Ulrich Koszinowski

Publications of Ulrich Koszinowski

• Recombinant mouse cytomegalovirus expressing a ligand for the NKG2D receptor is attenuated and has improved vaccine properties.

  Authors: Irena Slavuljica, Andreas Busche, Marina Babić, Maja Mitrović, Iva Gašparović, Durđica Cekinović, Elitza Markova Car, Ester Pernjak Pugel, Ana Ciković, Vanda Juranić Lisnić, William J Britt, Ulrich Koszinowski, Martin Messerle, Astrid Krmpotić, Stipan Jonjić

  The Journal of clinical investigation. 12/2010; 120(12):4532-45.

  Human CMV (HCMV) is a major cause of morbidity and mortality in both congenitally infected and immunocompromised individuals. Development of an effective HCMV vaccine would help protect theseHuman CMV (HCMV) is a major cause of morbidity and mortality in both congenitally infected and immunocompromised individuals. Development of an effective HCMV vaccine would help protect these vulnerable groups. NK group 2, member D (NKG2D) is a potent activating receptor expressed by cells of the innate and adaptive immune systems. Its importance in HCMV immune surveillance is indicated by the elaborative evasion mechanisms evolved by the virus to avoid NKG2D. In order to study this signaling pathway, we engineered a recombinant mouse CMV expressing the high-affinity NKG2D ligand RAE-1γ (RAE-1γMCMV). Expression of RAE-1γ by MCMV resulted in profound virus attenuation in vivo and lower latent viral DNA loads. RAE-1γMCMV infection was efficiently controlled by immunodeficient hosts, including mice lacking type I interferon receptors or immunosuppressed by sublethal γ-irradiation. Features of MCMV infection in neonates were also diminished. Despite tight innate immune control, RAE-1γMCMV infection elicited strong and long-lasting protective immunity. Maternal RAE-1γMCMV immunization protected neonatal mice from MCMV disease via placental transfer of antiviral Abs. Despite strong selective pressure, the RAE-1γ transgene did not exhibit sequence variation following infection. Together, our results indicate that use of a recombinant virus encoding the ligand for an activating NK cell receptor could be a powerful approach to developing a safe and immunogenic HCMV vaccine.

• A spread-deficient cytomegalovirus for assessment of first-target cells in vaccination.

  Authors: Christian Andreas Mohr, Jurica Arapovic, Hermine Mühlbach, Marc Panzer, Annelies Weyn, Lars Dölken, Astrid Krmpotic, David Voehringer, Zsolt Ruzsics, Ulrich Koszinowski, Torsten Sacher

  Journal of virology. 05/2010; 84(15):7730-42.

  Human cytomegalovirus (HCMV) is a human pathogen that causes severe disease primarily in the immunocompromised or immunologically immature individual. To date, no vaccine is available. We describeHuman cytomegalovirus (HCMV) is a human pathogen that causes severe disease primarily in the immunocompromised or immunologically immature individual. To date, no vaccine is available. We describe use of a spread-deficient murine CMV (MCMV) as a novel approach for betaherpesvirus vaccination. To generate a spread-deficient MCMV, the conserved, essential gene M94 was deleted. Immunization with MCMV-DeltaM94 is apathogenic and protective against wild-type challenge even in highly susceptible IFNalphabetaR(-/-) mice. MCMV-DeltaM94 was able to induce a robust CD4(+) and CD8(+) T-cell response as well as a neutralizing antibody response comparable to that induced by wild-type infection. Endothelial cells were identified as activators of CD8(+) T cells in vivo. Thus, the vaccination with a spread-deficient betaherpesvirus is a safe and protective strategy and allows the linkage between cell tropism and immunogenicity. Furthermore, genomes of MCMV-DeltaM94 were present in lungs 12 months after infection, revealing first-target cells as sites of genome maintenance.

• The m74 gene product of murine cytomegalovirus (MCMV) is a functional homolog of human CMV gO and determines the entry pathway of MCMV.

