsics, Mark Lötzerich, Margot Iglesias, Ila R Singh, Ulrich H. Koszinowski

  Journal of virology. 09/2004; 78(15):8026-35.

  Essential viral proteins perform vital functions during morphogenesis via a complex interaction with other viral and cellular gene products. Here, we present a novel approach to comprehensiveEssential viral proteins perform vital functions during morphogenesis via a complex interaction with other viral and cellular gene products. Here, we present a novel approach to comprehensive mutagenesis of essential cytomegalovirus genes and biological analysis in the 230-kbp-genome context. A random Tn7-based mutagenesis procedure at the single-gene level was combined with site-specific recombination via the FLP/FLP recognition target site system for viral genome reconstitution. We show the function of more than 100 mutants from a larger library of M50/p35, a protein involved in capsid egress from the nucleus. This protein recruits other viral proteins and cellular enzymes to the inner nuclear membrane. Our approach enabled us to rapidly discriminate between essential and nonessential regions within the coding sequence. Based on the prediction of the screen, we were able to map a site essential for viral protein-protein interaction at the amino acid level.

• Gain of virulence caused by loss of a gene in murine cytomegalovirus.

  Authors: Ivan Bubić, Markus Wagner, Astrid Krmpotić, Tanja Saulig, Sungjin Kim, Wayne M Yokoyama, Stipan Jonjić, Ulrich H. Koszinowski

  Journal of virology. 08/2004; 78(14):7536-44.

  Mouse strains are either resistant or susceptible to murine cytomegalovirus (MCMV). Resistance is determined by the Cmv1(r) (Ly49h) gene, which encodes the Ly49H NK cell activation receptor. TheMouse strains are either resistant or susceptible to murine cytomegalovirus (MCMV). Resistance is determined by the Cmv1(r) (Ly49h) gene, which encodes the Ly49H NK cell activation receptor. The protein encoded by the m157 gene of MCMV has been defined as a ligand for Ly49H. To find out whether the m157 protein is the only Ly49H ligand encoded by MCMV, we constructed the m157 deletion mutant and a revertant virus. Viruses were tested for susceptibility to NK cell control in Ly49H+ and Ly49H- mouse strains. Deletion of the m157 gene abolished the viral activation of Ly49H+ NK cells, resulting in higher virus virulence in vivo. Thus, in the absence of m157, Ly49H+ mice react like susceptible strains. 129/SvJ mice lack the Ly49H activation NK cell receptor but express the inhibitory Ly49I NK cell receptor that binds to the m157 protein. The Deltam157 inhibitory phenotype was weak because MCMV encodes a number of proteins that mediate NK inhibition, whose contribution could be shown by another mutant.

• Escape of mutant double-stranded DNA virus from innate immune control.

  Authors: Anthony R French, Jeanette T Pingel, Markus Wagner, Ivan Bubic, Liping Yang, Sungjin Kim, Ulrich Koszinowski, Stipan Jonjic, Wayne M Yokoyama

  Immunity. 07/2004; 20(6):747-56.

  As innate immune system components, natural killer (NK) cells respond rapidly to infections and effectively control replication of pathogens, including murine cytomegalovirus (MCMV), aAs innate immune system components, natural killer (NK) cells respond rapidly to infections and effectively control replication of pathogens, including murine cytomegalovirus (MCMV), a double-stranded DNA beta-herpesvirus. In the absence of NK cell control, MCMV infection results in early mortality due to uncontrolled viral replication. However, here we show that even in the face of initial NK cell control, there is late recrudescence of disease and mortality in immunodeficient mice due to the outgrowth of MCMV mutants that escape recognition by innate NK cells. These data suggest that viral infections in certain clinical settings also may be due to viral escape from innate immunity.

• Murine cytomegalovirus interference with antigen presentation has little effect on the size or the effector memory phenotype of the CD8 T cell response.

  Authors: Marielle C Gold, Michael W Munks, Markus Wagner, Christopher W McMahon, Ann Kelly, Daniel G Kavanagh, Mark K Slifka, Ulrich H. Koszinowski, David H Raulet, Ann B Hill

  Journal of immunology (Baltimore, Md. : 1950). 07/2004; 172(11):6944-53.

