Ulrich H. Koszinowski

Publications of Ulrich H. Koszinowski

• A Cell Free Protein Fragment Complementation Assay for Monitoring the Core Interaction of the Human Cytomegalovirus Nuclear Egress Complex.

  Authors: Margit Schnee, Felicia Wagner, Ulrich H Koszinowski, Zsolt Ruzsics

  Antiviral research. 05/2012;

  Certain viral protein-protein interactions provide attractive targets for antiviral drug development. Recently, we described a β-lactamase based protein fragment complementation assay (PCA) to studyCertain viral protein-protein interactions provide attractive targets for antiviral drug development. Recently, we described a β-lactamase based protein fragment complementation assay (PCA) to study the core interaction of the nuclear egress complex (NEC) of different herpesviruses in cells. Now, to have a cell free assay for inhibitor screens, we expressed split β-lactamase tagged interaction domains of the viral pUL50 and pUL53 proteins representing the NEC of human cytomegalovirus (HCMV) in bacteria. After validation and basic characterization of this NEC-PCA, we tested peptide inhibitors of the pUL50-pUL53 complex. We show that peptides resembling sequences of the first conserved region of pUL53 can inhibit the NEC-PCA. This, on one hand, indicated that the core interaction in the HCMV NEC is mediated by a linear motif. On the other hand it proved that this new pUL50-pUL53 interaction assay allows a simple cell free test for small molecular inhibitors.

• Degradation of cellular mir-27 by a novel, highly abundant viral transcript is important for efficient virus replication in vivo.

  Authors: Lisa Marcinowski, Mélanie Tanguy, Astrid Krmpotic, Bernd Rädle, Vanda J Lisnić, Lee Tuddenham, Béatrice Chane-Woon-Ming, Zsolt Ruzsics, Florian Erhard, Corinna Benkartek, Marina Babic, Ralf Zimmer, Joanne Trgovcich, Ulrich H Koszinowski, Stipan Jonjic, Sébastien Pfeffer, Lars Dölken

  PLoS pathogens. 02/2012; 8(2):e1002510.

  Cytomegaloviruses express large amounts of viral miRNAs during lytic infection, yet, they only modestly alter the cellular miRNA profile. The most prominent alteration upon lytic murineCytomegaloviruses express large amounts of viral miRNAs during lytic infection, yet, they only modestly alter the cellular miRNA profile. The most prominent alteration upon lytic murine cytomegalovirus (MCMV) infection is the rapid degradation of the cellular miR-27a and miR-27b. Here, we report that this regulation is mediated by the ∼1.7 kb spliced and highly abundant MCMV m169 transcript. Specificity to miR-27a/b is mediated by a single, apparently optimized, miRNA binding site located in its 3'-UTR. This site is easily and efficiently retargeted to other cellular and viral miRNAs by target site replacement. Expression of the 3'-UTR of m169 by an adenoviral vector was sufficient to mediate its function, indicating that no other viral factors are essential in this process. Degradation of miR-27a/b was found to be accompanied by 3'-tailing and -trimming. Despite its dramatic effect on miRNA stability, we found this interaction to be mutual, indicating potential regulation of m169 by miR-27a/b. Most interestingly, three mutant viruses no longer able to target miR-27a/b, either due to miRNA target site disruption or target site replacement, showed significant attenuation in multiple organs as early as 4 days post infection, indicating that degradation of miR-27a/b is important for efficient MCMV replication in vivo.

• Shedding light on the elusive role of endothelial cells in cytomegalovirus dissemination.

  Authors: Torsten Sacher, Joachim Andrassy, Aivars Kalnins, Lars Dölken, Stefan Jordan, Jürgen Podlech, Zsolt Ruzsics, Karl-Walter Jauch, Matthias J Reddehase, Ulrich H Koszinowski

  PLoS pathogens. 11/2011; 7(11):e1002366.

  Cytomegalovirus (CMV) is frequently transmitted by solid organ transplantation and is associated with graft failure. By forming the boundary between circulation and organ parenchyma, endothelialCytomegalovirus (CMV) is frequently transmitted by solid organ transplantation and is associated with graft failure. By forming the boundary between circulation and organ parenchyma, endothelial cells (EC) are suited for bidirectional virus spread from and to the transplant. We applied Cre/loxP-mediated green-fluorescence-tagging of EC-derived murine CMV (MCMV) to quantify the role of infected EC in transplantation-associated CMV dissemination in the mouse model. Both EC- and non-EC-derived virus originating from infected Tie2-cre(+) heart and kidney transplants were readily transmitted to MCMV-naïve recipients by primary viremia. In contrast, when a Tie2-cre(+) transplant was infected by primary viremia in an infected recipient, the recombined EC-derived virus poorly spread to recipient tissues. Similarly, in reverse direction, EC-derived virus from infected Tie2-cre(+) recipient tissues poorly spread to the transplant. These data contradict any privileged role of EC in CMV dissemination and challenge an indiscriminate applicability of the primary and secondary viremia concept of virus dissemination.

• Virus progeny of murine cytomegalovirus bacterial artificial chromosome pSM3fr show reduced growth in salivary Glands due to a fixed mutation of MCK-2.

  Authors: Stefan Jordan, Johannes Krause, Adrian Prager, Maja Mitrovic, Stipan Jonjic, Ulrich H Koszinowski, Barbara Adler

  Journal of virology. 08/2011; 85(19):10346-53.

  Murine cytomegalovirus (MCMV) Smith strain has been cloned as a bacterial artificial chromosome (BAC) named pSM3fr and used for analysis of virus gene functions in vitro and in vivo. When sequencingMurine cytomegalovirus (MCMV) Smith strain has been cloned as a bacterial artificial chromosome (BAC) named pSM3fr and used for analysis of virus gene functions in vitro and in vivo. When sequencing the complete BAC genome, we identified a frameshift mutation within the open reading frame (ORF) encoding MCMV chemokine homologue MCK-2. This mutation would result in a truncated MCK-2 protein. When mice were infected with pSM3fr-derived virus, we observed reduced virus production in salivary glands, which could be reverted by repair of the frameshift mutation. When looking for the source of the mutation, we consistently found that virus stocks of cell culture-passaged MCMV Smith strain are mixtures of viruses with or without the MCK-2 mutation. We conclude that the MCK-2 mutation in the pSM3fr BAC is the result of clonal selection during the BAC cloning procedure.

• M94 is essential for the secondary envelopment of murine cytomegalovirus.

  Authors: Silke Maninger, Jens Bernhard Bosse, Frederic Lemnitzer, Madlen Pogoda, Christian A Mohr, Jens von Einem, Paul Walther, Ulrich H Koszinowski, Zsolt Ruzsics

  Journal of virology. 06/2011; 85(18):9254-67.

  The gene M94 of murine cytomegalovirus (MCMV) as well as its homologues UL16 in alphaherpesviruses is involved in viral morphogenesis. For a better understanding of its role in the viral life cycle,The gene M94 of murine cytomegalovirus (MCMV) as well as its homologues UL16 in alphaherpesviruses is involved in viral morphogenesis. For a better understanding of its role in the viral life cycle, a library of random M94 mutants was generated by modified transposon-based linker scanning mutagenesis. A comprehensive set of M94 mutants was reinserted into the MCMV genome and tested for their capacity to complement the M94 null mutant. Thereby, 34 loss-of-function mutants of M94 were identified, which were tested in a second screen for their capacity to inhibit virus replication. This analysis identified two N-terminal insertion mutants of M94 with a dominant negative effect. We compared phenotypes induced by the conditional expression of these dominant negative M94 alleles with the null phenotype of the M94 deletion. The viral gene expression cascade and the nuclear morphogenesis steps were not affected in either setting. In both cases, however, secondary envelopment did not proceed in the absence of functional M94, and capsids subsequently accumulated in the center of the cytoplasmic assembly complex. In addition, deletion of M94 resulted in a block of cell-to-cell spread. Moreover, the dominant negative mutant of M94 demonstrated a defect in interacting with M99, the UL11 homologue of MCMV.

