Project area Z:
Dr. Jochen Wilhelm
Univ. of Gießen Lung Center
German Lung Research Center
Phone: 0641-99 42545
PD Dr. Torsten Hain
German Center of Infection
Institut für Medizinische Mikrobiologie
Phone: 0641-99 39860
Research area: Molecular Virology
Complete viral genome analyses and transcriptomics studies of virus-infected host cells have become key technologies in the field of RNA virus research. Recent advances in next-generation sequencing (NGS) and microarray technologies resulted in higher throughput, improved accuracy and lower costs. The technical improvements and the wealth of information obtained from these technologies make them extremely valuable tools for studying RNA viruses and virus-host interactions in vitro and in vivo.
The microarray facility of Z02 will provide the infrastructure to plan and perform whole transcriptome analyses using the Agilent microarray platform. The available microarrays encompass all known protein-coding genes and more than 30,000 unique long non-coding RNAs (lncRNA), the latter being suggested to play important roles in virus replication. Also, expression profiles obtained in these analyses will be linked to information on viral and cellular non-coding RNA-associated interactions provided by recently developed databases such as ViRBase.
The NGS infrastructure of Z02 project will perform (i) whole transcriptome analyses using RNA-seq (ii) targeted resequencing (including amplicon sequencing), (iii) tRNA-seq and (iv) RNA virus genome sequencing for all members of the CRC. Furthermore (v), viral RNA genome quasispecies will be analyzed using recently established protocols suitable to generate “PCR and sequencing error bias free” NGS datasets and study true genetic variants of RNA virus genomes by circle sequencing (Cir-seq).
NGS via RNA-seq of host cells will be used to (vi) analyze samples for which appropriate DNA microarrays are not (yet) available or (vii) obtain parallel sequence information for both host cell and virus (Dual-seq) including single-cell RNA sequencing (scRNA-seq) of virus-infected/mock-infected cells.
The Z02 project will provide biostatistical support in the planning of microarray and NGS experiments, generate genome sequencing and expression profiling data and apply bioinformatics methods, such as over-representation and gene-set enrichment analyses, to identify specific signalling pathways and regulatory networks relevant to specific RNA virus infections. Together with other CRC1021 researchers, we will provide additional training (sample preparation, bioinformatics) to PhD students and postdocs using these technologies in their projects. Also, we will contribute to developing hypotheses and writing manuscripts arising from data generated in the Z02 project.
Project-related publications of the investigators:
- MacKenzie B, Henneke I, Hezel S, Al Alam D, El Agha E, Chao C-M, Quantius J, Wilhelm J, Jones M, Goth K, Li X, Seeger W, Königshoff M, Herold S, Rizvanov AA, Günther A, Bellusci S. 2015. Attenuating endogenous Fgfr2b ligands during bleomycin-induced lung fibrosis does not compromise murine lung repair. Am J Physiol Lung Cell Mol Physiol 308:L1014–24.
- Wilhelm J, Hoffmann J, Marsh LM, Ghanim B, Klepetko W, Kovacs G, Olschewski H, Olschewski A, Kwapiszewska G. 2014. Distinct differences in gene expression patterns in pulmonary arteries of patients with chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis with pulmonary hypertension. Am J Respir Crit Care Med 190:98–111.
- Weisel FC, Kloepping C, Pichl A, Sydykov A, Kojonazarov B, Wilhelm J, Roth M, Ridge KM, Igarashi K, Nishimura K, Maison W, Wackendorff C, Klepetko W, Jaksch P, Ghofrani HA, Grimminger F, Seeger W, Schermuly RT, Weissmann N, Kwapiszewska G. 2014. Impact of S-adenosylmethionine decarboxylase 1 on pulmonary vascular remodeling. Circulation 129:1510–1523.
