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:
- Shrestha A, Carraro G, Nottet N, Vazquez-Armendariz AI, Herold S, Cordero J, Singh I, Wilhelm J, Barreto G, Morty R, El Agha E, Mari B, Chen C, Zhang JS, Chao CM, Bellusci S. A critical role for miR-142 in alveolar epithelial lineage formation in mouse lung development. Cell Mol Life Sci. 2019;76(14):2817-2832.
- Hu P, Wilhelm J, Gerresheim GK, Shalamova LA, Niepmann M. Lnc-ITM2C-1 and GPR55 are proviral host factors for hepatitis C virus. Viruses. 2019;11(6). pii: E549.
- Poppe M, Wittig S, Jurida L, Bartkuhn M, Wilhelm J, Müller H, Beuerlein K, Karl N, Bhuju S, Ziebuhr J, Schmitz ML, Kracht M. The NF-κB-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229E-infected cells. PLoS Pathog. 2017;13(3):e1006286.
- Rodríguez-Gil A, Ritter O, Saul VV, Wilhelm J, Yang CY, Grosschedl R, Imai Y, Kuba K, Kracht M, Schmitz ML. The CCR4-NOT complex contributes to repression of major histocompatibility complex class II transcription. Sci Rep. 2017;7(1):3547.
- Ehmann R, Kristen-Burmann C, Bank-Wolf B, König M, Herden C, Hain T, Thiel HJ, Ziebuhr J, Tekes G. Reverse Genetics for Type I Feline coronavirus field isolate to study the molecular pathogenesis of feline infectious peritonitis. mBio. 2018;9(4). pii: e01422-18.
- Whitmer SLM, Strecker T, Cadar D, Dienes HP, Faber K, Patel K, Brown SM, Davis WG, Klena JD, Rollin PE, Schmidt-Chanasit J, Fichet-Calvet E, Noack B, Emmerich P, Rieger T, Wolff S, Fehling SK, Eickmann M, Mengel JP, Schultze T, Hain T, Ampofo W, Bonney K, Aryeequaye JND, Ribner B, Varkey JB, Mehta AK, Lyon GM 3rd, Kann G, De Leuw P, Schuettfort G, Stephan C, Wieland U, Fries JWU, Kochanek M, Kraft CS, Wolf T, Nichol ST, Becker S, Ströher U, Günther S. New Lineage of Lassa Virus, Togo, 2016. Emerg Infect Dis. 2018;24(3):599-602.
- ReferenceSeeker: rapid determination of appropriate reference genomes. Schwengers O, Hain T, Chakraborty T, Goesmann A. Journal of Open Source Software. 2020;5(46), 1994.
- ASA³P: an automatic and scalable pipeline for the assembly, annotation and higher level analysis of closely related bacterial isolates. Schwengers O, Hoek A, Fritzenwanker M, Falgenhauer L, Hain T, Chakraborty T, Goesmann A, PLoS Computational Biology, 2020; in press.
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:
- Weber A, Dam S, Saul VV, Kuznetsova I, Muller C, Fritz-Wolf K, Becker K, Linne U, Gu H, Stokes MP, Pleschka S, Kracht M, Schmitz ML. 2019. Phosphoproteome Analysis of Cells Infected with Adapted and Nonadapted Influenza A Virus Reveals Novel Pro- and Antiviral Signaling Networks. J Virol 93(13). pii: e00528-19. doi: 10.1128
- Weiterer SS, Meier-Soelch J, Georgomanolis T, Mizi A, Beyerlein A, Weiser H, Brant L, Mayr-Buro C, Jurida L, Beuerlein K, Muller H, Weber A, Tenekeci U, Dittrich-Breiholz O, Bartkuhn M, Nist A, Stiewe T, van IWF, Riedlinger T, Schmitz ML, Papantonis A, Kracht M. 2019. Distinct IL-1alpha-responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner. EMBO J 39(1):e101533. doi: 10.15252.
- Meier-Soelch J, Jurida L, Weber A, Newel D, Kim J, Braun T, Schmitz ML, Kracht M. 2018. RNAi-Based Identification of Gene-Specific Nuclear Cofactor Networks Regulating Interleukin-1 Target Genes. Frontiers in Immunology 9:775. doi: 10.3389.
- Schmitz ML, Shaban MS, Albert BV, Gokcen A, Kracht M. 2018. The Crosstalk of Endoplasmic Reticulum (ER) Stress Pathways with NF-kappaB: Complex Mechanisms Relevant for Cancer, Inflammation and Infection. Biomedicines 6(2). pii: E58. doi: 10.3390.
- Poppe M, Wittig S, Jurida L, Bartkuhn M, Wilhelm J, Muller H, Beuerlein K, Karl N, Bhuju S, Ziebuhr J, Schmitz ML, Kracht M. 2017. The NF-kappaB-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229E-infected cells. PLoS Pathog 13(3):e1006286. doi: 10.1371.
- Brühl J, Trautwein J, Schäfer A, Linne U, Bouazoune K. 2019 The DNA repair protein SHPRH is a nucleosome-stimulated ATPase and a nucleosome-E3 ubiquitin ligase. Epigenetics Chromatin. 12(1):52. doi:10.1186.
- Robledo M, Schlüter JP, Loehr LO, Linne U, Albaum SP, Jiménez-Zurdo JI, Becker A 2018. An sRNA and Cold Shock Protein Homolog-Based Feedforward Loop Post-transcriptionally Controls Cell Cycle Master Regulator CtrA. Front Microbiol. 9:763. doi:10.3389.
- Nickel AI, Wäber NB, Gößringer M, Lechner M, Linne U, Toth U, Rossmanith W, Hartmann RK 2017. Minimal and RNA-free RNase P in Aquifex aeolicus. Proc Natl Acad Sci U S A. 114(42):11121–11126. doi:10.1073.
- Hajipour MJ, Ghasemi F, Aghaverdi H, Raoufi M, Linne U, Atyabi F, Nabipour I, Azhdarzadeh M, Derakhshankhah H, Lotfabadi A, Bargahi A, Alekhamis Z, Aghaie A, Hashemi E, Tafakhori A, Aghamollaii V, Mashhadi MM, Sheibani S, Vani H, Mahmoudi M 2017. Sensing of Alzheimer's Disease and Multiple Sclerosis Using Nano-Bio Interfaces. J Alzheimers Dis. 59(4):1187–1202. doi:10.3233.
- Bertram K, Valcu CM, