Heinrich Pette Institute for Experimental Virology and Immunology

Hamburg, Germany

Heinrich Pette Institute for Experimental Virology and Immunology

Hamburg, Germany
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Hildebrand J.,Beiersdorf AG | Rutze M.,Beiersdorf AG | Walz N.,Heinrich Pette Institute for Experimental Virology and Immunology | Gallinat S.,Beiersdorf AG | And 4 more authors.
Journal of Investigative Dermatology | Year: 2011

Here, we report a comprehensive investigation of changes in microRNA (miRNA) expression profiles on human keratinocyte (HK) differentiation in vitro and in vivo. We have monitored expression patterns of 377 miRNAs during calcium-induced differentiation of primary HKs, and have compared these patterns with miRNA expression profiles of epidermal stem cells, transient amplifying cells, and terminally differentiated HKs from human skin. Apart from the previously described miR-203, we found an additional nine miRNAs (miR-23b, miR-95, miR-210, miR-224, miR-26a, miR-200a, miR-27b, miR-328, and miR-376a) that are associated with HK differentiation in vitro and in vivo. In situ hybridization experiments confirmed miR-23b as a marker of HK differentiation in vivo. Additionally, gene ontology analysis and functional validation of predicted miRNA targets using 3′-untranslated region-luciferase assays suggest that multiple miRNAs that are upregulated on HK differentiation cooperate to regulate gene expression during skin development. Our results thus provide the basis for further analysis of miRNA functions during epidermal differentiation. © 2011 The Society for Investigative Dermatology.

Polo S.E.,University of Cambridge | Blackford A.N.,University of Birmingham | Chapman J.R.,University of Cambridge | Baskcomb L.,University of Cambridge | And 13 more authors.
Molecular Cell | Year: 2012

DNA double-strand break (DSB) signaling and repair are critical for cell viability, and rely on highly coordinated pathways whose molecular organization is still incompletely understood. Here, we show that heterogeneous nuclear ribonucleoprotein U-like (hnRNPUL) proteins 1 and 2 play key roles in cellular responses to DSBs. We identify human hnRNPUL1 and -2 as binding partners for the DSB sensor complex MRE11-RAD50-NBS1 (MRN) and demonstrate that hnRNPUL1 and -2 are recruited to DNA damage in an interdependent manner that requires MRN. Moreover, we show that hnRNPUL1 and -2 stimulate DNA-end resection and promote ATR-dependent signaling and DSB repair by homologous recombination, thereby contributing to cell survival upon exposure to DSB-inducing agents. Finally, we establish that hnRNPUL1 and -2 function downstream of MRN and CtBP-interacting protein (CtIP) to promote recruitment of the BLM helicase to DNA breaks. Collectively, these results provide insights into how mammalian cells respond to DSBs. © 2012 Elsevier Inc..

Surendranath V.,Max Planck Institute of Molecular Cell Biology and Genetics | Chusainow J.,Max Planck Institute of Molecular Cell Biology and Genetics | Hauber J.,Heinrich Pette Institute for Experimental Virology and Immunology | Buchholz F.,Max Planck Institute of Molecular Cell Biology and Genetics | And 2 more authors.
Nucleic Acids Research | Year: 2010

Site-specific recombinases have become a resourceful tool for genome engineering, allowing sophisticated in vivo DNA modifications and rearrangements, including the precise removal of integrated retroviruses from host genomes. In a recent study, a mutant form of Cre recombinase has been used to excise the provirus of a specific HIV-1 strain from the human genome. To achieve provirus excision, the Cre recombinase had to be evolved to recombine an asymmetric locus of recombination (lox)-like sequence present in the long terminal repeat (LTR) regions of a HIV-1 strain. One pre-requisite for this type of work is the identification of degenerate lox-like sites in genomic sequences. Given their nature-two inverted repeats flanking a spacer of variable length-existing search tools like BLAST or RepeatMasker perform poorly. To address this lack of available algorithms, we have developed the web-server SeLOX, which can identify degenerate lox-like sites within genomic sequences. SeLOX calculates a position weight matrix based on lox-like sequences, which is used to search genomic sequences. For computational efficiency, we transform sequences into binary space, which allows us to use a bit-wise AND Boolean operator for comparisons. Next to finding lox-like sites for Cre type recombinases in HIV LTR sequences, we have used SeLOX to identify lox-like sites in HIV LTRs for six yeast recombinases. We finally demonstrate the general usefulness of SeLOX in identifying lox-like sequences in large genomes by searching Cre type recombination sites in the entire human genome. SeLOX is freely available at http://selox.mpi-cbg .de/cgi-bin/selox/index. © The Author(s) 2010. Published by Oxford University Press.

Seto E.,Helmholtz Center for Environmental Research | Moosmann A.,Ludwig Maximilians University of Munich | Gromminger S.,Helmholtz Center Munich | Walz N.,Heinrich Pette Institute for Experimental Virology and Immunology | And 2 more authors.
PLoS Pathogens | Year: 2010

Cellular and viral microRNAs (miRNAs) are involved in many different processes of key importance and more than 10,000 miRNAs have been identified so far. In general, relatively little is known about their biological functions in mammalian cells because their phenotypic effects are often mild and many of their targets still await identification. The recent discovery that Epstein-Barr virus (EBV) and other herpesviruses produce their own, barely conserved sets of miRNAs suggests that these viruses usurp the host RNA silencing machinery to their advantage in contrast to the antiviral roles of RNA silencing in plants and insects. We have systematically introduced mutations in EBV's precursor miRNA transcripts to prevent their subsequent processing into mature viral miRNAs. Phenotypic analyses of these mutant derivatives of EBV revealed that the viral miRNAs of the BHRF1 locus inhibit apoptosis and favor cell cycle progression and proliferation during the early phase of infected human primary B cells. Our findings also indicate that EBV's miRNAs are not needed to control the exit from latency. The phenotypes of viral miRNAs uncovered by this genetic analysis indicate that they contribute to EBV-associated cellular transformation rather than regulate viral genes of EBV's lytic phase. © 2010 Seto et al.

Banning C.,Heinrich Pette Institute for Experimental Virology and Immunology | Votteler J.,Friedrich - Alexander - University, Erlangen - Nuremberg | Hoffmann D.,Heinrich Pette Institute for Experimental Virology and Immunology | Koppensteiner H.,Heinrich Pette Institute for Experimental Virology and Immunology | And 6 more authors.
PLoS ONE | Year: 2010

Background: Försters resonance energy transfer (FRET) microscopy is widely used for the analysis of protein interactions in intact cells. However, FRET microscopy is technically challenging and does not allow assessing interactions in large cell numbers. To overcome these limitations we developed a flow cytometry-based FRET assay and analysed interactions of human and simian immunodeficiency virus (HIV and SIV) Nef and Vpu proteins with cellular factors, as well as HIV Rev multimer-formation. Results: Amongst others, we characterize the interaction of Vpu with CD317 (also termed Bst-2 or tetherin), a host restriction factor that inhibits HIV release from infected cells and demonstrate that the direct binding of both is mediated by the Vpu membrane-spanning region. Furthermore, we adapted our assay to allow the identification of novel protein interaction partners in a high-throughput format. Conclusion: The presented combination of FRET and FACS offers the precious possibility to discover and define protein interactions in living cells and is expected to contribute to the identification of novel therapeutic targets for treatment of human diseases. © 2010 Banning et al.

Fischer N.,University of Hamburg | Brandner J.,University of Hamburg | Fuchs F.,University of Hamburg | Moll I.,University of Hamburg | Grundhoff A.,Heinrich Pette Institute for Experimental Virology and Immunology
International Journal of Cancer | Year: 2010

The recently discovered human polyomavirus (MCPyV) is frequently found in Merkel cell carcinoma (MCC) tissue and is believed to be causally linked to MCC pathogenesis. While cell lines established from MCC represent a valuable tool to study the contribution of MCPyV to MCC pathogenesis, hitherto only 1 MCPyV-positive line has been described. We have analyzed 7 MCC cell lines for the presence, integration pattern and copy number of MCPyV. In 5 cell lines, MCPyV specific sequences were detected. In 3 of these lines, multiple copies of viral genomes per cell were detected, and sequencing of PCR amplificates identified distinct mutations predicted to lead to the expression of a truncated large T-Antigen (LT-Ag). In 1 cell line, clonal integration of concatamerized viral genomes was confirmed by Southern Blotting. MCC cell lines are conventionally categorized as "classic" or "variant" and further divided into 4 subtypes, based on expression of neuroendocrine markers and morphology. While it has been suggested that the presence of MCPyV might promote a classic phenotype, such a notion is not supported by our data. Instead, we find MCPyV-positive as well as-negative lines of the classic variety, indicating that the distinguishing features are either inherently independent of viral infection or have become so in the course of tumorigenesis and/or cell line establishment. We therefore suggest a novel classification scheme based on MCPyV presence, integration patterns and T-Ag mutations. The cell lines described here extend the repertoire of available MCPyV-positive MCC-lines and should aid in the elucidation of the role of MCPyV in the pathogenesis of MCC. © 2009 UICC.

Grundhoff A.,Heinrich Pette Institute for Experimental Virology and Immunology
Methods in Molecular Biology | Year: 2011

While cloning and/or massive parallel sequencing of small RNAs represent powerful tools for the discovery of novel miRNAs, computational miRNA prediction represents a valuable alternative which can be performed with comparably little technical effort. This is especially true for viruses, as the number of predicted candidates generally remains low and thus within a range that may be readily confirmed by experimental means. Here, we provide a detailed protocol for the prediction of putative miRNA genes using VMir, an ab initio prediction program which we have recently designed specifically to identify pre-miRNAs in viral genomes. © Springer Science+Business Media, LLC 2011.

