Wolfram Brune

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• Construction of a lytically replicating Kaposi's sarcoma-associated herpesvirus (KSHV)

  Authors: Matthias Budt, Tsvetana Histozova, Georg Hille, Katrin Berger, Wolfram Brune

  Journal of virology. 07/2011;

  KSHV is found predominantly in a latent state in most cell types, impeding investigations of the lytic replication cycle. Here we engineered the cloned KSHV genome, BAC36, to enforce constitutiveKSHV is found predominantly in a latent state in most cell types, impeding investigations of the lytic replication cycle. Here we engineered the cloned KSHV genome, BAC36, to enforce constitutive expression of the main lytic switch regulator, RTA (ORF50). The resulting virus, KSHV-lyt, activated by default the lytic cycle and replicated to high titers in various cells. Using KSHV-lyt we showed that ORF33 (encoding a tegument protein) is essential for lytic KSHV replication in cell culture, but ORF73 (encoding the latent nuclear antigen, LANA) is not. Thus, KSHV-lyt should be highly useful to study viral gene function during lytic replication.

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• Inhibition of programmed cell death by cytomegaloviruses

  Authors: Wolfram Brune

  Virus research. 10/2010;

  The elimination of infected cells by programmed cell death (PCD) is one of the most ancestral defense mechanisms against infectious agents. This mechanism should be most effective againstThe elimination of infected cells by programmed cell death (PCD) is one of the most ancestral defense mechanisms against infectious agents. This mechanism should be most effective against intracellular parasites, such as viruses, which depend on the host cell for their replication. However, even large and slowly replicating viruses like the cytomegaloviruses (CMVs) can prevail and persist in face of cellular suicide programs and other innate defense mechanisms. During evolution, these viruses have developed an impressive set of countermeasures against premature demise of the host cell. In the last decade, several genes encoding suppressors of apoptosis and necrosis have been identified in the genomes of human und murine CMV (HCMV and MCMV). Curiously, most of the gene products are not homologous to cellular antiapoptotic proteins, suggesting that the CMVs did not capture the genes from the host cell genome. This review summarizes our current understanding of how the CMVs suppress PCD and which signaling pathways they target.

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• Mutations in the M112/M113 coding region facilitate murine cytomegalovirus replication in human cells

  Authors: Uwe Schumacher, Wiebke Handke, Igor Jurak, Wolfram Brune

  Journal of virology. 06/2010;

  Cytomegaloviruses, representatives of the Betaherpesvirinae, cause opportunistic infections in immunocompromised hosts. They infect various cells and tissues in their natural host, but are highlyCytomegaloviruses, representatives of the Betaherpesvirinae, cause opportunistic infections in immunocompromised hosts. They infect various cells and tissues in their natural host, but are highly species specific. For instance, human cytomegalovirus (HCMV) does not replicate in mouse cells, and human cells are not permissive for murine cytomegalovirus (MCMV). However, the underlying molecular mechanisms are so far poorly understood. In the present study we isolated and characterized a spontaneously occurring MCMV mutant that has gained the capacity to replicate rapidly and to high titers in human cells. Compared to the parental wildtype (wt) virus, this mutant formed larger nuclear replication compartments and replicated viral DNA more efficiently. It also disrupted PML nuclear domains with greater efficiency, but caused less apoptosis than wt MCMV. Sequence analysis of the mutant virus genome revealed mutations in the M112-M113 coding region. This region is homologous to the HCMV UL112-113 region and encodes the viral early-1 (E1) proteins, which are known to play an important role in viral DNA replication. By introducing the M112/113 mutations into wt MCMV we demonstrated that they are sufficient to facilitate MCMV replication in human cells and are, at least in part, responsible for the efficient replication capability of the spontaneously adapted virus. However, additional mutations probably contribute as well. These results reveal a previously unrecognized role of the viral E1 proteins in regulating viral replication in different cells and provide new insights into the mechanisms of the cytomegalovirus species specificity.