• The role of cell types in cytomegalovirus infection in vivo.

  Authors: Torsten Sacher, Christian A Mohr, Annelies Weyn, Christina Schlichting, Ulrich H Koszinowski, Zsolt Ruzsics

  European journal of cell biology. 04/2011; 91(1):70-7.

  Human cytomegalovirus (HCMV) is the major viral cause of morbidity in immune compromised patients and of pre- and perinatal pathology in newborns. The clinical manifestations are highly variable andHuman cytomegalovirus (HCMV) is the major viral cause of morbidity in immune compromised patients and of pre- and perinatal pathology in newborns. The clinical manifestations are highly variable and the principles which govern these differences cannot be understood without detailed knowledge on tissue specific aspects of HCMV infection. For decades the role of individual cell types during cytomegalovirus infection and disease has been discussed. The pathogenesis of mouse cytomegalovirus (MCMV) mirrors the human infection in many aspects. Although only MCMV infection is studied extensively at the level of organs, the relative contribution of specific cell types to viral pathogenesis in vivo has remained enigmatic. Here we discuss new approaches based on the cre/loxP-system to label nascent virus progeny or to lift a replication block. The salient aspect of this technique is the change of viral genome properties selectively in cells that express cre during infection in vivo. This allowed us to study the role of endothelial cells and hepatocytes for virus dissemination and will permit detailed studies on innate and adaptive immune responses to CMV.

• 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.

• Dominant negative mutants of the murine cytomegalovirus M53 gene block nuclear egress and inhibit capsid maturation.

  Authors: Mirela Popa, Zsolt Ruzsics, Mark Lötzerich, Lars Dölken, Christopher Buser, Paul Walther, Ulrich H Koszinowski

  Journal of virology. 09/2010; 84(18):9035-46.

  The alphaherpesvirus proteins UL31 and UL34 and their homologues in other herpesvirus subfamilies cooperate at the nuclear membrane in the export of nascent herpesvirus capsids. We studied theThe alphaherpesvirus proteins UL31 and UL34 and their homologues in other herpesvirus subfamilies cooperate at the nuclear membrane in the export of nascent herpesvirus capsids. We studied the respective betaherpesvirus proteins M53 and M50 in mouse cytomegalovirus (MCMV). Recently, we established a random approach to identify dominant negative (DN) mutants of essential viral genes and isolated DN mutants of M50 (B. Rupp, Z. Ruzsics, C. Buser, B. Adler, P. Walther and U. H. Koszinowski, J. Virol 81:5508-5517). Here, we report the identification and phenotypic characterization of DN alleles of its partner, M53. While mutations in the middle of the M53 open reading frame (ORF) resulted in DN mutants inhibiting MCMV replication by approximately 100-fold, mutations at the C terminus resulted in up to 1,000,000-fold inhibition of virus production. C-terminal DN mutants affected nuclear distribution and steady-state levels of the nuclear egress complex and completely blocked export of viral capsids. In addition, they induced a marked maturation defect of viral capsids, resulting in the accumulation of nuclear capsids with aberrant morphology. This was associated with a two-thirds reduction in the total amount of unit length genomes, indicating an accessory role for M53 in DNA packaging.

• Intact NKG2D-independent function of NK cells chronically stimulated with the NKG2D ligand Rae-1.

  Authors: Marine Champsaur, Joshua N Beilke, Kouetsu Ogasawara, Ulrich H Koszinowski, Stipan Jonjic, Lewis L Lanier

  Journal of immunology (Baltimore, Md. : 1950). 07/2010; 185(1):157-65.

  Human tumors frequently express membrane-bound or soluble NK group 2, member D (NKG2D) ligands. This results in chronic engagement of NKG2D on the surfaces of NK and CD8(+) T cells and rapidHuman tumors frequently express membrane-bound or soluble NK group 2, member D (NKG2D) ligands. This results in chronic engagement of NKG2D on the surfaces of NK and CD8(+) T cells and rapid internalization of the receptor. Although it is well appreciated that this phenomenon impairs NKG2D-dependent function, careful analysis of NKG2D-independent functions in cells chronically stimulated through NKG2D is lacking. Using a mouse model of chronic NKG2D ligand expression, we show that constant exposure to NKG2D ligands does not functionally impair NK cells and CD8(+) T cells in the context of viral infection.