Gunther T.,Heinrich Pette Institute for Experimental Virology and Immunology | Grundhoff A.,Heinrich Pette Institute for Experimental Virology and Immunology
PLoS Pathogens | Year: 2010

Herpesvirus latency is generally thought to be governed by epigenetic modifications, but the dynamics of viral chromatin at early timepoints of latent infection are poorly understood. Here, we report a comprehensive spatial and temporal analysis of DNA methylation and histone modifications during latent infection with Kaposi Sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi Sarcoma and primary effusion lymphoma (PEL). By use of high resolution tiling microarrays in conjunction with immunoprecipitation of methylated DNA (MeDIP) or modified histones (chromatin IP, ChIP), our study revealed highly distinct landscapes of epigenetic modifications associated with latent KSHV infection in several tumor-derived cell lines as well as de novo infected endothelial cells. We find that KSHV genomes are subject to profound methylation at CpG dinucleotides, leading to the establishment of characteristic global DNA methylation patterns. However, such patterns evolve slowly and thus are unlikely to control early latency. In contrast, we observed that latency-specific histone modification patterns were rapidly established upon a de novo infection. Our analysis furthermore demonstrates that such patterns are not characterized by the absence of activating histone modifications, as H3K9/K14-ac and H3K4-me3 marks were prominently detected at several loci, including the promoter of the lytic cycle transactivator Rta. While these regions were furthermore largely devoid of the constitutive heterochromatin marker H3K9-me3, we observed rapid and widespread deposition of H3K27-me3 across latent KSHV genomes, a bivalent modification which is able to repress transcription in spite of the simultaneous presence of activating marks. Our findings suggest that the modification patterns identified here induce a poised state of repression during viral latency, which can be rapidly reversed once the lytic cycle is induced. © 2010 Günther, Grundhoff.

Wimmer P.,Heinrich Pette Institute for Experimental Virology and Immunology | Schreiner S.,Heinrich Pette Institute for Experimental Virology and Immunology | Everett R.D.,Institute of Virology | Sirma H.,University of Hamburg | And 2 more authors.
Oncogene | Year: 2010

The E1B-55K product from human adenovirus is a substrate of the small ubiquitin-related modifier (SUMO)-conjugation system. SUMOylation of E1B-55K is required to transform primary mammalian cells in cooperation with adenovirus E1A and to repress p53 tumour suppressor functions. The biochemical consequences of SUMO1 conjugation of 55K have so far remained elusive. Here, we report that E1B-55K physically interacts with different isoforms of the tumour suppressor protein promyelocytic leukaemia (PML). We show that E1B-55K binds to PML isoforms IV and V in a SUMO1-dependent and-independent manner. Interaction with PML-IV promotes the localization of 55K to PML-containing subnuclear structures (PML-NBs). In virus-infected cells, this process is negatively regulated by other viral proteins, indicating that binding to PML is controlled through reversible SUMOylation in a timely coordinated manner. These results together with earlier work are consistent with the idea that SUMOylation regulates targeting of E1B-55K to PML-NBs, known to control transcriptional regulation, tumour suppression, DNA repair and apoptosis. Furthermore, they suggest that SUMO1-dependent modulation of p53-dependent growth suppression through E1B-55K PML-IV interaction has a key role in adenovirus-mediated cell transformation. © 2010 Macmillan Publishers Limited All rights reserved.

Chemnitz J.,Heinrich Pette Institute for Experimental Virology and Immunology | Turza N.,Friedrich - Alexander - University, Erlangen - Nuremberg | Hauber I.,Heinrich Pette Institute for Experimental Virology and Immunology | Steinkasserer A.,Friedrich - Alexander - University, Erlangen - Nuremberg | Hauber J.,Heinrich Pette Institute for Experimental Virology and Immunology
Immunobiology | Year: 2010

Dendritic cells (DC) are the most potent antigen-presenting cells (APC) of the immune system and are specialized to activate T as well as B cell-dependent immune responses. Mature DC are characterized by expression of CD83, a surface molecule that has been postulated to be required for efficient DC activity. Here we show that Leptomycin B (LMB), a highly specific inhibitor of the nuclear export receptor CRM1, abrogates the ability of DC to stimulate T cells in an allogeneic mixed lymphocyte reaction. Interestingly, this effect correlates with down-regulation of CD83, CD80 and CD86 surface expression during DC maturation, whereas other investigated DC surface molecules, such as MHC class I and II molecules are not significantly affected. Analysis of RNA distribution reveals that particularly the stimulated expression of CD83 depended on a functional CRM1 export receptor. Taken together, the presented data show a critical involvement of the CRM1 transport receptor in DC maturation, most likely by enabling efficient nucleo-cytoplasmic translocation of specific mRNAs. Thus, interference with this pathway may provide new strategies to modulate DC function and, subsequently, DC-mediated immune responses. © 2009 Elsevier GmbH.

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