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• Deletion of the rat cytomegalovirus immediate early 1 gene results in a virus capable of establishing latency but with lower levels of acute virus replication and latency that compromise reactivation efficiency

  Authors: Gordon R. Sandford, Uwe Schumacher, Jakob Ettinger, Wolfram Brune, Gary S Hayward, William H Burns, Sebastian Voigt

  The Journal of general virology. 11/2009;

  The IE1 and IE2 proteins encoded by the major immediate-early (MIE) transcription unit of cytomegaloviruses are thought to play key roles in the switch between latent and lytic cycle infection.The IE1 and IE2 proteins encoded by the major immediate-early (MIE) transcription unit of cytomegaloviruses are thought to play key roles in the switch between latent and lytic cycle infection. Whilst IE2 is essential for triggering the lytic cycle, the exact roles of IE1 have not been resolved. An MIE-exon 4 deleted rat cytomegalovirus (DeltaIE1) failed to synthesize the IE1 protein and did not disperse promyelocytic leukemia bodies (PML) early post-infection, but it was still capable of normal replication in fibroblast cell culture. However, DeltaIE1 had diminished ability to infect salivary glands persistently in vivo and to reactivate from spleen explant cultures ex vivo. Quantitation of viral genomes in spleens of infected animals revealed a reduced amount of DeltaIE1 virus produced during acute infection, suggesting a role for IE1 as a regulator in establishing a chronic or persistent infection, rather than in more directly influencing the latency or reactivation processes.

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• Essential role for either TRS1 or IRS1 in human cytomegalovirus replication

  Authors: Emily E Marshall, Craig J Bierle, Wolfram Brune, Adam P Geballe

  Journal of virology. 03/2009;

  Viral infections often produce double-stranded RNA (dsRNA), which in turn triggers potent antiviral responses including the global repression of protein synthesis mediated by protein kinase R (PKR)Viral infections often produce double-stranded RNA (dsRNA), which in turn triggers potent antiviral responses including the global repression of protein synthesis mediated by protein kinase R (PKR) and 2'-5' oligoadenylate synthetase (OAS). As a consequence, many viruses have evolved genes, such as those encoding dsRNA-binding proteins, which counteract these pathways. Human cytomegalovirus (HCMV) encodes two related proteins, pTRS1 and pIRS1, which bind dsRNA and can prevent activation of the PKR and OAS pathways. HCMV mutants lacking either IRS1 or TRS1 replicate at least moderately well in cell culture. However, as we demonstrate in the present study, an HCMV mutant lacking both IRS1 and TRS1 (HCMV[DeltaI/DeltaT]) has a severe replication defect. Infection with HCMV[DeltaI/DeltaT] results in a profound inhibition of overall and viral protein synthesis, as well as increased phosphorylation of eukaryotic initiation factor 2alpha. The vaccinia virus E3L gene can substitute for IRS1 or TRS1, enabling HCMV replication. Despite the accumulation of dsRNA in HCMV infected cells, the OAS pathway remains inactive, even in HCMV[DeltaI/DeltaT]-infected cells. These results suggest that PKR-mediated phosphorylation of eIF2alpha is the dominant dsRNA-activated pathway responsible for inhibition of protein synthesis and HCMV replication in the absence of both IRS1 and TRS1, and that the requirement for evasion of the PKR pathway likely explains the necessity of IRS1 or TRS1 for productive infection.

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• Tinkering with a viral ribonucleotide reductase

  Authors: David Lembo, Wolfram Brune

  Trends in biochemical sciences. 12/2008;

  Ribonucleotide reductase (RNR), a crucial enzyme for nucleotide anabolism, is encoded by all living organisms and by large DNA viruses such as the herpesviruses. Surprisingly, the beta-herpesvirusRibonucleotide reductase (RNR), a crucial enzyme for nucleotide anabolism, is encoded by all living organisms and by large DNA viruses such as the herpesviruses. Surprisingly, the beta-herpesvirus subfamily RNR R1 subunit homologues are catalytically inactive and their function remained enigmatic for many years. Recent work sheds light on the function of M45, the murine cytomegalovirus R1 homologue; during viral evolution, M45 apparently lost its original RNR activity but gained the ability, via inhibiting RIP1, a cellular adaptor protein, to block cellular signaling pathways involved in innate immunity and inflammation. The discovery of this novel mechanism of viral immune subversion provides further support to the concept of evolutionary tinkering.

