Martin Messerle

Publications of Martin Messerle

• Temporal profiling of the coding and noncoding murine cytomegalovirus transcriptomes.

  Authors: Paul Lacaze, Thorsten Forster, Alan Ross, Lorraine E Kerr, Eliane Salvo-Chirnside, Vanda Juranic Lisnic, Guillermo H López-Campos, José J García-Ramírez, Martin Messerle, Joanne Trgovcich, Ana Angulo, Peter Ghazal

  Journal of virology. 04/2011; 85(12):6065-76.

  The global transcriptional program of murine cytomegalovirus (MCMV), involving coding, noncoding, and antisense transcription, remains unknown. Here we report an oligonucleotide custom microarrayThe global transcriptional program of murine cytomegalovirus (MCMV), involving coding, noncoding, and antisense transcription, remains unknown. Here we report an oligonucleotide custom microarray platform capable of measuring both coding and noncoding transcription on a genome-wide scale. By profiling MCMV wild-type and immediate-early mutant strains in fibroblasts, we found rapid activation of the transcriptome by 6.5 h postinfection, with absolute dependency on ie3, but not ie1 or ie2, for genomic programming of viral gene expression. Evidence is also presented to show, for the first time, genome-wide noncoding and bidirectional transcription at late stages of MCMV infection.

• Genetic labeling reveals altered turnover and stability of innate lymphocytes in latent mouse cytomegalovirus infection.

  Authors: Andreas Busche, Susanne Schmitz, Henrike Fleige, Scott H Robbins, Thierry Walzer, Charles A Stewart, Reinhold Förster, Martin Messerle, Immo Prinz

  Journal of immunology (Baltimore, Md. : 1950). 03/2011; 186(5):2918-25.

  Mouse CMV (MCMV) infection rapidly induces the proliferation of NK cells, which correlates with immunological protection. Whether NK cells primed during acute response against MCMV are maintained forMouse CMV (MCMV) infection rapidly induces the proliferation of NK cells, which correlates with immunological protection. Whether NK cells primed during acute response against MCMV are maintained for the long term is not known. In this study, we used TcrdH2BeGFP mice in which maturing NK cells are genetically labeled with a pulse of very stable histone-2B-eGFP. In this system, we found that the reporter protein was diluted out upon NK cell division during acute MCMV infection. At the same time, mature NK cells in uninfected mice showed only very limited turnover in vivo. Three months after primary infection when MCMV latency was established, the majority of peripheral NK cells still displayed a higher record of proliferation than NK cells in mock-infected controls. This observation included both Ly49H(+) and Ly49H(-) NK cells. Conversely, naive NK cells did not show more proliferation after transfer into latently MCMV-infected mice than that after transfer into mock-infected control mice. This indicated that the observed alterations of the NK cell compartment in MCMV latency were "legacy" (i.e., resulting from prior events during the initial immune response). Together, these results suggest that antiviral immune responses induce sustained alterations of innate lymphocyte populations that extend far beyond the first days of acute infection.

• The activator protein 1 binding motifs within the human cytomegalovirus major immediate-early enhancer are functionally redundant and act in a cooperative manner with the NF-{kappa}B sites during acute infection.

  Authors: Elena Isern, Montse Gustems, Martin Messerle, Eva Borst, Peter Ghazal, Ana Angulo

  Journal of virology. 02/2011; 85(4):1732-46.

