Zsolt Ruzsics

Publications of Zsolt Ruzsics

• 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.

• Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution.

  Authors: Lukas Windhager, Thomas Bonfert, Kaspar Burger, Zsolt Ruzsics, Stefan Krebs, Stefanie Kaufmann, Georg Malterer, Anne L'hernault, Markus Schilhabel, Stefan Schreiber, Philip Rosenstiel, Ralf Zimmer, Dirk Eick, Caroline C Friedel, Lars Dölken

  Genome research. 04/2012;

  RNA synthesis and decay rates determine the steady-state levels of cellular RNAs. Metabolic tagging of newly transcribed RNA by 4-thiouridine (4sU) can reveal the relative contributions of RNARNA synthesis and decay rates determine the steady-state levels of cellular RNAs. Metabolic tagging of newly transcribed RNA by 4-thiouridine (4sU) can reveal the relative contributions of RNA synthesis and decay rates. The kinetics of RNA processing, however, so far remained unresolved. Here, we show that ultra-short 4sU-tagging not only provides snap-shot pictures of eukaryotic gene expression but, when combined with progressive 4sU-tagging and RNA-seq, reveals global RNA processing kinetics at nucleotide resolution. Using this method, we identified classes of rapidly and slowly spliced/degraded introns. Interestingly, each class of splicing kinetics was characterized by a distinct association with intron length, gene length and splice site strength. For a large group of introns, we also observed long lasting retention in the primary transcript, but efficient secondary splicing or degradation at later time points. Finally, we show that processing of most, but not all small nucleolar (sno)RNA-containing introns is remarkably inefficient with the majority of introns being spliced and degraded rather than processed into mature snoRNAs. In summary, our study yields unparalleled insights into the kinetics of RNA processing and provides the tools to study molecular mechanisms of RNA processing and their contribution to the regulation of gene expression.

• 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.

• 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.

• 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.

• 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.

• 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.

• 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.

• 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.

• 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.

• 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.

• 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.

• Targeted deletion of regions rich in immune-evasive genes from the cytomegalovirus genome as a novel vaccine strategy.

  Authors: Luka Cicin-Sain, Ivan Bubić, Margit Schnee, Zsolt Ruzsics, Christian Mohr, Stipan Jonjić, Ulrich H. Koszinowski

  Journal of virology. 01/2008; 81(24):13825-34.

  Human cytomegalovirus (CMV), a ubiquitous human pathogen, is a leading cause of congenital infections and represents a serious health risk for the immunosuppressed patient. A vaccine against CMV isHuman cytomegalovirus (CMV), a ubiquitous human pathogen, is a leading cause of congenital infections and represents a serious health risk for the immunosuppressed patient. A vaccine against CMV is currently not available. CMV is characterized by its large genome and by multiple genes modulating the immunity of the host, which cluster predominantly at genome termini. Here, we tested whether the deletion of gene blocks rich in immunomodulatory genes could be used as a novel concept in the generation of immunogenic but avirulent, herpesvirus vaccines. To generate an experimental CMV vaccine, we selectively deleted 32 genes from the mouse cytomegalovirus (MCMV) genome. The resulting mutant grew to titers similar to that of wild-type MCMV in vitro. In vivo, the mutant was 10,000-fold attenuated and well tolerated, even by highly susceptible mice deficient for B, T, and NK cells or for the interferon type I receptor. Equally relevant for safety concerns, immune suppression did not lead to the mutant's reactivation from latency. Immunization with the replication-competent mutant, but not with inactivated virus, resulted in protective immunity, which increased over time. Vaccination induced MCMV-specific antibodies and a strong T-cell response. We propose that a targeted and rational approach can improve future herpesvirus vaccines and vaccine vectors.

• 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.