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• Specific inhibition of the PKR-mediated antiviral response by the murine cytomegalovirus proteins m142 and m143

  Authors: Matthias Budt, Lars Niederstadt, Ralitsa S Valchanova, Stipan Jonjic, Wolfram Brune

  Journal of virology. 12/2008;

  Double-stranded (ds) RNA produced during viral infection activates several cellular antiviral responses. Among the best characterized is the shutoff of protein synthesis mediated by theDouble-stranded (ds) RNA produced during viral infection activates several cellular antiviral responses. Among the best characterized is the shutoff of protein synthesis mediated by the dsRNA-dependent protein kinase (PKR) and the oligoadenylate synthetase (OAS)/RNase L system. As viral replication depends on protein synthesis, many viruses have evolved mechanisms for counteracting the PKR and OAS/RNase L pathways. The murine cytomegalovirus (MCMV) proteins m142 and m143 have been characterized as dsRNA binding proteins that inhibit PKR activation, phosphorylation of the translation initiation factor eIF2alpha, and a subsequent protein synthesis shutoff. In the present study we analyzed the contribution of the PKR- and the OAS-dependent pathways to the control of MCMV replication in the absence or presence of m142 and m143. We show that the induction of eIF2alpha phosphorylation during infection with an m142/m143-deficient MCMV is specifically mediated by PKR, not by the related eIF2alpha kinases PERK or GCN2. PKR antagonists of vaccinia virus (E3L) or herpes simplex virus (gamma34.5) rescued the replication defect of an m142/m143-deficient MCMV. Moreover, m142 and m143 bound to each other and interacted with PKR. By contrast, an activation of the OAS/RNase L pathway by MCMV was not detected in the presence or absence of m142 and m143, suggesting that these viral proteins have little or no influence on this pathway. Consistently, an m142/m143-deficient MCMV replicated to high titers in fibroblasts lacking PKR, but did not replicate in cells lacking RNase L. Hence, the PKR-mediated antiviral response is responsible for the essentiality of m142 and m143.

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• Murine cytomegalovirus m38.5 protein inhibits Bax-mediated cell death

  Authors: Igor Jurak, Uwe Schumacher, Hrvoje Simic, Sebastian Voigt, Wolfram Brune

  Journal of virology. 06/2008; 82(10):4812-22.

  Many viruses encode proteins that inhibit the induction of programmed cell death at the mitochondrial checkpoint. Murine cytomegalovirus (MCMV) encodes the m38.5 protein, which localizes toMany viruses encode proteins that inhibit the induction of programmed cell death at the mitochondrial checkpoint. Murine cytomegalovirus (MCMV) encodes the m38.5 protein, which localizes to mitochondria and protects human HeLa cells and fibroblasts from apoptosis triggered by proteasome inhibitors but not from Fas-induced apoptosis. However, the ability of this protein to suppress the apoptosis of murine cells and its role during MCMV infection have not been investigated previously. Here we show that m38.5 is expressed at early time points during MCMV infection. Cells infected with MCMVs lacking m38.5 showed increased sensitivity to cell death induced by staurosporine, MG132, or the viral infection itself compared to the sensitivity of cells infected with wild-type MCMV. This defect was eliminated when an m38.5 or Bcl-X(L) gene was inserted into the genome of a deletion mutant. Using fibroblasts deficient in the proapoptotic Bcl-2 family proteins Bak and/or Bax, we further demonstrated that m38.5 protected from Bax- but not Bak-mediated apoptosis and interacted with Bax in infected cells. These results consolidate the role of m38.5 as a viral mitochondrion-localized inhibitor of apoptosis and its functional similarity to the human cytomegalovirus UL37x1 gene product. Although the m38.5 gene is not homologous to the UL37x1 gene at the sequence level, m38.5 is conserved among rodent cytomegaloviruses. Moreover, the fact that MCMV-infected cells are protected from both Bak- and Bax-mediated cell death suggests that MCMV possesses an additional, as-yet-unidentified mechanism to block Bak-mediated apoptosis.