• Systematic analysis of viral and cellular microRNA targets in cells latently infected with human gamma-herpesviruses by RISC immunoprecipitation assay.

  Authors: Lars Dölken, Georg Malterer, Florian Erhard, Sheila Kothe, Caroline C Friedel, Guillaume Suffert, Lisa Marcinowski, Natalie Motsch, Stephanie Barth, Michaela Beitzinger [......] Susanne M Bailer, Reinhard Hoffmann, Zsolt Ruzsics, Elisabeth Kremmer, Sébastien Pfeffer, Ralf Zimmer, Ulrich H Koszinowski, Friedrich Grässer, Gunter Meister, Jürgen Haas

  Cell host & microbe. 04/2010; 7(4):324-34.

  The mRNA targets of microRNAs (miRNAs) can be identified by immunoprecipitation of Argonaute (Ago) protein-containing RNA-induced silencing complexes (RISCs) followed by microarray analysisThe mRNA targets of microRNAs (miRNAs) can be identified by immunoprecipitation of Argonaute (Ago) protein-containing RNA-induced silencing complexes (RISCs) followed by microarray analysis (RIP-Chip). Here we used Ago2-based RIP-Chip to identify transcripts targeted by Kaposi's sarcoma-associated herpesvirus (KSHV) miRNAs (n = 114), Epstein-Barr virus (EBV) miRNAs (n = 44), and cellular miRNAs (n = 2337) in six latently infected or stably transduced human B cell lines. Of the six KSHV miRNA targets chosen for validation, four showed regulation via their 3'UTR, while two showed regulation via binding sites within coding sequences. Two genes governing cellular transport processes (TOMM22 and IPO7) were confirmed to be targeted by EBV miRNAs. A significant number of viral miRNA targets were upregulated in infected cells, suggesting that viral miRNAs preferentially target cellular genes induced upon infection. Transcript half-life both of cellular and viral miRNA targets negatively correlated with recruitment to RISC complexes, indicating that RIP-Chip offers a quantitative estimate of miRNA function.

• Virology. A vaccine monkey wrench?

  Authors: Hartmut Hengel, Ulrich H Koszinowski

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

• Cytomegalovirus microRNAs facilitate persistent virus infection in salivary glands.

  Authors: Lars Dölken, Astrid Krmpotic, Sheila Kothe, Lee Tuddenham, Mélanie Tanguy, Lisa Marcinowski, Zsolt Ruzsics, Naama Elefant, Yael Altuvia, Hanah Margalit, Ulrich H Koszinowski, Stipan Jonjic, Sébastien Pfeffer

  PLoS pathogens. 01/2010; 6(10):e1001150.

  Micro (mi)RNAs are small non-coding RNAs that regulate the expression of their targets' messenger RNAs through both translational inhibition and regulation of target RNA stability. Recently, a numberMicro (mi)RNAs are small non-coding RNAs that regulate the expression of their targets' messenger RNAs through both translational inhibition and regulation of target RNA stability. Recently, a number of viruses, particularly of the herpesvirus family, have been shown to express their own miRNAs to control both viral and cellular transcripts. Although some targets of viral miRNAs are known, their function in a physiologically relevant infection remains to be elucidated. As such, no in vivo phenotype of a viral miRNA knock-out mutant has been described so far. Here, we report on the first functional phenotype of a miRNA knock-out virus in vivo. During subacute infection of a mutant mouse cytomegalovirus lacking two viral miRNAs, virus production is selectively reduced in salivary glands, an organ essential for virus persistence and horizontal transmission. This phenotype depends on several parameters including viral load and mouse genetic background, and is abolished by combined but not single depletion of natural killer (NK) and CD4+ T cells. Together, our results point towards a miRNA-based immunoevasion mechanism important for long-term virus persistence.

• Dominant-negative proteins in herpesviruses - from assigning gene function to intracellular immunization.

  Authors: Hermine Mühlbach, Christian A Mohr, Zsolt Ruzsics, Ulrich H Koszinowski

  Viruses. 12/2009; 1(3):420-40.

  Investigating and assigning gene functions of herpesviruses is a process, which profits from consistent technical innovation. Cloning of bacterial artificial chromosomes encoding herpesvirus genomesInvestigating and assigning gene functions of herpesviruses is a process, which profits from consistent technical innovation. Cloning of bacterial artificial chromosomes encoding herpesvirus genomes permits nearly unlimited possibilities in the construction of genetically modified viruses. Targeted or randomized screening approaches allow rapid identification of essential viral proteins. Nevertheless, mapping of essential genes reveals only limited insight into function. The usage of dominant-negative (DN) proteins has been the tool of choice to dissect functions of proteins during the viral life cycle. DN proteins also facilitate the analysis of host-virus interactions. Finally, DNs serve as starting-point for design of new antiviral strategies.

• Conserved principles of mammalian transcriptional regulation revealed by RNA half-life.

  Authors: Caroline C Friedel, Lars Dölken, Zsolt Ruzsics, Ulrich H. Koszinowski, Ralf Zimmer

  Nucleic acids research. 07/2009;

  RNA levels in a cell are regulated by the relative rates of RNA synthesis and decay. We recently developed a new approach for measuring both RNA synthesis and decay in a single experimental settingRNA levels in a cell are regulated by the relative rates of RNA synthesis and decay. We recently developed a new approach for measuring both RNA synthesis and decay in a single experimental setting by biosynthetic labeling of newly transcribed RNA. Here, we show that this provides measurements of RNA half-lives from microarray data with a so far unreached accuracy. Based on such measurements of RNA half-lives for human B-cells and mouse fibroblasts, we identified conserved regulatory principles for a large number of biological processes. We show that different regulatory patterns between functionally similar proteins are characterized by differences in the half-life of the corresponding transcripts and can be identified by measuring RNA half-life. We identify more than 100 protein families which show such differential regulatory patterns in both species. Additionally, we provide strong evidence that the activity of protein complexes consisting of subunits with overall long transcript half-lives can be regulated by transcriptional regulation of individual key subunits with short-lived transcripts. Based on this observation, we predict more than 100 key regulatory subunits for human complexes of which 28% could be confirmed in mice (P < 10(-9)). Therefore, this atlas of transcript half-lives provides new fundamental insights into many cellular processes.

• Differential susceptibility of RAE-1 isoforms to mouse cytomegalovirus.

  Authors: Jurica Arapovic, Tihana Lenac, Ronald Antulov, Bojan Polic, Zsolt Ruzsics, Leonidas N. Carayannopoulos, Ulrich H. Koszinowski, Astrid Krmpotic, Stipan Jonjic

  Journal of virology. 07/2009;

  The NKG2D receptor is one of the most potent activating NK cell receptors involved in antiviral responses. Mouse NKG2D ligands, MULT-1, RAE-1 and H60 are regulated by mouse cytomegalovirus (MCMV)The NKG2D receptor is one of the most potent activating NK cell receptors involved in antiviral responses. Mouse NKG2D ligands, MULT-1, RAE-1 and H60 are regulated by mouse cytomegalovirus (MCMV) proteins m145, m152 and m155, respectively. In addition, the m138 protein interferes with the expression of both MULT-1 and H60. Here we show that one of five RAE-1 isoforms, RAE-1delta, is resistant to down-regulation by MCMV and that this escape has functional importance in vivo. Although m152 retained newly synthesized RAE-1delta and RAE-1gamma in the endoplasmic reticulum, no viral regulator was able to affect the mature RAE-1delta form which remains expressed on the surface of infected cells. This differential susceptibility to down-regulation by MCMV is not a consequence of faster maturation of RAE-1delta as compared to RAE-1gamma but rather an intrinsic property of the mature surface-resident protein. This difference can be attributed to the absence of a PLWY motif from RAE-1delta. Altogether, these findings provide evidence for a novel mechanism of host escape from viral immunoevasion of NKG2D-dependent control.