  Authors: Laura Scrivano, Jasmina Esterlechner, Hermine Mühlbach, Nicole Ettischer, Christoph Hagen, Kay Grünewald, Christian A Mohr, Zsolt Ruzsics, Ulrich Koszinowski, Barbara Adler

  Journal of virology. 02/2010; 84(9):4469-80.

  The glycoprotein gO (UL74) of human cytomegalovirus (HCMV) forms a complex with gH/gL. Virus mutants with a deletion of gO show a defect in secondary envelopment with the consequence that virusThe glycoprotein gO (UL74) of human cytomegalovirus (HCMV) forms a complex with gH/gL. Virus mutants with a deletion of gO show a defect in secondary envelopment with the consequence that virus spread is restricted to a cell-associated pathway. Here we report that the positional homolog of HCMV gO, m74 of mouse CMV (MCMV), codes for a glycosylated protein which also forms a complex with gH (M75). m74 knockout mutants of MCMV show the same spread phenotype as gO knockout mutants of HCMV, namely, a shift from supernatant-driven to cell-associated spread. We could show that this phenotype is due to a reduction of infectious virus particles in cell culture supernatants. m74 knockout mutants enter fibroblasts via an energy-dependent and pH-sensitive pathway, whereas in the presence of an intact m74 gene product, entry is neither energy dependent nor pH sensitive. This entry phenotype is shared by HCMV expressing or lacking gO. Our data indicate that the m74 and UL74 gene products both codetermine CMV spread and CMV entry into cells. We postulate that MCMV, like HCMV, expresses alternative gH/gL complexes which govern cell-to-cell spread of the virus.

• Evolutionarily conserved herpesviral protein interaction networks.

  Authors: Even Fossum, Caroline C Friedel, Seesandra V Rajagopala, Björn Titz, Armin Baiker, Tina Schmidt, Theo Kraus, Thorsten Stellberger, Christiane Rutenberg, Silpa Suthram [......] Christine Zeretzke, Yu-An Dong, Hélène Boulet, Manfred Koegl, Susanne M Bailer, Ulrich Koszinowski, Trey Ideker, Peter Uetz, Ralf Zimmer, Jürgen Haas

  PLoS pathogens. 10/2009; 5(9):e1000570.

  Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encodingHerpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species.

• Virally Infected Mouse Liver Endothelial Cells Trigger CD8+ T-cell Immunity.

  Authors: Michaela Kern, Alexey Popov, Kai Scholz, Beatrix Schumak, Dominik Djandji, Andreas Limmer, Daniela Eggle, Torsten Sacher, Rainer Zawatzky, Rafaela Holtappels, Matthias J. Reddehase, Gunther Hartmann, Svenja Debey-Pascher, Linda Diehl, Ulrich Kalinke, Ulrich Koszinowski, Joachim Schultze, Percy Knolle

  Gastroenterology. 09/2009;

  BACKGROUND & AIMS:: Dendritic cell activation through ligation of pattern-recognition receptors leading to full functional maturation causes induction of CD8(+) T-cell immunity through increasedBACKGROUND & AIMS:: Dendritic cell activation through ligation of pattern-recognition receptors leading to full functional maturation causes induction of CD8(+) T-cell immunity through increased delivery of costimulatory signals instead of tolerance. Here we investigate whether organ-resident antigen-presenting cells, such as liver sinusoidal endothelial cells (LSEC), also switch from tolerogenic to immunogenic CD8(+) T-cell activation upon such stimulation. METHODS AND RESULTS:: We demonstrate that LSEC expressed numerous pattern-recognition receptors that attributed sentinel function, but ligand-induced activation of these receptors was not sufficient to overcome tolerance induction of CD8(+) T-cells. Importantly, viral infection with murine cytomegalovirus (MCMV) caused functional maturation of antigen-presenting LSEC and was sufficient to promote antigen-specific differentiation into effector CD8(+) T-cells in the absence of dendritic cells and independent of CD80/86. CONCLUSIONS:: These results shed new light on the generation of organ-specific immunity and may contribute to overcome tolerance in relevant situations such as cancer.

• Cytomegalovirus microRNAs.