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• Inhibition of proinflammatory and innate immune signaling pathways by a cytomegalovirus RIP1-interacting protein

  Authors: Claudia Mack, Albert Sickmann, David Lembo, Wolfram Brune

  Proceedings of the National Academy of Sciences of the United States of America. 03/2008; 105(8):3094-9.

  TNFalpha is an important cytokine in antimicrobial immunity and inflammation. The receptor-interacting protein RIP1 is an essential component of the TNF receptor 1 signaling pathway that mediates theTNFalpha is an important cytokine in antimicrobial immunity and inflammation. The receptor-interacting protein RIP1 is an essential component of the TNF receptor 1 signaling pathway that mediates the activation of NF-kappaB, MAPKs, and programmed cell death. It also transduces signals derived from Toll-like receptors and intracellular sensors of DNA damage and double-stranded RNA. Here, we show that the murine CMV M45 protein binds to RIP1 and inhibits TNFalpha-induced activation of NF-kappaB, p38 MAPK, and caspase-independent cell death. M45 also inhibited NF-kappaB activation upon stimulation of Toll-like receptor 3 and ubiquitination of RIP1, which is required for NF-kappaB activation. Hence, M45 functions as a viral inhibitor of RIP1-mediated signaling. The results presented here reveal a mechanism of viral immune subversion and demonstrate how a viral protein can simultaneously block proinflammatory and innate immune signaling pathways by interacting with a central mediator molecule.

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• 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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• Murine cytomegalovirus m142 and m143 are both required to block protein kinase R-mediated shutdown of protein synthesis

  Authors: Ralitsa S Valchanova, Marcus Picard-Maureau, Matthias Budt, Wolfram Brune

  Journal of virology. 11/2006; 80(20):10181-90.

  Cytomegaloviruses carry the US22 family of genes, which have common sequence motifs but diverse functions. Only two of the 12 US22 family genes of murine cytomegalovirus (MCMV) are essential forCytomegaloviruses carry the US22 family of genes, which have common sequence motifs but diverse functions. Only two of the 12 US22 family genes of murine cytomegalovirus (MCMV) are essential for virus replication, but their functions have remained unknown. In the present study, we deleted the essential US22 family genes, m142 and m143, from the MCMV genome and propagated the mutant viruses on complementing cells. The m142 and the m143 deletion mutants were both unable to replicate in noncomplementing cells at low and high multiplicities of infection. In cells infected with the deletion mutants, viral immediate-early and early proteins were expressed, but viral DNA replication and synthesis of the late-gene product glycoprotein B were inhibited, even though mRNAs of late genes were present. Global protein synthesis was impaired in these cells, which correlated with phosphorylation of the double-stranded RNA-dependent protein kinase R (PKR) and its target protein, the eukaryotic translation initiation factor 2alpha, suggesting that m142 and m143 are necessary to block the PKR-mediated shutdown of protein synthesis. Replication of the m142 and m143 knockout mutants was partially restored by expression of the human cytomegalovirus TRS1 gene, a known double-stranded-RNA-binding protein that inhibits PKR activation. These results indicate that m142 and m143 are both required for inhibition of the PKR-mediated host antiviral response.

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• Induction of apoptosis limits cytomegalovirus cross-species infection

  Authors: Igor Jurak, Wolfram Brune

  The EMBO journal. 07/2006; 25(11):2634-42.

  Cross-species infections are responsible for the majority of emerging and re-emerging viral diseases. However, little is known about the mechanisms that restrict viruses to a certain host species,Cross-species infections are responsible for the majority of emerging and re-emerging viral diseases. However, little is known about the mechanisms that restrict viruses to a certain host species, and the factors viruses need to cross the species barrier and replicate in a different host. Cytomegaloviruses (CMVs) are representatives of the beta-herpesviruses that are highly species specific. They replicate only in cells of their own or a closely related species. In this study, the molecular mechanism underlying the cytomegalovirus species specificity was investigated. We show that infection of human cells with the murine cytomegalovirus (MCMV) triggers the intrinsic apoptosis pathway involving caspase-9 activation. MCMV can break the species barrier and replicate in human cells if apoptosis is blocked by Bcl-2 or a functionally analogous protein. A single gene of the human cytomegalovirus encoding a mitochondrial inhibitor of apoptosis is sufficient to allow MCMV replication in human cells. Moreover, the same principle facilitates replication of the rat cytomegalovirus in human cells. Thus, induction of apoptosis serves as an innate immune defense to inhibit cross-species infections of rodent CMVs.