  Human cytomegalovirus (HCMV) infection causes a rapid induction of c-Fos and c-Jun, the major subunits of activator protein 1 (AP-1), which in turn have been postulated to activate the viralHuman cytomegalovirus (HCMV) infection causes a rapid induction of c-Fos and c-Jun, the major subunits of activator protein 1 (AP-1), which in turn have been postulated to activate the viral immediate-early (IE) genes. Accordingly, the major IE promoter (MIEP) enhancer, a critical control region for initiating lytic HCMV infection and reactivation from the latent state, contains one well-characterized AP-1 site and a second candidate interaction site. In this study we explored the role of these AP-1 elements in the context of the infection. We first show that the distal candidate AP-1 motif binds c-Fos/c-Jun heterodimers (AP-1 complex) and confers c-Fos/c-Jun-mediated activity to a core promoter. Site-directed mutagenesis studies indicate that both AP-1 response elements are critical for 12-O-tetradecanoylphorbol-13-acetate (TPA)-enhanced MIEP activity in transient-transfection assays. In marked contrast to the results obtained with the isolated promoter, disruption of the AP-1 recognition sites of the MIEP in the context of the infectious HCMV genome has no significant influence on the expression of the MIE protein IE1 or viral replication in different cell types. Moreover, a chimeric murine CMV driven by the HCMV MIEP (hMCMV-ES) with the two AP-1 binding sites mutated is not compromised in virulence, is able to grow and disseminate to different organs of the newborn mice as efficiently as the parental virus, and is competent in reactivation. We show, however, that combined inactivation of the enhancer AP-1 and NF-κB recognition sites leads to an attenuation of the hMCMV-ES in the neonatal murine infection model, not observed when each single element is abolished. Altogether, these results underline the functional redundancy of the MIEP elements, highlighting the plasticity of this region, which probably evolved to ensure maximal transcriptional performance across many diverse environments.

• Single cell detection of latent cytomegalovirus reactivation in host tissue.

  Authors: Anja Marquardt, Stephan Halle, Christof K Seckert, Niels A W Lemmermann, Tibor Z Veres, Armin Braun, Ulrich A Maus, Reinhold Förster, Matthias J Reddehase, Martin Messerle, Andreas Busche

  The Journal of general virology. 02/2011; 92(Pt 6):1279-91.

  The molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need toThe molecular mechanisms leading to reactivation of latent cytomegalovirus are not well understood. To study reactivation, the few cells in an organ tissue that give rise to reactivated virus need to be identified, ideally at the earliest possible time point in the process. To this end, mouse cytomegalovirus (MCMV) reporter mutants were designed to simultaneously express the red fluorescent protein mCherry and the secreted Gaussia luciferase (Gluc). Whereas Gluc can serve to assess infection at the level of individual mice by measuring luminescence in blood samples or by in vivo imaging, mCherry fluorescence offers the advatage of detection of infection at the single cell level. To visualize cells in which MCMV was being reactivated, precision-cut lung slices (PCLS) that preserve tissue microanatomy were prepared from the lungs of latently infected mice. By day 3 of cultivation of the PCLS, reactivation was revealed by Gluc expression, preceding the detection of infectious virus by approximately 4 days. Reactivation events in PCLS could be identified when they were still confined to single cells. Notably, using fractalkine receptor-GFP reporter mice, we never observed reactivation originating from CX3CR1(+) monocytes or pulmonary dendritic cells derived therefrom. Furthermore, latent viral genome in the lungs was not enriched in sorted bone-marrow-derived cells expressing CD11b. Taken together, these complementary approaches suggest that CD11b(+) and CX3CR1(+) subsets of the myeloid differentiation lineage are not the main reservoirs and cellular sites of MCMV latency and reactivation in the lungs.

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

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

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

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

• Conditional and reversible disruption of essential herpesvirus proteins.

  Authors: Mandy Glass, Andreas Busche, Karen Wagner, Martin Messerle, Eva Maria Borst

  Nature methods. 09/2009; 6(8):577-9.

  Elucidating the function of essential proteins of complex pathogenic viruses is impeded by a paucity of complementing systems. By fusing a destabilizing domain of the FK506-binding protein toElucidating the function of essential proteins of complex pathogenic viruses is impeded by a paucity of complementing systems. By fusing a destabilizing domain of the FK506-binding protein to essential cytomegalovirus proteins, we generated virus mutants in which amounts of fusion proteins and viral growth can be regulated by the synthetic ligand shield-1. This conditional approach will greatly facilitate the analysis of gene functions of herpesviruses and viruses of other families.

• The mouse cytomegalovirus immediate-early 1 gene is not required for establishment of latency or for reactivation in the lungs.

  Authors: Andreas Busche, Anja Marquardt, Andre Bleich, Peter Ghazal, Ana Angulo, Martin Messerle

  Journal of virology. 03/2009;

  The immediate-early protein IE1 of human and mouse cytomegalovirus (MCMV) is one of the first proteins expressed during the productive infection cycle and upon reactivation from latency. The CMV IE1The immediate-early protein IE1 of human and mouse cytomegalovirus (MCMV) is one of the first proteins expressed during the productive infection cycle and upon reactivation from latency. The CMV IE1 proteins were found to inhibit histone deacetylases, suggesting a role in epigenetic regulation of viral gene expression. Consequently, the IE1 protein is considered to have a profound effect on reactivation, because small amounts of IE1 may be decisive for the switch to lytic replication. Here, we asked if an MCMV Deltaie1 mutant is able to both establish latency and to reactivate from the lungs of latently infected mice. Since the Deltaie1 mutant was known to be attenuated during acute infection, we first defined conditions that led to comparable levels of viral genomes during latent infection of the mutant and wild-type (wt) MCMV. Viral genome copy numbers dropped considerably at the onset of the latent infection but then remained steady for both viruses even after several months. Reactivation of the Deltaie1 mutant and of wt MCMV from latency occurred with a similar incidence in lung explant cultures at 4, 7 and 12 months post infection. The increase in the frequency of a subset of MCMV-specific memory T cells, a possible indicator of frequent transcriptional reactivation events during latency, was in a comparable range for both viruses. Recurrence of the Deltaie1 virus infection in vivo could also be induced by hematoablative treatment of latently infected mice. We conclude that the ie1 gene is not essential for establishment of latency or for reactivation of MCMV.

• Tumor-specific activity of cellular regulatory elements is down-regulated upon insertion into the herpes simplex virus genome.

  Authors: Mandy Glass, Ariane Soling, Martin Messerle

  Journal of neurovirology. 11/2008;

  Transcriptional targeting of viral genes is a promising strategy to achieve tumor-specific replication of oncolytic viruses. Due to its natural tropism, herpes simplex virus type 1 (HSV-1) may be anTranscriptional targeting of viral genes is a promising strategy to achieve tumor-specific replication of oncolytic viruses. Due to its natural tropism, herpes simplex virus type 1 (HSV-1) may be an ideal tool for oncolytic therapy of brain tumors such as malignant glioblastoma. To study whether glioma-specific gene expression can be accomplished within the HSV-1 genome, four cellular regulatory elements were exemplarily studied. Whereas the human telomerase reverse transcriptase (hTERT) and survivin promoters and the nestin and vascular endothelial growth factor A (VEGF-A) enhancers displayed pronounced glioma specificity after plasmid transfection, only the nestin enhancer conferred a certain selectivity for glioma cells and notable activity when transferred into the viral genome. The nestin enhancer was also found to be highly useful for tumor cell-specific expression of a therapeutically relevant gene (interleukin-2) when tested in combination with the hTERT or simian virus 40 (SV40) early promoter in the HSV-1 genome. Because activity of the chosen promoter in a tumor is a prerequisite for the successful application of an oncolytic virus, we examined whether the activity of a promoter can be deduced from the amounts of cellular mRNA or protein expressed under its control. We found little correlation between promoter activity and mRNA levels of the corresponding gene, whereas protein expression was more closely related to promoter activity. We conclude that the cellular elements are differently regulated in the viral and cellular genomes. Mechanistic insight into the differential regulation is required to improve and refine the design of transcriptionally targeted HSV vectors.

• In vivo competence of murine cytomegalovirus under the control of the human cytomegalovirus major immediate-early enhancer in the establishment of latency and reactivation.

  Authors: Montse Gustems, Andreas Busche, Martin Messerle, Peter Ghazal, Ana Angulo

  Journal of virology. 09/2008;

  The human cytomegalovirus (HCMV) major immediate-early enhancer has been postulated to play a pivotal role in the control of latency and reactivation. However, the absence of an animal model hasThe human cytomegalovirus (HCMV) major immediate-early enhancer has been postulated to play a pivotal role in the control of latency and reactivation. However, the absence of an animal model has obstructed a direct test of this hypothesis. Here we report on the establishment of an in vivo experimentally tractable system for quantitatively investigating physiological functions of the HCMV enhancer. Using a neonate BALB/c mice model, we show that a chimeric murine CMV under control of the HCMV enhancer is competent in vivo: replicating in key organs of the CMV acute infection and exhibiting latency/reactivation features mostly comparable to those of the parental and revertant viruses.

• Phenotypes of major immediate-early gene mutants of mouse cytomegalovirus.

  Authors: Andreas Busche, Ana Angulo, Penelope Kay-Jackson, Peter Ghazal, Martin Messerle

  Medical microbiology and immunology. 07/2008; 197(2):233-40.

  Immediate-early (IE) genes are the first genes to be transcribed during the lytic replication cycle of cytomegaloviruses (CMV), and encode nonstructural proteins, which are assumed to have mainlyImmediate-early (IE) genes are the first genes to be transcribed during the lytic replication cycle of cytomegaloviruses (CMV), and encode nonstructural proteins, which are assumed to have mainly regulatory functions. The IE proteins may play important roles in the pathogenesis of CMV in vivo, for instance during the establishment of latency and during reactivation. We constructed mouse CMV mutants with disruptions in the major IE genes, ie1 and ie3, to study the roles of these genes in the context of the viral infection. Here we summarize the current results on the characterization of these mutants and give a perspective of the future research in this field.

• The essential human cytomegalovirus gene UL52 is required for cleavage-packaging of the viral genome.

  Authors: Eva Maria Borst, Karen Wagner, Anne Binz, Beate Sodeik, Martin Messerle

  Journal of virology. 04/2008; 82(5):2065-78.

  Replication of human cytomegalovirus (HCMV) produces large DNA concatemers of head-to-tail-linked viral genomes that upon packaging into capsids are cut into unit-length genomes. The mechanismsReplication of human cytomegalovirus (HCMV) produces large DNA concatemers of head-to-tail-linked viral genomes that upon packaging into capsids are cut into unit-length genomes. The mechanisms underlying cleavage-packaging and the subsequent steps prior to nuclear egress of DNA-filled capsids are incompletely understood. The hitherto uncharacterized product of the essential HCMV UL52 gene was proposed to participate in these processes. To investigate the function of pUL52, we constructed a DeltaUL52 mutant as well as a complementing cell line. We found that replication of viral DNA was not impaired in noncomplementing cells infected with the DeltaUL52 virus, but viral concatemers remained uncleaved. Since the subnuclear localization of the known cleavage-packaging proteins pUL56, pUL89, and pUL104 was unchanged in DeltaUL52-infected fibroblasts, pUL52 does not seem to act via these proteins. Electron microscopy studies revealed only B capsids in the nuclei of DeltaUL52-infected cells, indicating that the mutant virus has a defect in encapsidation of viral DNA. Generation of recombinant HCMV genomes encoding epitope-tagged pUL52 versions showed that only the N-terminally tagged pUL52 supported viral growth, suggesting that the C terminus is crucial for its function. pUL52 was expressed as a 75-kDa protein with true late kinetics. It localized preferentially to the nuclei of infected cells and was found to enclose the replication compartments. Taken together, our results demonstrate an essential role for pUL52 in cleavage-packaging of HCMV DNA. Given its unique subnuclear localization, the function of pUL52 might be distinct from that of other cleavage-packaging proteins.

• Nuclear egress and envelopment of herpes simplex virus capsids analyzed with dual-color fluorescence HSV1(17+).

  Authors: Claus-Henning Nagel, Katinka Döhner, Mojgan Fathollahy, Tanja Strive, Eva Maria Borst, Martin Messerle, Beate Sodeik

  Journal of virology. 04/2008; 82(6):3109-24.

  To analyze the assembly of herpes simplex virus type 1 (HSV1) by triple-label fluorescence microscopy, we generated a bacterial artificial chromosome (BAC) and inserted eukaryotic Cre recombinase, asTo analyze the assembly of herpes simplex virus type 1 (HSV1) by triple-label fluorescence microscopy, we generated a bacterial artificial chromosome (BAC) and inserted eukaryotic Cre recombinase, as well as beta-galactosidase expression cassettes. When the BAC pHSV1(17(+))blueLox was transfected back into eukaryotic cells, the Cre recombinase excised the BAC sequences, which had been flanked with loxP sites, from the viral genome, leading to HSV1(17(+))blueLox. We then tagged the capsid protein VP26 and the envelope protein glycoprotein D (gD) with fluorescent protein domains to obtain HSV1(17(+))blueLox-GFPVP26-gDRFP and -RFPVP26-gDGFP. All HSV1 BACs had variations in the a-sequences and lost the oriL but were fully infectious. The tagged proteins behaved as their corresponding wild type, and were incorporated into virions. Fluorescent gD first accumulated in cytoplasmic membranes but was later also detected in the endoplasmic reticulum and the plasma membrane. Initially, cytoplasmic capsids did not colocalize with viral glycoproteins, indicating that they were naked, cytosolic capsids. As the infection progressed, they were enveloped and colocalized with the viral membrane proteins. We then analyzed the subcellular distribution of capsids, envelope proteins, and nuclear pores during a synchronous infection. Although the nuclear pore network had changed in ca. 20% of the cells, an HSV1-induced reorganization of the nuclear pore architecture was not required for efficient nuclear egress of capsids. Our data are consistent with an HSV1 assembly model involving primary envelopment of nuclear capsids at the inner nuclear membrane and primary fusion to transfer capsids into the cytosol, followed by their secondary envelopment on cytoplasmic membranes.

• 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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• Protection from CMV infection in immunodeficient hosts by adoptive transfer of memory B cells.

  Authors: Karin Klenovsek, Florian Weisel, Andrea Schneider, Uwe Appelt, Stipan Jonjic, Martin Messerle, Birgit Bradel-Tretheway, Thomas H Winkler, Michael Mach

  Blood. 12/2007; 110(9):3472-9.

  Severe disease associated with cytomegalovirus (CMV) infection is still a major problem in patients who undergo transplantation. Support of the patients' immune defense against the virus is a majorSevere disease associated with cytomegalovirus (CMV) infection is still a major problem in patients who undergo transplantation. Support of the patients' immune defense against the virus is a major goal in transplantation medicine. We have used the murine model of CMV (MCMV) to investigate the potential of a cell-based strategy to support the humoral antiviral immune response. Immunocompetent C57BL/6 mice were infected with MCMV, and memory B cells from the immune animals were adoptively transferred into T-cell- and B-cell-deficient RAG-1(-/-) mice. Following MCMV infection, a virus-specific IgG response developed within 4 to 7 days in the recipient animals. Concomitantly, a significant reduction in viral titers and DNA copies in several organs was observed. In addition, the memory B-cell transfer provided long-term protection from the lethal course of the infection that is invariably seen in immunodeficient animals. Transfer of memory B cells was also effective in protecting from an already ongoing viral infection, indicating a therapeutic potential of virus-specific memory B cells. T cells were not involved in this process. Our data provide evidence that a cell-based strategy to support the humoral immune response can be effective to combat infectious pathogens in severely immunodeficient hosts.

• Use of bacterial artificial chromosomes in generating targeted mutations in human and mouse cytomegaloviruses.

  Authors: Eva Maria Borst, Corinna Benkartek, Martin Messerle

  Current protocols in immunology / edited by John E. Coligan ... [et al.]. 06/2007; Chapter 10:Unit 10.32.

  Cloning of cytomegalovirus (CMV) genomes as bacterial artificial chromosomes (BAC) in E. coli and their manipulation using the techniques of bacterial genetics has greatly facilitated theCloning of cytomegalovirus (CMV) genomes as bacterial artificial chromosomes (BAC) in E. coli and their manipulation using the techniques of bacterial genetics has greatly facilitated the construction of CMV mutants. This unit describes easily applicable procedures that allow rapid introduction of any kind of targeted mutation into BAC-cloned CMV genomes. Protocols for the reconstitution of virus from isolated BAC DNA, preparation of a virus stock, and isolation and characterization of viral DNA are also included. Special emphasis is laid on description of critical steps and thorough characterization of the altered BACs.

• Regulation of the transcription and replication cycle of human cytomegalovirus is insensitive to genetic elimination of the cognate NF-kappaB binding sites in the enhancer.

  Authors: Montse Gustems, Eva Borst, Chris A Benedict, Carmen Pérez, Martin Messerle, Peter Ghazal, Ana Angulo

  Journal of virology. 11/2006; 80(19):9899-904.

  The role of NF-kappaB in regulating human cytomegalovirus (HCMV) replication and gene transcription remains controversial. Multiple, functional NF-kappaB response elements exist in the majorThe role of NF-kappaB in regulating human cytomegalovirus (HCMV) replication and gene transcription remains controversial. Multiple, functional NF-kappaB response elements exist in the major immediate-early promoter (MIEP) enhancer of HCMV, suggesting a possible requirement for this transcription factor in lytic viral replication. Here we demonstrate by generating and analyzing HCMVs with alterations in the MIEP-enhancer that, although this region is essential for HCMV growth, none of the four NF-kappaB response elements contained within the enhancer are required for MIE gene expression or HCMV replication in multiple cell types. These data reveal the robustness of the regulatory network controlling the MIEP enhancer.

• The herpesviral Fc receptor fcr-1 down-regulates the NKG2D ligands MULT-1 and H60.

  Authors: Tihana Lenac, Matthias Budt, Jurica Arapovic, Milena Hasan, Albert Zimmermann, Hrvoje Simic, Astrid Krmpotic, Martin Messerle, Zsolt Ruzsics, Ulrich H. Koszinowski, Hartmut Hengel, Stipan Jonjic

  The Journal of experimental medicine. 09/2006; 203(8):1843-50.

  Members of the alpha- and beta-subfamily of herpesviridae encode glycoproteins that specifically bind to the Fc part of immunoglobulin (Ig)G. Plasma membrane resident herpesviral Fc receptors seem toMembers of the alpha- and beta-subfamily of herpesviridae encode glycoproteins that specifically bind to the Fc part of immunoglobulin (Ig)G. Plasma membrane resident herpesviral Fc receptors seem to prevent virus-specific IgG from activating antibody-dependent effector functions. We show that the mouse cytomegalovirus (MCMV) molecule fcr-1 promotes a rapid down-regulation of NKG2D ligands murine UL16-binding protein like transcript (MULT)-1 and H60 from the cell surface. Deletion of the m138/fcr-1 gene from the MCMV genome attenuates viral replication to natural killer (NK) cell response in an NKG2D-dependent manner in vivo. A distinct N-terminal module within the fcr-1 ectodomain in conjunction with the fcr-1 transmembrane domain was required to dispose MULT-1 to degradation in lysosomes. In contrast, down-modulation of H60 required the complete fcr-1 ectodomain, implying independent modes of fcr-1 interaction with the NKG2D ligands. The results establish a novel viral strategy for down-modulating NK cell responses and highlight the impressive diversity of Fc receptor functions.

• Identification of the interaction domain of the small terminase subunit pUL89 with the large subunit pUL56 of human cytomegalovirus.