  Authors: Lars Dölken, Sébastien Pfeffer, Ulrich Koszinowski

  Virus genes. 04/2009;

  MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression at a post-transcriptional level in virtually all eukaryotic organisms and some viruses, particularlyMicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression at a post-transcriptional level in virtually all eukaryotic organisms and some viruses, particularly herpesviruses. miRNAs are non-immunogenic, stealthy tools for viruses to regulate their as well as host gene expression. The human cytomegalovirus (HCMV) is the major cause of morbidity in immunocompromised patients and allogenic bone-marrow or organ-transplant recipients and the leading cause of congenital birth defects. HCMV miRNAs may provide valuable targets for new urgently needed antiviral drugs. This review focuses on recent findings for viral miRNAs expressed by cytomegaloviruses (CMV) including data from human, chimpanzee, and murine CMV. These are discussed in the context of findings for other viruses to highlight potentially conserved roles exerted by viral miRNAs.

• UL74 of human cytomegalovirus contributes to virus release by promoting secondary envelopment of virions.

  Authors: Xiao Jing Jiang, Barbara Adler, Kerstin Laib Sampaio, Margarete Digel, Gerhard Jahn, Nicole Ettischer, York-Dieter Stierhof, Laura Scrivano, Ulrich Koszinowski, Michael Mach, Christian Sinzger

  Journal of virology. 04/2008; 82(6):2802-12.

  The glycoprotein (g) complex gH/gL represents an essential part of the herpesvirus fusion machinery mediating entry of cell-free virions and cell-associated viral spread. In some herpesvirusesThe glycoprotein (g) complex gH/gL represents an essential part of the herpesvirus fusion machinery mediating entry of cell-free virions and cell-associated viral spread. In some herpesviruses additional proteins are associated with gH/gL contributing to the cell tropism of the respective virus. Human cytomegalovirus (HCMV) gH/gL forms complexes with either gO (UL74) or proteins of the UL128-131A gene locus. While a contribution of UL128-131A to endothelial cell tropism is known, the role of gO is less clear. We studied the role of gH/gL-associated proteins in HCMV replication in human foreskin fibroblasts (HFF) and human umbilical vein endothelial cells (HUVEC). Deletions of UL74 alone or in combination with mutations of the UL128-131A gene region were introduced into bacterial artificial chromosome vectors derived from the endotheliotropic strain TB40/E. Deletion of UL74 caused a profound defect regarding virus release from infected HFF and HUVEC. Large numbers of capsids accumulated in the cytoplasm of infected HFF but failed to acquire an envelope. Clear cell type differences were observed in the cell-associated spread of the UL74-defective virus. In HFF, focal growth was severely impaired, whereas it was normal in HUVEC. Deletion of UL131A abolished focal growth in endothelial cells. UL74/UL128-131A dual mutants showed severely impaired reconstitution efficiency. Our data suggest that gO plays a critical role in secondary envelopment and release of cell-free virions independent of the cell type but affects cell-associated growth specifically in HFF, whereas UL128-131A contributes to cell-associated spread in HFF and HUVEC.

• Cloning and sequencing of a highly productive, endotheliotropic virus strain derived from human cytomegalovirus TB40/E.

  Authors: Christian Sinzger, Gabriele Hahn, Margarete Digel, Ruth Katona, Kerstin Laib Sampaio, Martin Messerle, Hartmut Hengel, Ulrich Koszinowski, Wolfram Brune, Barbara Adler

  The Journal of general virology. 03/2008; 89(Pt 2):359-68.