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• Manipulating cytomegalovirus genomes by BAC mutagenesis: Strategies and applications

  Authors: Wolfram Brune, Martin Messerle

  Cytomegaloviruses: Molecular Biology and Immunology

  edited by Matthias J. Reddehase, 01/2006: pages 61-69; Caister Academic Press.

  ISBN: 1-904455-02-6

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• SUMOylation of the human cytomegalovirus 72-kilodalton IE1 protein facilitates expression of the 86-kilodalton IE2 protein and promotes viral replication

  Authors: Michael Nevels, Wolfram Brune, Thomas Shenk

  Journal of virology. 08/2004; 78(14):7803-12.

  The 72-kDa immediate-early 1 protein (IE1-72kDa) of human cytomegalovirus has been previously shown to be posttranslationally modified by covalent conjugation to the ubiquitin-related protein SUMO-1.The 72-kDa immediate-early 1 protein (IE1-72kDa) of human cytomegalovirus has been previously shown to be posttranslationally modified by covalent conjugation to the ubiquitin-related protein SUMO-1. Using an infectious bacterial artificial chromosome clone of human cytomegalovirus, we constructed a mutant virus (BADpmIE1-K450R) that is deficient for SUMOylation of IE1-72 kDa due to a single amino acid exchange in the SUMO-1 attachment site. Compared to wild-type virus, this mutant grew more slowly and generated a reduced yield in infected human fibroblasts, indicating that SUMO modification is required for the full activity of IE1-72 kDa. The lack of SUMOylation did not affect the intranuclear localization of IE1-72 kDa, including its ability to target to and disrupt PML bodies and to bind to mitotic chromatin. Likewise, SUMOylation-deficient IE1-72 kDa activated several viral promoters as efficiently as the wild-type protein. However, the failure to modify IE1-72 kDa resulted in substantially reduced levels of the IE2 transcript and its 86-kDa protein (IE2-86 kDa). These observations suggest that SUMO modification of IE1-72 kDa contributes to efficient HCMV replication by promoting the accumulation of IE2-86 kDa.

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• 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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• Murine cytomegalovirus m41 open reading frame encodes a Golgi-localized antiapoptotic protein

  Authors: Wolfram Brune, Michael Nevels, Thomas Shenk

  Journal of virology. 12/2003; 77(21):11633-43.

  Viruses have evolved various strategies to prevent premature apoptosis of infected host cells. Some of the viral genes mediating antiapoptotic functions have been identified by their homology toViruses have evolved various strategies to prevent premature apoptosis of infected host cells. Some of the viral genes mediating antiapoptotic functions have been identified by their homology to cellular genes, but others are structurally unrelated to genes of known function. In this study, we used a random, unbiased approach to identify such genes in the murine cytomegalovirus genome. From a library of random transposon insertion mutants, a mutant virus that caused premature cell death was isolated. The transposon was inserted within open reading frame m41. An independently constructed m41 deletion mutant showed the same phenotype, whereas deletion mutants lacking the adjacent genes m40 and M42 did not. Apoptosis occurred in different cell types, could be blocked by caspase inhibitors, and did not require p53. Within the murine cytomegalovirus genome, m41, m40, and m39 form a small cluster of genes of unknown function. They are homologous to r41, r40, and r39 of rat cytomegalovirus, but lack sequence homology to UL41, UL40, and UL37 exon 1 (UL37x1) which are located at the corresponding positions of the human cytomegalovirus genome. Unlike UL37x1 of human cytomegalovirus, which encodes a mitochondrion-localized inhibitor of apoptosis that is essential for virus replication, m41 encodes a protein that localizes to the Golgi apparatus. The murine cytomegalovirus m41 product is the first example of a Golgi-localized protein that prevents premature apoptosis and thus extends the life span of infected cells.