  As with most herpesviruses, CMVs encode viral genes that inhibit Ag presentation to CD8 T cells (VIPRs). VIPR function has been assumed to be essential for CMV to establish its characteristicAs with most herpesviruses, CMVs encode viral genes that inhibit Ag presentation to CD8 T cells (VIPRs). VIPR function has been assumed to be essential for CMV to establish its characteristic lifetime infection of its host. We compared infection of C57BL/6 mice with wild-type murine CMV (MCMV) and a virus lacking each of MCMV's three known VIPRs: m4, m6, and m152. During acute infection, there was very little difference between the two viruses with respect to the kinetics of viral replication and clearance, or in the size and kinetics of the virus-specific CD8 T cell response. During chronic infection, a large, effector memory, virus-specific CD8 T cell population (CD8(low)CD62L(-)CD11c(+)NKG2A(+)) was maintained in both infections; the size and phenotype of the CD8 T cell response to both viruses was remarkably similar. The characteristic effector memory phenotype of the CD8 T cells suggested that both wild-type and Deltam4+m6+m152 virus continued to present Ag to CD8 T cells during the chronic phase of infection. During the chronic phase of infection, MCMV cannot be isolated from immunocompetent mice. However, upon immunosuppression, both Deltam4+m6+m152 and wild-type virus could be reactivated from mice infected for 6 wk. Thus, restoring the ability of CD8 T cells to detect MCMV had little apparent effect on the course of MCMV infection and on the CD8 T cell response to it. These results challenge the notion that VIPR function is necessary for CMV persistence in the host.

• Expansion of protective CD8+ T-cell responses driven by recombinant cytomegaloviruses.

  Authors: Urs Karrer, Markus Wagner, Sophie Sierro, Annette Oxenius, Hartmut Hengel, Tilman Dumrese, Stefan Freigang, Ulrich H. Koszinowski, Rodney E Phillips, Paul Klenerman

  Journal of virology. 04/2004; 78(5):2255-64.

  CD8(+) T cells are critical for the control of many persistent viral infections, such as human immunodeficiency virus, hepatitis C virus, Epstein-Barr virus, and cytomegalovirus (CMV). In mostCD8(+) T cells are critical for the control of many persistent viral infections, such as human immunodeficiency virus, hepatitis C virus, Epstein-Barr virus, and cytomegalovirus (CMV). In most infections, large CD8(+)-T-cell populations are induced early but then contract and are maintained thereafter at lower levels. In contrast, CD8(+) T cells specific for murine CMV (MCMV) have been shown to gradually accumulate after resolution of primary infection. This unique behavior is restricted to certain epitopes, including an immunodominant epitope derived from the immediate-early 1 (IE1) gene product. To explore the mechanism behind this further, we measured CD8(+)-T-cell-mediated immunity induced by recombinant MCMV-expressing epitopes derived from influenza A virus or lymphocytic choriomeningitis virus placed under the control of an IE promoter. We observed that virus-specific CD8(+)-T-cell populations were induced and that these expanded gradually over time. Importantly, these CD8(+) T cells provided long-term protection against challenge without boosting. These results demonstrate a unique pattern of accumulating T cells, which provide long-lasting immune protection, that is independent of the initial immunodominance of the epitope and indicates the potential of T-cell-inducing vaccines based on persistent vectors.

• Mutagenesis of viral BACs with linear PCR fragments (ET recombination).

  Authors: Markus Wagner, Ulrich H. Koszinowski

  Methods in molecular biology (Clifton, N.J.). 02/2004; 256:257-68.

• Cytomegalovirus misleads its host by priming of CD8 T cells specific for an epitope not presented in infected tissues.

  Authors: Rafaela Holtappels, Jürgen Podlech, Marcus-Folker Pahl-Seibert, Markus Jülch, Doris Thomas, Christian O Simon, Markus Wagner, Matthias J. Reddehase

  The Journal of experimental medicine. 02/2004; 199(1):131-6.

