Matthias Budt

Publications of Matthias Budt

• Construction of a lytically replicating Kaposi's sarcoma-associated herpesvirus.

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

  Journal of virology. 07/2011; 85(19):10415-20.

  Karposi's sarcoma-associated herpesvirus (KSHV) is found predominantly in a latent state in most cell types, impeding investigations of the lytic replication cycle. Here, we engineered the clonedKarposi's sarcoma-associated herpesvirus (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, bacterial artificial chromosome 36 (BAC36), to enforce constitutive expression of the main lytic switch regulator, the replication and transcription activator (RTA) (open reading frame 50 [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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• 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 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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• 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.

• Expansion of CD94/NKG2C+ NK cells in response to human cytomegalovirus-infected fibroblasts.

  Authors: Mónica Gumá, Matthias Budt, Andrea Sáez, Tamara Brckalo, Hartmut Hengel, Ana Angulo, Miguel López-Botet

  Blood. 06/2006; 107(9):3624-31.

  CD94/NKG2C(+) natural killer (NK) cells are increased in healthy individuals infected with human cytomegalovirus (HCMV), suggesting that HCMV infection may shape the NK cell receptor repertoire. ToCD94/NKG2C(+) natural killer (NK) cells are increased in healthy individuals infected with human cytomegalovirus (HCMV), suggesting that HCMV infection may shape the NK cell receptor repertoire. To address this question, we analyzed the distribution of NK cell subsets in peripheral blood lymphocytes (PBLs) cocultured with HCMV-infected fibroblasts. A substantial increase of NK cells was detected by day 10 in samples from a group of HCMV(+) donors, and CD94/NKG2C(+) cells outnumbered the CD94/NKG2A(+) subset. Fibroblast infection was required to induce the preferential expansion of CD94/NKG2C(+) NK cells that was comparable with allogeneic or autologous fibroblasts, and different virus strains. A CD94-specific monoclonal antibody (mAb) abrogated the effect, supporting an involvement of the lectinlike receptor. Purified CD56(+) populations stimulated with HCMV-infected cells did not proliferate, but the expansion of the CD94/NKG2C(+) subset was detected in the presence of interleukin-15 (IL-15). Experiments with HCMV deletion mutants indicated that the response of CD94/NKG2C(+) NK cells was independent of the UL16, UL18, and UL40 HCMV genes, but was impaired when cells were infected with a mutant lacking the US2-11 gene region. Taken together the data support that the interaction of CD94/NKG2C with HCMV-infected fibroblasts, concomitant to the inhibition of human leukocyte antigen (HLA) class I expression, promotes an outgrowth of CD94/NKG2C(+) NK cells.

• Herpesviral Fcgamma receptors: culprits attenuating antiviral IgG?

  Authors: Matthias Budt, Henrike Reinhard, Arndt Bigl, Hartmut Hengel

  International immunopharmacology. 10/2004; 4(9):1135-48.

  Production of IgG in response to virus infection is central to antiviral immune effector functions and a hallmark of B cell memory. Antiviral antibodies (Abs) recognising viral glycoproteins orProduction of IgG in response to virus infection is central to antiviral immune effector functions and a hallmark of B cell memory. Antiviral antibodies (Abs) recognising viral glycoproteins or protein antigen displayed on the surface of virions or virus-infected cells are crucial in rendering the virus noninfectious and in eliminating viruses or infected cells, either acting alone or in conjunction with complement. In many instances, passive transfer of Abs is sufficient to protect from viral infection. Herpesviruses (HV) are equipped with a large array of immunomodulatory functions which increase the efficiency of infection by dampening the antiviral immunity. Members of the alpha- and beta-subfamily of the Herpesviridae are distinct in encoding transmembrane glycoproteins which selectively bind IgG via its Fc domain. The Fc-binding proteins constitute viral Fcgamma receptors (vFcgammaRs) which are expressed on the cell surface of infected cells. Moreover, vFcgammaRs are abundantly incorporated into the envelope of virions. Despite their molecular and structural heterogeneity, the vFcgammaRs generally interfere with IgG-mediated effector functions like antibody (Ab)-dependent cellular cytolysis, complement activation and neutralisation of infectivity of virions. vFcgammaRs may thus contribute to the limited therapeutic potency of antiherpesviral IgG in clinical settings. A detailed molecular understanding of vFcgammaRs opens up the possibility to design recombinant IgG molecules resisting vFcgammaRs. Engineering IgG with a better antiviral efficiency represents a new therapeutic option against herpesviral diseases.

• Clustering-induced signaling of CEACAM1 in PC12 cells.

  Authors: Matthias Budt, Iwona Cichocka, Werner Reutter, Lothar Lucka

  Biological chemistry. 06/2002; 383(5):803-12.

  Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), an Ig-like transmembrane protein, functions in cell adhesion, angiogenesis and epithelial cell morphogenesis, and has beenCarcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), an Ig-like transmembrane protein, functions in cell adhesion, angiogenesis and epithelial cell morphogenesis, and has been identified as a tumor suppressor. For all of these functions, CEACAM1 requires signaling capabilities. However, the mechanisms of CEACAM1-mediated signaling are only poorly understood. Here we characterized for the first time CEACAM1 expression and signaling in the neuroendocrine rat pheochromocytoma PC12 cell line. Stimulation of CEACAM1 by ligation on the cell surface with antibodies induced formation of large CEACAM1 clusters and a rapid and transient CEACAM1 tyrosine dephosphorylation. Functionally, this dephosphorylation correlated with a reduced association between CEACAM1 and the tyrosine phosphatase SHP2. Clustering also stimulated binding of CEACAM1 to the actin cytoskeleton, measured by a partial translocation of CEACAM1 into the insoluble fraction after detergent extraction. Both tyrosine dephosphorylation and interaction with the cytoskeleton were sensitive to neuronal differentiation of PC12 cells. The first detected downstream activation of the mitogen-activated protein kinases ERK1 and ERK2, but not of JNK or p38, describes a novel target of CEACAM1-mediated signaling and contributes to the understanding of how CEACAM1 regulates cellular function.

• Secreted CEACAM1 splice variants in rat cell lines and in vivo in rat serum.

  Authors: Matthias Budt, Beate Michely, Mario M Müller, Werner Reutter, Lothar Lucka

  Biochemical and biophysical research communications. 05/2002; 292(3):749-55.

  The widely expressed adhesion receptor CEACAM1 is a member of the carcinoembryonic antigen (CEA) family within the immunoglobulin (Ig) superfamily of glycoproteins. While the expression ofThe widely expressed adhesion receptor CEACAM1 is a member of the carcinoembryonic antigen (CEA) family within the immunoglobulin (Ig) superfamily of glycoproteins. While the expression of transmembrane isoforms has been described in detail, only little is known about soluble isoforms. By RT-PCR characterization of rat pheochromocytoma PC12 and mammary adenocarcinoma MTC cell lines, two novel splice variants, designated CEACAM1-4C1 and CEACAM1-4C2, lacking the transmembrane region, were identified. In addition, we demonstrate the expression of transmembrane CEACAM1-4L and CEACAM1-4S with a truncated cytoplasmic domain. The C-termini of CEACAM1-4C2 and CEACAM1-L are identical, which allowed the specific in vitro and in vivo detection of the soluble CEACAM1-4C2 protein by an antiserum generated against the CEACAM1-L cytoplasmic part. Functionally, soluble CEACAM1 could inhibit CEACAM1-mediated aggregation of CHO cells. In conclusion, our data define a new mechanism for the appearance of functionally active rat CEACAM1 protein in body fluids.