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• Vaccination of mice with bacteria carrying a cloned herpesvirus genome reconstituted in vivo

  Authors: Luka Cicin-Sain, Wolfram Brune, Ivan Bubic, Stipan Jonjic, Ulrich H. Koszinowski

  Journal of virology. 09/2003; 77(15):8249-55.

  Bacterial delivery systems are gaining increasing interest as potential vaccination vectors to deliver either proteins or nucleic acids for gene expression in the recipient. Bacterial deliveryBacterial delivery systems are gaining increasing interest as potential vaccination vectors to deliver either proteins or nucleic acids for gene expression in the recipient. Bacterial delivery systems for gene expression in vivo usually contain small multicopy plasmids. We have shown before that bacteria containing a herpesvirus bacterial artificial chromosome (BAC) can reconstitute the virus replication cycle after cocultivation with fibroblasts in vitro. In this study we addressed the question of whether bacteria containing a single plasmid with a complete viral genome can also reconstitute the viral replication process in vivo. We used a natural mouse pathogen, the murine cytomegalovirus (MCMV), whose genome has previously been cloned as a BAC in Escherichia coli. In this study, we tested a new application for BAC-cloned herpesvirus genomes. We show that the MCMV BAC can be stably maintained in certain strains of Salmonella enterica serovar Typhimurium as well and that both serovar Typhimurium and E. coli harboring the single-copy MCMV BAC can reconstitute a virus infection upon injection into mice. By this procedure, a productive virus infection is regenerated only in immunocompromised mice. Virus reconstitution in vivo causes elevated titers of specific anti-MCMV antibodies, protection against lethal MCMV challenge, and strong expression of additional genes introduced into the viral genome. Thus, the reconstitution of infectious virus from live attenuated bacteria presents a novel concept for multivalent virus vaccines launched from bacterial vectors.

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

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

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

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

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• Random transposon mutagenesis of large DNA molecules in Escherichia coli

  Authors: Wolfram Brune

  Methods in molecular biology (Clifton, N.J.). 02/2002; 182:165-71.

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• Viral genes involved in immune evasion of cytomegalovirus

  Authors: Ulrich H. Koszinowski, Wolfram Brune

  Nova Acta Leopoldina. 01/1999; 78:199-213.

  Cytomegaloviruses (CMVs) are prototypes of the beta-subgroup of herpesviruses. After immune control of primary CMV infection, viral genomes persist in a latent state for lifetime of the infectedCytomegaloviruses (CMVs) are prototypes of the beta-subgroup of herpesviruses. After immune control of primary CMV infection, viral genomes persist in a latent state for lifetime of the infected host. From the non-productive state of latency, CMV replication is occasionally reactivated folowed by virus shedding and transmission to other hosts. To replicate in the face of a repeatedly primed immune system and despite the protracted replication cycle of their large DNA genomes expressing more than 200 potentially antigenic proteins, CMVs have devised a multitude of fascinating mechanisms to evade immunity. Among these strategies are methods of blocking pathways of antigen presentation to cytotoxic T lymphocytes as well as inhibition of signalling pathways of antivoiral cytokines. More than 20 CMV genes have been identified which code for immune modulators, and this collection is certainly incomplete. In addition, CMVs even exploit immune functions utilized by the host for their own advantage. These sophisticated and highly adapted strategies secure the permanent coexistence of CMVs with their host and provide the opportunity to sspread to naive individuals.

• Cytomegaloviruses inhibit Bak- and Bax-mediated apoptosis with two separate viral proteins