• Ly49P recognition of cytomegalovirus-infected cells expressing H2-Dk and CMV-encoded m04 correlates with the NK cell antiviral response.

  Authors: Agnieszka Kielczewska, Michal Pyzik, Tianhe Sun, Astrid Krmpotic, Melissa B Lodoen, Michael W Munks, Marina Babic, Ann B Hill, Ulrich H. Koszinowski, Stipan Jonjic, Lewis L Lanier, Silvia M Vidal

  The Journal of experimental medicine. 04/2009; 206(3):515-23.

  Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49PNatural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-D(k) and the MCMV m04 protein. Using H2 chimeras between H2-D(b) and -D(k), we demonstrate that the H2-D(k) peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04-deletion mutant (Deltam04) virus infection. MA/My mice, which express Ly49P and H2-D(k), are resistant to MCMV; however, infection with Deltam04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Deltam04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatibility complex class I-restricted recognition of virally infected cells by an activating NK cell receptor.

• High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay.

  Authors: Lars Dölken, Zsolt Ruzsics, Bernd Rädle, Caroline C Friedel, Ralf Zimmer, Jörg Mages, Reinhard Hoffmann, Paul Dickinson, Thorsten Forster, Peter Ghazal, Ulrich H. Koszinowski

  RNA (New York, N.Y.). 09/2008; 14(9):1959-72.

  RNA levels in a cell are determined by the relative rates of RNA synthesis and decay. State-of-the-art transcriptional analyses only employ total cellular RNA. Therefore, changes in RNA levels cannotRNA levels in a cell are determined by the relative rates of RNA synthesis and decay. State-of-the-art transcriptional analyses only employ total cellular RNA. Therefore, changes in RNA levels cannot be attributed to RNA synthesis or decay, and temporal resolution is poor. Recently, it was reported that newly transcribed RNA can be biosynthetically labeled for 1-2 h using thiolated nucleosides, purified from total cellular RNA and subjected to microarray analysis. However, in order to study signaling events at molecular level, analysis of changes occurring within minutes is required. We developed an improved approach to separate total cellular RNA into newly transcribed and preexisting RNA following 10-15 min of metabolic labeling. Employing new computational tools for array normalization and half-life determination we simultaneously study short-term RNA synthesis and decay as well as their impact on cellular transcript levels. As an example we studied the response of fibroblasts to type I and II interferons (IFN). Analysis of RNA transcribed within 15-30 min at different times during the first three hours of interferon-receptor activation resulted in a >10-fold increase in microarray sensitivity and provided a comprehensive profile of the kinetics of IFN-mediated changes in gene expression. We identify a previously undisclosed highly connected network of short-lived transcripts selectively down-regulated by IFNgamma in between 30 and 60 min after IFN treatment showing strong associations with cell cycle and apoptosis, indicating novel mechanisms by which IFNgamma affects these pathways.

• Conditional gene expression systems to study herpesvirus biology in vivo.

  Authors: Torsten Sacher, Stefan Jordan, Christian A Mohr, Aurore Vidy, Annelies M G Weyn, Zsolt Ruszics, Ulrich H. Koszinowski

  Medical microbiology and immunology. 07/2008; 197(2):269-76.