  Human cytomegalovirus (HCMV) strain TB40/E, replicates efficiently, exhibits a broad cell tropism and is widely used for infection of endothelial cells and monocyte-derived cells yet has not beenHuman cytomegalovirus (HCMV) strain TB40/E, replicates efficiently, exhibits a broad cell tropism and is widely used for infection of endothelial cells and monocyte-derived cells yet has not been available in a phenotypically homogeneous form compatible with genetic analysis. To overcome this problem, we cloned the TB40/E strain into a bacterial artificial chromosome (BAC) vector. Both highly endotheliotropic and poorly endotheliotropic virus clones, representing three distinct restriction fragment patterns, were reconstituted after transfection of BAC clones derived from previously plaque-purified strain TB40/E. For one of the highly endotheliotropic clones, TB40-BAC4, we provide the genome sequence. Two BACs with identical restriction fragment patterns but different cell tropism were further analysed in the UL128-UL131A gene region. Sequence analysis revealed one coding-relevant adenine insertion at position 332 of UL128 in the BAC of the poorly endotheliotropic virus, which caused a frameshift in the C-terminal part of the coding sequence. Removal of this insertion by markerless mutagenesis restored the highly endotheliotropic phenotype, indicating that the loss of endothelial cell tropism was caused by this insertion. In conclusion, HCMV strain TB40/E, which combines the high endothelial cell tropism of a clinical isolate with the high titre growth of a cell culture adapted strain, is now available as a BAC clone suitable for genetic engineering. The results also suggest BAC cloning as a suitable method for selection of genetically defined virus clones.

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• Mouse cytomegalovirus microRNAs dominate the cellular small RNA profile during lytic infection and show features of posttranscriptional regulation.

  Authors: Lars Dölken, Jonathan Perot, Valérie Cognat, Abdelmalek Alioua, Matthias John, Jürgen Soutschek, Zsolt Ruzsics, Ulrich Koszinowski, Olivier Voinnet, Sébastien Pfeffer

  Journal of virology. 01/2008; 81(24):13771-82.

  MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Originally identified in a variety of organisms ranging from plants to mammals,MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Originally identified in a variety of organisms ranging from plants to mammals, miRNAs have recently been identified in several viruses. Viral miRNAs may play a role in modulating both viral and host gene expression. Here, we report on the identification and characterization of 18 viral miRNAs from mouse fibroblasts lytically infected with the murine cytomegalovirus (MCMV). The MCMV miRNAs are expressed at early times of infection and are scattered in small clusters throughout the genome with up to four distinct miRNAs processed from a single transcript. No significant homologies to human CMV-encoded miRNAs were found. Remarkably, as soon as 24 h after infection, MCMV miRNAs constituted about 35% of the total miRNA pool, and at 72 h postinfection, this proportion was increased to more than 60%. However, despite the abundance of viral miRNAs during the early phase of infection, the expression of some MCMV miRNAs appeared to be regulated. Hence, for three miRNAs we observed polyuridylation of their 3' end, coupled to subsequent degradation. Individual knockout mutants of two of the most abundant MCMV miRNAs, miR-m01-4 and miR-M44-1, or a double knockout mutant of miR-m21-1 and miR-M23-2, incurred no or only a very mild growth deficit in murine embryonic fibroblasts in vitro.

• Role of human cytomegalovirus UL131A in cell type-specific virus entry and release.

  Authors: Barbara Adler, Laura Scrivano, Zsolt Ruzcics, Brigitte Rupp, Christian Sinzger, Ulrich Koszinowski

  The Journal of general virology. 10/2006; 87(Pt 9):2451-60.

  The human cytomegalovirus (HCMV) genes UL128, UL130 and UL131A are essential for endothelial cell infection. Complementation of the defective UL131A gene of the non-endotheliotropic HCMV strain AD169The human cytomegalovirus (HCMV) genes UL128, UL130 and UL131A are essential for endothelial cell infection. Complementation of the defective UL131A gene of the non-endotheliotropic HCMV strain AD169 with wild-type UL131A in cis in an ectopic position restored endothelial cell tropism. The UL131A protein was found in virions in a complex with gH. Coinfection of fibroblasts with UL131A-negative and -positive viruses restored the endothelial cell tropism of UL131A-negative virions by complementing the virions with UL131A protein. Virus entry into endothelial cells, but not into fibroblasts, was blocked by an antipeptide antiserum to pUL131A. AD169, cis-complemented with wild-type UL131A, showed an impaired release of infectious particles from fibroblasts. A comparable defect in virus release was observed when UL131A was expressed ectopically in a virus background already expressing an intact copy of UL131A. In contrast, virus release from infected endothelial cells was not affected by UL131A. These data suggest a dual role for pUL131A in virus entry and virus exit from infected cells.