  Authors: M Çam, Handke Wiebke, M Picard-Maureau, Wolfram Brune

  Apoptosis of infected cells can limit virus replication and serves as an innate defense mechanism against viral infections. Consequently, viruses delay apoptosis by expressing antiapoptotic proteins,Apoptosis of infected cells can limit virus replication and serves as an innate defense mechanism against viral infections. Consequently, viruses delay apoptosis by expressing antiapoptotic proteins, many of which structurally resemble the cellular antiapoptotic protein Bcl-2. Like Bcl-2, the viral analogs inhibit apoptosis by preventing activation and/or oligomerization of the proapoptotic mitochondrial proteins Bax and Bak. Here we show that cytomegaloviruses (CMVs) have adopted a different strategy. They encode two separate mitochondrial proteins that lack obvious sequence similarities to Bcl-2-family proteins and specifically counteract either Bax or Bak. We identified a small mitochondrion-localized protein encoded by the murine CMV open reading frame (ORF) m41.1, which functions as a viral inhibitor of Bak oligomerization (vIBO). It blocks Bak-mediated cytochrome c release and Bak-dependent induction of apoptosis. It protects cells from cell death-inducing stimuli together with the previously identified Bax-specific inhibitor viral mitochondria-localized inhibitor of apoptosis (vMIA) (encoded by ORF m38.5). Similar vIBO proteins are encoded by CMVs of rats, and possibly by other CMVs as well. These results suggest a non-redundant function of Bax and Bak during viral infection, and a benefit for CMVs derived from the ability to inhibit Bak and Bax separately with two viral proteins.

• Novel GLRA1 Missense Mutation (P250T) in Dominant Hyperekplexia Defines an Intracellular Determinant of Glycine Receptor Channel Gating

  Authors: Brigitta Saul, Thomas Kuner, Diana Sobetzko, Wolfram Brune, Folker Hanefeld, Hans-Michael Meinck, Cord-Michael Becker

  Journal of Neuroscience, v.19, 869-877 (1999).

  Missense mutations as well as a null allele of the human glycine receptor alpha 1 subunit gene GLRA1 result in the neurological disorder hyperekplexia [startle disease, stiff baby syndrome, MendelianMissense mutations as well as a null allele of the human glycine receptor alpha 1 subunit gene GLRA1 result in the neurological disorder hyperekplexia [startle disease, stiff baby syndrome, Mendelian Inheritance in Man (MIM) #149400]. In a pedigree showing dominant transmission of hyperekplexia, we identified a novel point mutation C1128A of GLRA1. This mutation encodes an amino acid substitution (P250T) in the cytoplasmic loop linking transmembrane regions M1 and M2 of the mature alpha 1 polypeptide. After recombinant expression, homomeric alpha 1P250T subunit channels showed a strong reduction of maximum whole-cell chloride currents and an altered desensitization, consistent with a prolonged recovery from desensitization. Apparent glycine binding was less affected, yielding an approximately fivefold increase in Ki values. Topological analysis predicts that the substitution of proline 250 leads to the loss of an angular polypeptide structure, thereby destabilizing open channel conformations. Thus, the novel GLRA1 mutant allele P250T defines an intracellular determinant of glycine receptor channel gating.

• Epithelial Differentiation Fails to Support Replication of Cloned Human Papillomavirus Type 16 DNA in Transfected Keratinocytes

  Authors: Wolfram Brune, Matthias Dürst

• Rapid identification of essential and nonessential herpesvirus genesby direct transposon mutagenesis

  Authors: Wolfram Brune, Carine Ménard, Urs Hobom, Stefan Odenbreit, Martin Messerle, Ulrich H. Koszinowski

• Fast Screening Procedures for Random Transposon Libraries of Cloned Herpesvirus Genomes: Mutational Analysis of Human Cytomegalovirus Envelope Glycoprotein Genes

  Authors: Urs Hobom, Wolfram Brune, Martin Messerle, Gabriele Hahn, Ulrich H. Koszinowski

  We have cloned the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli. Here, we have subjected the HCMV BAC to random transposon (Tn)We have cloned the human cytomegalovirus (HCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli. Here, we have subjected the HCMV BAC to random transposon (Tn) mutagenesis using a Tn1721-derived insertion sequence and have provided the conditions for excision of the BAC cassette. We report on a fast and efficient screening procedure for a Tn insertion library. Bacterial clones containing randomly mutated full-length HCMV genomes were transferred into 96-well microtiter plates. A PCR screening method based on two Tn primers and one primer specific for the desired genomic position of the Tn insertion was established. Within three consecutive rounds of PCR a Tn insertion of interest can be assigned to a specific bacterial clone. We applied this method to retrieve mutants of HCMV envelope glycoprotein genes. To determine the infectivities of the mutant HCMV genomes, the DNA of the identified BACs was transfected into permissive fibroblasts. In contrast to BACs with mutations in the genes coding for gB, gH, gL, and gM, which did not yield infectious virus, BACs with disruptions of open reading frame UL4 (gp48) or UL74 (gO) were viable, although gO-deficient viruses showed a severe growth deficit. Thus, gO (UL74), a component of the glycoprotein complex III, is dispensable for viral growth. We conclude that our approach of PCR screening for Tn insertions will greatly facilitate the functional analysis of herpesvirus genomes.