  Cytomegalovirus (CMV), a prototypic beta-herpesvirus, is an important human pathogen causing protean clinical manifestations in immature and immunocompromised patients. Mechanisms of infection can beCytomegalovirus (CMV), a prototypic beta-herpesvirus, is an important human pathogen causing protean clinical manifestations in immature and immunocompromised patients. Mechanisms of infection can be studied in a mouse model. Mouse cytomegalovirus (MCMV) resembles in pathogenesis its human counterpart in many ways. Although MCMV infection is studied extensively on the level of organs, the contribution of specific cell types to viral replication in vivo is still elusive. Here we describe our approach based on the the Cre/loxP-system to investigate MCMV infection at the level of cell types in vivo. Using bacterial artificial chromosome (BAC)-technology, we created an MCMV virus containing an enhanced green fluorescent protein (egfp) reporter-gene which is not expressed due to a 'Stop' cassette flanked by two loxP-sites between promoter and coding sequence. Infection of cre-transgenic mice with this reporter virus results in the deletion of the 'Stop' cassette and expression of EGFP in a cell type-specific manner. Using this conditional gene expression system we are able to quantify viral productivity in specific cell types and to determine their contribution to viral dissemination in vivo. Furthermore, the deletion of viral genes can be used to screen for cell type-specificity of viral gene functions. Hence, conditional MCMV mutants allow the study of herpesvirus biology on the level of cell types in vivo.

• The major virus-producing cell type during murine cytomegalovirus infection, the hepatocyte, is not the source of virus dissemination in the host.

  Authors: Torsten Sacher, Jürgen Podlech, Christian A Mohr, Stefan Jordan, Zsolt Ruzsics, Matthias J. Reddehase, Ulrich H. Koszinowski

  Cell host & microbe. 05/2008; 3(4):263-72.

  The course of systemic viral infections is determined by the virus productivity of infected cell types and the efficiency of virus dissemination throughout the host. Here, we used aThe course of systemic viral infections is determined by the virus productivity of infected cell types and the efficiency of virus dissemination throughout the host. Here, we used a cell-type-specific virus labeling system to quantitatively track virus progeny during murine cytomegalovirus infection. We infected mice that expressed Cre recombinase selectively in vascular endothelial cells or hepatocytes with a murine cytomegalovirus for which Cre-mediated recombination would generate a fluorescently labeled virus. We showed that endothelial cells and hepatocytes produced virus after direct infection. However, in the liver, the main contributor to viral load in the mouse, most viruses were produced by directly infected hepatocytes. Remarkably, although virus produced in hepatocytes spread to hepatic endothelial cells (and vice versa), there was no significant spread from the liver to other organs. Thus, the cell type producing the most viruses was not necessarily the one responsible for virus dissemination within the host.

• Dominant-negative FADD rescues the in vivo fitness of a cytomegalovirus lacking an antiapoptotic viral gene.

  Authors: Luka Cicin-Sain, Zsolt Ruzsics, Juergen Podlech, Ivan Bubić, Carine Menard, Stipan Jonjić, Matthias J. Reddehase, Ulrich H. Koszinowski

  Journal of virology. 04/2008; 82(5):2056-64.

  Genes that inhibit apoptosis have been described for many DNA viruses. Herpesviruses often contain even more than one gene to control cell death. Apoptosis inhibition by viral genes is postulated toGenes that inhibit apoptosis have been described for many DNA viruses. Herpesviruses often contain even more than one gene to control cell death. Apoptosis inhibition by viral genes is postulated to contribute to viral fitness, although a formal proof is pending. To address this question, we studied the mouse cytomegalovirus (MCMV) protein M36, which binds to caspase-8 and blocks death receptor-induced apoptosis. The growth of MCMV recombinants lacking M36 (DeltaM36) was attenuated in vitro and in vivo. In vitro, caspase inhibition by zVAD-fmk blocked apoptosis in DeltaM36-infected macrophages and rescued the growth of the mutant. In vivo, DeltaM36 infection foci in liver tissue contained significantly more apoptotic hepatocytes and Kupffer cells than did revertant virus foci, and apoptosis occurred during the early phase of virus replication prior to virion assembly. To further delineate the mode of M36 function, we replaced the M36 gene with a dominant-negative FADD (FADD(DN)) in an MCMV recombinant. FADD(DN) was expressed in cells infected with the recombinant and blocked the death-receptor pathway, replacing the antiapoptotic function of M36. Most importantly, FADD(DN) rescued DeltaM36 virus replication, both in vitro and in vivo. These findings have identified the biological role of M36 and define apoptosis inhibition as a key determinant of viral fitness.