• 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.

• 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.

• Cloning of the varicella-zoster virus genome as an infectious bacterial artificial chromosome in Escherichia coli.

  Authors: Kazuhiro Nagaike, Yasuko Mori, Yasuyuki Gomi, Hironori Yoshii, Michiaki Takahashi, Markus Wagner, Ulrich Koszinowski, Koichi Yamanishi

  Vaccine. 10/2004; 22(29-30):4069-74.

  The complete genome of the varicella-zoster virus (VZV) Oka strain has been cloned as a bacterial artificial chromosome (BAC). Following electroporation into Escherichia coli (E. coli) strain DH10B,The complete genome of the varicella-zoster virus (VZV) Oka strain has been cloned as a bacterial artificial chromosome (BAC). Following electroporation into Escherichia coli (E. coli) strain DH10B, the VZV BAC was stably propagated over multiple generations of its host. Human embryonic lung (HEL) cells transfected with VZV BAC DNA recovered from DH10B showed cytopathic effect (CPE), and virus spread to neighbouring cells was observed. BAC vector sequences are flanked by loxP sites and, coinfection of the reconstituted virus, with a recombinant adenovirus expressing Cre recombinase removed the bacterial sequences. The resulting recombinant rV02 grew as well as the parental virus in HEL cells. The recombinant VZV will promote VZV research and increase use of the viral genome as an investigative tool.

• 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.

• 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.

• The ribonucleotide reductase R1 homolog of murine cytomegalovirus is not a functional enzyme subunit but is required for pathogenesis.

  Authors: David Lembo, Manuela Donalisio, Anders Hofer, Maura Cornaglia, Wolfram Brune, Ulrich Koszinowski, Lars Thelander, Santo Landolfo

  Journal of virology. 05/2004; 78(8):4278-88.

  Ribonucleotide reductase (RNR) is the key enzyme in the biosynthesis of deoxyribonucleotides. Alpha- and gammaherpesviruses express a functional enzyme, since they code for both the R1 and the R2Ribonucleotide reductase (RNR) is the key enzyme in the biosynthesis of deoxyribonucleotides. Alpha- and gammaherpesviruses express a functional enzyme, since they code for both the R1 and the R2 subunits. By contrast, betaherpesviruses contain an open reading frame (ORF) with homology to R1, but an ORF for R2 is absent, suggesting that they do not express a functional RNR. The M45 protein of murine cytomegalovirus (MCMV) exhibits the sequence features of a class Ia RNR R1 subunit but lacks certain amino acid residues believed to be critical for enzymatic function. It starts to be expressed independently upon the onset of viral DNA synthesis at 12 h after infection and accumulates at later times in the cytoplasm of the infected cells. Moreover, it is associated with the virion particle. To investigate direct involvement of the virally encoded R1 subunit in ribonucleotide reduction, recombinant M45 was tested in enzyme activity assays together with cellular R1 and R2. The results indicate that M45 neither is a functional equivalent of an R1 subunit nor affects the activity or the allosteric control of the mouse enzyme. To replicate in quiescent cells, MCMV induces the expression and activity of the cellular RNR. Mutant viruses in which the M45 gene has been inactivated are avirulent in immunodeficient SCID mice and fail to replicate in their target organs. These results suggest that M45 has evolved a new function that is indispensable for virus replication and pathogenesis in vivo.

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• Rapid enzymatic test for phenotypic HIV protease drug resistance.

  Authors: Dieter Hoffmann, Irmgard Assfalg-Machleidt, Hans Nitschko, Klaus von der Helm, Ulrich Koszinowski, Werner Machleidt

  Biological chemistry. 08/2003; 384(7):1109-17.