• Specific Inhibition of the PKR-Mediated Antiviral Response by the Murine Cytomegalovirus Proteins m142 and m143

  Authors: Matthias Budt, Lars Niederstadt, Ralitsa S Valchanova, Stipan Jonjić, Wolfram Brune

  Double-stranded RNA (dsRNA) produced during viral infection activates several cellular antiviral responses. Among the best characterized is the shutoff of protein synthesis mediated by theDouble-stranded RNA (dsRNA) produced during viral infection activates several cellular antiviral responses. Among the best characterized is the shutoff of protein synthesis mediated by the dsRNA-dependent protein kinase (PKR) and the oligoadenylate synthetase (OAS)/RNase L system. As viral replication depends on protein synthesis, many viruses have evolved mechanisms for counteracting the PKR and OAS/RNase L pathways. The murine cytomegalovirus (MCMV) proteins m142 and m143 have been characterized as dsRNA binding proteins that inhibit PKR activation, phosphorylation of the translation initiation factor eIF2α, and a subsequent protein synthesis shutoff. In the present study we analyzed the contribution of the PKR- and the OAS-dependent pathways to the control of MCMV replication in the absence or presence of m142 and m143. We show that the induction of eIF2α phosphorylation during infection with an m142- and m143-deficient MCMV is specifically mediated by PKR, not by the related eIF2α kinases PERK or GCN2. PKR antagonists of vaccinia virus (E3L) or herpes simplex virus (γ34.5) rescued the replication defect of an MCMV strain with deletions of both m142 and m143. Moreover, m142 and m143 bound to each other and interacted with PKR. By contrast, an activation of the OAS/RNase L pathway by MCMV was not detected in the presence or absence of m142 and m143, suggesting that these viral proteins have little or no influence on this pathway. Consistently, an m142- and m143-deficient MCMV strain replicated to high titers in fibroblasts lacking PKR but did not replicate in cells lacking RNase L. Hence, the PKR-mediated antiviral response is responsible for the essentiality of m142 and m143.

• Mutations in the M112/M113-Coding Region Facilitate Murine Cytomegalovirus Replication in Human Cells

  Authors: Uwe Schumacher, Wiebke Handke, Igor Jurak, Wolfram Brune

  Cytomegaloviruses, representatives of the Betaherpesvirinae, cause opportunistic infections in immunocompromised hosts. They infect various cells and tissues in their natural host but are highlyCytomegaloviruses, representatives of the Betaherpesvirinae, cause opportunistic infections in immunocompromised hosts. They infect various cells and tissues in their natural host but are highly species specific. For instance, human cytomegalovirus (HCMV) does not replicate in mouse cells, and human cells are not permissive for murine cytomegalovirus (MCMV) infection. However, the underlying molecular mechanisms are so far poorly understood. In the present study we isolated and characterized a spontaneously occurring MCMV mutant that has gained the capacity to replicate rapidly and to high titers in human cells. Compared to the parental wild-type (wt) virus, this mutant formed larger nuclear replication compartments and replicated viral DNA more efficiently. It also disrupted promyelocytic leukemia (PML) protein nuclear domains with greater efficiency but caused less apoptosis than did wt MCMV. Sequence analysis of the mutant virus genome revealed mutations in the M112/M113-coding region. This region is homologous to the HCMV UL112-113 region and encodes the viral early 1 (E1) proteins, which are known to play an important role in viral DNA replication. By introducing the M112/M113 mutations into wt MCMV, we demonstrated that they are sufficient to facilitate MCMV replication in human cells and are, at least in part, responsible for the efficient replication capability of the spontaneously adapted virus. However, additional mutations probably contribute as well. These results reveal a previously unrecognized role of the viral E1 proteins in regulating viral replication in different cells and provide new insights into the mechanisms of the species specificity of cytomegaloviruses.