- Zakrzewicz, A., Krasteva, G., Wilhelm, J., Dietrich, H., Wilker, S., Padberg, W., Wygrecka, M., and Grau, V. Reduced expression of arrestin beta 2 by graft monocytes during acute rejection of rat kidneys. 2011. Immunobiology. 216(7): 854-61
- Mikulski, Z., Zaslona, Z., Cakarova, L., Hartmann, P., Wilhelm, J., Tecott, L.H., Lohmeyer, J., and Kummer, W. Serotonin activates murine alveolar macrophages through 5-HT2C receptors. 2010. Am J Physiol Lung Cell Mol Physiol. 299(2): L272-80
- Hecker, M., Zaslona, Z., Kwapiszewska, G., Niess, G., Zakrzewicz, A., Hergenreider, E., Wilhelm, J., Marsh, L.M., et al. Dysregulation of the IL-13 receptor system: a novel pathomechanism in pulmonary arterial hypertension. 2010. Am J Respir Crit Care Med. 182(6): 805-18
- Konigshoff, M., Kramer, M., Balsara, N., Wilhelm, J., Amarie, O.V., Jahn, A., Rose, F., Fink, L., et al. WNT1-inducible signaling protein-1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis. 2009. J Clin Invest. 119(4): 772-87
- Kwapiszewska, G., Wygrecka, M., Marsh, L.M., Schmitt, S., Trosser, R., Wilhelm, J., Helmus, K., Eul, B., et al. Fhl-1, a new key protein in pulmonary hypertension. 2008. Circulation. 118(11): 1183-94
- Wilhelm, J., Muyal, J.P., Best, J., Kwapiszewska, G., Stein, M.M., Seeger, W., Bohle, R.M., and Fink, L. Systematic comparison of the T7-IVT and SMART-based RNA preamplification techniques for DNA microarray experiments. 2006. Clin Chem. 52(6): 1161-7
- Mengel JP, Lissin A, Biedenkopf N, Schultze T, Mannala GK, Schudt G, Kann G, Wolf T, Eickmann M, Becker S, Cemic F, Hain T.
- Wolff S, Schultze T, Fehling S, Mengel JP, Kann G, Wolf T, Eickmann M, Becker S, Hain T, and Strecker T. 2016. Genome sequence of Lassa virus isolated from the first domestically acquired case in Germany. Genome Announc. accepted
- Mengel JP, Lissin A, Biedenkopf B, Schultze T, Mannala GK, Schudt G, Kann G, Wolf T, Eickmann M, Becker S, Cemic F, and Hain T. 2016. Complete genome sequence of an Ebola virus isolate from an imported case in Germany ex Sierra Leone determined by circle sequencing (Cir-seq). Genome Announc, accepted
- Schultze T, Hilker R, Mannala GK, Gentil K, Weigel M, Farmani N, Windhorst AC, Goesmann A, Chakraborty T, Hain T. 2015. A detailed view of the intracellular transcriptome of Listeria monocytogenes in murine macrophages using RNA-seq. Front Microbiol 6:1–13.
- Behrens S, Widder S, Mannala GK, Qing X, Madhugiri R, Kefer N, Mraheil MA, Rattei T, Hain T. 2014. Ultra deep sequencing of Listeria monocytogenes sRNA transcriptome revealed new antisense RNAs. PLoS One 9:1–10.
- Wehner S, Mannala GK, Qing X, Madhugiri R, Chakraborty T, Mraheil MA, Hain T, Marz M. 2014. Detection of very long antisense transcripts by whole transcriptome RNA-seq analysis of listeria monocytogenes by semiconductor sequencing technology. PLoS One 9:1–10.
- Hain, T., R. Ghai, A. Billion, C. T. Kuenne, C. Steinweg, B. Izar, W. Mohamed, M. Mraheil, E. Domann, S. Schaffrath, U. Kärst, A. Goesmann, S. Oehm, A. Pühler, R. Merkl, S. Vorwerk, P. Glaser, P. Garrido, C. Rusniok, C. Buchrieser, W. Goebel, and T. Chakraborty. 2012. Comparative genomics and transcriptomics of lineages I, II, and III strains of Listeria monocytogenes. BMC Genomics, accepted.
- Izar, B., G. K. Mannala, M. A. Mraheil, T. Chakraborty, and T. Hain. 2012. microRNA Response to Listeria monocytogenes infection in epithelial cells. Int. J Mol. Sci. 13:1173-1185.
- Mraheil, M. A., A. Billion, W. Mohamed, K. Mukherjee, C. Kuenne, J. Pischimarov, C. Krawitz, J. Retey, T. Hartsch, T. Chakraborty, and T. Hain. 2011. The intracellular sRNA transcriptome of Listeria monocytogenes during growth in macrophages. Nucleic Acids Res 39:4235-4248.
- Kuenne, C., S. Voget, J. Pischimarov, S. Oehm, A. Goesmann, R. Daniel, T. Hain, and T. Chakraborty. 2010. Comparative analysis of plasmids in the genus Listeria. PLoS. One. 5.
- Kuenne, C. T., R. Ghai, T. Chakraborty, and T. Hain. 2007. GECO--linear visualization for comparative genomics. Bioinformatics. 23:125-126.
- Hain, T., C. Steinweg, C. T. Kuenne, A. Billion, R. Ghai, S. S. Chatterjee, E. Domann, U. Karst, A. Goesmann, T. Bekel, D. Bartels, O. Kaiser, F. Meyer, A. Puhler, B. Weisshaar, J. Wehland, C. Liang, T. Dandekar, R. Lampidis, J. Kreft, W. Goebel, and T. Chakraborty. 2006. Whole-genome sequence of Listeria welshimeri reveals common steps in genome reduction with Listeria innocua as compared to Listeria monocytogenes. J Bacteriol. 188:7405-7415.
Prof. Dr. Michael Kracht
Rudolf-Buchheim-Institut für Pharmakologie
Phone: 0641-99 47601
Dr. Uwe Linne
Phone: 06421-28 25618
Research area: Proteomics, PTM-analysis
Viral infection of target cells results in multiple alterations at the level of cellular signaling, transcription and translation to enable viral replication and to fight off cellular antiviral responses. Viral proteins interact with specific subsets of host cell proteins and are post-translationally modified by host cell enzymes. In turn, the virus infection affects expression rates of host cell proteins and their modification status either directly or indirectly. Changes at the level of the proteome precede or are a (direct) consequence of transcriptome changes. Additionally, both events can occur uncoupled. Thus, understanding proteome changes in relation to transcriptome alterations is instrumental to develop a holistic view on virus/cell interactions. The central aim of the Z03 project is to enable CRC1021 projects to investigate these aspects at a proteome-wide level quantitatively and with high resolution. To achieve this goal, the expertise from the group of M.Kracht in application of proteomics techniques to RNA virus biology will be combined with the expertise of U.Linne, who is heading a high-end mass spectrometry facility at the Faculty of Chemistry, Marburg. The facility is fully equipped with state of the art mass spectrometers including Orbitraps Velos Pro and XL (Thermo Scientific) and a Synapt G2Si, all connected to nanoHPLCs. Z03 will provide standardized work flows to study (i) protein-protein interactions, (ii) protein expression levels and (iii) post-translational modifications (PTM) of proteins. Additionally, mass spectrometric standard methods for quality control, amino acid sequence and mass determination of purified proteins will be provided to all members of CRC1021. These approaches can be used to study the interaction of viral components with host cells proteins or between host cell proteins including identification of proteins in samples derived from specific tagging strategies or the usage of cell-permeable crosslinkers . Quantitative changes of expression across entire proteomes will be analyzed using stable isotope labelling strategies or label-free quantification methods. Phosphorylated, ubiquitylated or acetylated peptides will be enriched from denatured lysates by antibodies recognizing K-ε-G-G motifs or acetylated lysines or by immobilized metal ion affinity chromatography (IMAC) and will be identified by LC-MS/MS. A specific further aim of the Z03 project is to provide sophisticated analysis work flows for the resulting large data sets. This includes summary tables, detailed statistics and visualizations of modified and regulated residues mapped to peptides and genes as well as identification of consensus motifs and de novo motif searches for regulated subgroups of peptides. A work flow that has already been established in the R biostatistics environment by Dr. Axel Weber in the Kracht group (C02) will enable identification of statistically enriched components of KEGG or GO pathways, pathway mapping, protein network analyses and publication-ready visualizations using Cytoscape, STRING and several additional bioinformatics tools.
Project-related publications of the investigators:
- Tenekeci, U, Poppe, M, Beuerlein, K, Buro, C, Müller, H, Weiser, H, Kettner-Buhrow, D, Porada, K, Newel, D, Xu, M, Chen, ZJ, Busch, J, Schmitz, ML and Kracht, M. 2016. K63-ubiquitylation and TRAF6 pathways regulate mammalian P-body formation and mRNA decapping. Mol.Cell, 62:943-957.
- Schuhmacher JS, Rossmann F, Dempwolff F, Knauer C, Altegoer F, Steinchen W, Dorrich AK, Klingl A, Stephan M, Linne U, Thormann KM, Bange G. 2015. MinD-like ATPase FlhG effects location and number of bacterial flagella during C-ring assembly. Proceedings of the National Academy of Sciences of the United States of America 112:3092-3097.
- Steinchen W, Schuhmacher JS, Altegoer F, Fage CD, Srinivasan V, Linne U, Marahiel MA, Bange G. 2015. Catalytic mechanism and allosteric regulation of an oligomeric (p)ppGpp synthetase by an alarmone. Proceedings of the National Academy of Sciences of the United States of America 112:13348-13353.
- Handschick K, Beuerlein K, Jurida L, Bartkuhn M, Muller H, Soelch J, Weber A, Dittrich-Breiholz O, Schneider H, Scharfe M, Jarek M, Stellzig J, Schmitz ML, Kracht M. 2014. Cyclin-dependent kinase 6 is a chromatin-bound cofactor for NF-kappaB-dependent gene expression. Mol Cell 53:193-208.
- Mahmoudi M, Abdelmonem AM, Behzadi S, Clement JH, Dutz S, Ejtehadi MR, Hartmann R, Kantner K, Linne U, Maffre P, Metzler S, Moghadam MK, Pfeiffer C, Rezaei M, Ruiz-Lozano P, Serpooshan V, Shokrgozar MA, Nienhaus GU, Parak WJ. 2013. Temperature: the "ignored" factor at the NanoBio interface. ACS nano 7:6555-6562.
- Ziesche E, Kettner-Buhrow D, Weber A, Wittwer T, Jurida L, Soelch J, Muller H, Newel D, Kronich P, Schneider H, Dittrich-Breiholz O, Bhaskara S, Hiebert SW, Hottiger MO, Li H, Burstein E, Schmitz ML, Kracht M. 2013. The coactivator role of histone deacetylase 3 in IL-1-signaling involves deacetylation of p65 NF-kappaB. Nucleic Acids Res 41:90-109.
- Imhof M, Rhinow D, Linne U, Hampp N. 2012. Two-Photon-Induced Selective Decarboxylation of Aspartic Acids D85 and D212 in Bacteriorhodopsin. The journal of physical chemistry letters 3:2991-2994.
- Buss H, Handschick K, Jurrmann N, Pekkonen P, Beuerlein K, Muller H, Wait R, Saklatvala J, Ojala PM, Schmitz ML, Naumann M, Kracht M. 2012. Cyclin-dependent kinase 6 phosphorylates NF-kappaB P65 at serine 536 and contributes to the regulation of inflammatory gene expression. PLoS One 7:e51847.
- Zirah S, Afonso C, Linne U, Knappe TA, Marahiel MA, Rebuffat S, Tabet JC. 2011. Topoisomer differentiation of molecular knots by FTICR MS: lessons from class II lasso peptides. Journal of the American Society for Mass Spectrometry 22:467-479.
- Weber A, Wasiliew P, Kracht M. 2010. Interleukin-1 (IL-1) pathway. Sci Signal 3:cm1.