  Authors: Corina Thoma, Eva Borst, Martin Messerle, Manuela Rieger, Jae-Seon Hwang, Elke Bogner

  Biochemistry. 08/2006; 45(29):8855-63.

  The small terminase subunit pUL89 of human cytomegalovirus (HCMV) is thought to be required for cleavage of viral DNA into unit-length genomes in the cleavage/packaging process. ImmunoprecipitationsThe small terminase subunit pUL89 of human cytomegalovirus (HCMV) is thought to be required for cleavage of viral DNA into unit-length genomes in the cleavage/packaging process. Immunoprecipitations with a UL89-specific antibody demonstrated that pUL89 occurs predominantly as a monomer of approximate M(r) 75.000 together with a dimer of approximate 150.000. This was confirmed by gel permeation chromatography. In view of its putative function, pUL89 needs to be transported into the nucleus. By use of laser scanning confocal microscopy, pUL89 was found to be predominantly localized throughout the nucleus and in particular in viral replication centers of infected cells. By immunofluorescence, we demonstrated that both terminase subunits co-localized in viral replication centers. Furthermore, analysis with pUL89 GST-fusion protein mutants showed that amino acids 580-600 may represent the interaction domain with pUL56. To verify this result, a recombinant HCMV genome was constructed in which the UL89 open reading frame was disrupted. By transfection of the deletion BACmid alone, we showed that it has a lethal phenotype. Cotransfection assays demonstrated that, in contrast to pUL89 wild-type, a plasmid construct encoding a pUL89 variant without aa 580-590 as well as one encoding a variant without aa 590-600 could not complement the HCMV-pUL89 null genome, thus, suggesting that the 20 aa sequence GRDKALAVEQFISRFNSGYIK is sufficient for the interaction with pUL56 and in conclusion required for DNA packaging.

• Manipulating cytomegalovirus genomes by BAC mutagenesis: Strategies and applications

  Authors: Wolfram Brune, Markus Wagner, Martin Messerle

  01/2006: pages 61-69;

  ISBN: 1-904455-02-6

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• Frequent coinfection of cells explains functional in vivo complementation between cytomegalovirus variants in the multiply infected host.

  Authors: Luka Cicin-Sain, Jürgen Podlech, Martin Messerle, Matthias J. Reddehase, Ulrich H. Koszinowski

  Journal of virology. 09/2005; 79(15):9492-502.

  In contrast to many other virus infections, primary cytomegalovirus (CMV) infection does not fully protect against reinfection. Accordingly, clinical data have revealed a coexistence of multipleIn contrast to many other virus infections, primary cytomegalovirus (CMV) infection does not fully protect against reinfection. Accordingly, clinical data have revealed a coexistence of multiple human CMV variants/strains in individual patients. Notably, the phenomenon of multiple infection was found to correlate with increased virus load and severity of CMV disease. Although of obvious medical relevance, the mechanism underlying this correlation is unknown. A weak immune response in an individual could be responsible for a more severe disease and for multiple infections. Alternatively, synergistic contributions of variants that differ in their biological properties can lead to qualitative changes in viral fitness by direct interactions such as genetic recombination or functional complementation within coinfected host cells. We have addressed this important question paradigmatically with the murine model by differently designed combinations of two viruses employed for experimental coinfection of mice. Specifically, a murine cytomegalovirus (MCMV) mutant expressing Cre recombinase was combined for coinfection with a mutant carrying Cre-inducible green fluorescent protein gene, and attenuated mutants were combined for coinfection with wild-type virus followed by two-color in situ hybridization studies visualizing the replication of the two viruses in infected host organs. These different approaches concurred in the conclusion that coinfection of host cells is more frequent than statistically predicted and that this coinfection alters virus fitness by functional trans-complementation rather than by genetic recombination. The reported findings make a major contribution to our molecular understanding of enhanced CMV pathogenicity in the multiply infected host.