  A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid one-step procedure asA phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid one-step procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIV-infected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for high-throughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (Ki). The Michaelis constant (Km) and the inhibition constant (Ki) of recombinant wild-type protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIV-positive patients scored from 'sensitive' to 'highly resistant' according to mutations detected by genotypic analysis. The measured Ki values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The non-infectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays.

• Serial detection of Epstein-Barr virus DNA in sera and peripheral blood leukocyte samples of pediatric renal allograft recipients with persistent mononucleosis-like symptoms defines patients at risk to develop post-transplant lymphoproliferative disease.

  Authors: Hartmut Campe, Gundula Jaeger, Claudia Abou-Ajram, Hans Nitschko, Martin Griebel, Carmen Montoya, Bernd Klare, Ulrich Koszinowski

  Pediatric transplantation. 03/2003; 7(1):46-52.

  We tested blood samples of 25 pediatric renal transplant recipients for Epstein-Barr virus (EBV) DNA load by quantitative polymerase chain reaction (PCR). Eleven of these transplant recipients showedWe tested blood samples of 25 pediatric renal transplant recipients for Epstein-Barr virus (EBV) DNA load by quantitative polymerase chain reaction (PCR). Eleven of these transplant recipients showed clinical persistent mononucleosis-like symptoms years after transplantation (Tx). A quantitation of EBV DNA by PCR in peripheral blood lymphocyte (PBL) and serum samples revealed variable EBV DNA titers. The majority of EBV PCR results in samples of the 14 asymptomatic transplant recipients was repeatedly below detection limit. In contrast, patients with mononucleosis-like symptoms showed persistent EBV genome titers over a period of 6 months, ranging from 75 to 18 750 copies/10 000 PBL and from 680 to 335 000 copies/mL serum, respectively. One child suffering from this mononucleosis-like condition developed an EBV-associated Burkitt-like lymphoma 29 months after Tx. Whereas clinical and histological investigations did not indicate a post-transplant lymphoproliferative disorder (PTLD) until tumor detection, EBV titers in PBL and serum had been high for at least 8 months. We propose that pediatric transplant recipients who show both, recurrent mononucleosis-like symptoms and a sustained high EBV genome load, are at increased risk for severe EBV-related post-transplant complications.

• Activated virus-specific T cells are early indicators of anti-CMV immune reactions in liver transplant patients.

  Authors: Christine Benz, Olaf Utermöhlen, Anna Wulf, Brigitte Villmow, Volker Dries, Tobias Goeser, Ulrich Koszinowski, Dirk H Busch

  Gastroenterology. 05/2002; 122(5):1201-15.

  BACKGROUND & AIMS: Cytomegalovirus (CMV) infection represents the most common infectious complication after liver transplantation. Because CMV-associated complications in liver transplantationBACKGROUND & AIMS: Cytomegalovirus (CMV) infection represents the most common infectious complication after liver transplantation. Because CMV-associated complications in liver transplantation patients are often liver-restricted and clinically unrecognized, diagnosis of early infection or reactivation is still very difficult. Because cytotoxic T cells (CTLs) are crucial for the immune control of CMV, analysis of virus-specific CTLs could contribute to diagnosis and management of CMV infection. METHODS: Major histocompatibility complex class I tetramers and intracellular cytokine staining were used to determine frequencies and phenotypes of peripheral blood CMV/pp65-specific CD8(+) T cells in HLA-A2, -B7, and -B35 positive liver transplantation patients and in healthy individuals. RESULTS: After liver transplantation (6-33 months after liver transplantation), frequencies of CMV-specific T cells were significantly elevated compared with healthy individuals. In contrast to immunoglobulin (Ig) M-negative patients and healthy blood donors, patients with increasing CMV IgM titers or IgG seroconversion had high percentages of activated (CD38(high)) CMV-specific T cells. In recently transplanted patients, activation of CMV-specific T cells was associated with increased transaminases and histopathological abnormalities in the absence of positive CMV-polymerase chain reaction results from peripheral blood. CONCLUSIONS: These data indicate that T-cell analysis based on MHC tetramer staining may be a valuable parameter in the early diagnosis of CMV-induced, liver-restricted complications after liver transplantation.