  Cytomegaloviruses (CMVs) code for several proteins that inhibit the presentation of antigenic peptides to CD8 T cells. Although the molecular mechanisms of CMV interference with the majorCytomegaloviruses (CMVs) code for several proteins that inhibit the presentation of antigenic peptides to CD8 T cells. Although the molecular mechanisms of CMV interference with the major histocompatibility complex class I pathway are long understood, surprisingly little evidence exists to support a role in vivo. Here we document the first example of the presentation of an antigenic peptide being blocked by a CMV immune evasion protein in organs relevant to CMV disease. Although this Db-restricted peptide, which is derived from the antiapoptotic protein M45 of murine CMV (mCMV), is classified as an immunodominant peptide based on response magnitude and long-term memory, adoptive transfer of M45 epitope-specific CD8 T cells did not protect against infection with wild-type mCMV. Notably, the same cells protected C57BL/6 mice infected with an mCMV mutant in which immune evasion protein m152/gp40 is deleted. These data indicate that direct presentation or cross-presentation of an antigenic peptide by professional antigen-presenting cells can efficiently prime CD8 T cells that fail in protection against CMV organ disease because m152/gp40 prevents presentation of this peptide in pathogenetically relevant tissue cells.

• Role of murine cytomegalovirus US22 gene family members in replication in macrophages.

  Authors: Carine Ménard, Markus Wagner, Zsolt Ruzsics, Karina Holak, Wolfram Brune, Ann E Campbell, Ulrich H. Koszinowski

  Journal of virology. 06/2003; 77(10):5557-70.

  The large cytomegalovirus (CMV) US22 gene family, found in all betaherpesviruses, comprises 12 members in both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV). Conserved sequenceThe large cytomegalovirus (CMV) US22 gene family, found in all betaherpesviruses, comprises 12 members in both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV). Conserved sequence motifs suggested a common ancestry and related functions for these gene products. Two members of this family, m140 and m141, were recently shown to affect MCMV replication on macrophages. To test the role of all US22 members in cell tropism, we analyzed the growth properties in different cell types of MCMV mutants carrying transposon insertions in all 12 US22 gene family members. When necessary, additional targeted mutants with gene deletions, ATG deletions, and ectopic gene revertants were constructed. Mutants with disruption of genes M23, M24, m25.1, m25.2, and m128 (ie2) showed no obvious growth phenotype, whereas growth of M43 mutants was reduced in a number of cell lines. Genes m142 and m143 were shown to be essential for virus replication. Growth of mutants with insertions into genes M36, m139, m140, and m141 in macrophages was severely affected. The common phenotype of the m139, m140, and m141 mutants was explained by an interaction at the protein level. The M36-dependent macrophage growth phenotype could be explained by the antiapoptotic function of the gene that was required for growth on macrophages but not for growth on other cell types. Together, the comprehensive set of mutants of the US22 gene family suggests that individual family members have diverged through evolution to serve a variety of functions for the virus.

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• Cloning of the genomes of human cytomegalovirus strains Toledo, TownevarRIT3, and Towne long as BACs and site-directed mutagenesis using a PCR-based technique.

  Authors: Gabriele Hahn, Dietlind Rose, Markus Wagner, Sylvia Rhiel, Michael A McVoy

  Virology. 04/2003; 307(1):164-77.

  The 230-kb human cytomegalovirus genome is among the largest of the known viruses. Experiments to determine the genetic determinants of attenuation, pathogenesis, and tissue tropism are underway;The 230-kb human cytomegalovirus genome is among the largest of the known viruses. Experiments to determine the genetic determinants of attenuation, pathogenesis, and tissue tropism are underway; however, a lack of complete sequence data for multiple strains and substantial problems with genetic instability during in vitro propagation create serious complications for such studies. For example, recent findings suggest that common laboratory strains Towne and AD169 passaged in cultured human fibroblasts are missing up to 15 kb of genetic information relative to clinical isolates. To establish standard, genetically stable genomes that can be sequenced, disseminated, and repeatedly reconstituted to produce virus stocks, we have undertaken to clone two variants of Towne, designated Towne(long) and Towne(short) (referred to as TownevarRIT3) (A., Proc. Natl. Acad. Sci. USA 98, 7829-7834), and the pathogenic strain Toledo into bacterial artificial chromosomes (BACs). We further demonstrate the ease with which mutagenesis can be achieved by deleting 13.5 kb from the Toledo genome using a PCR-based technique.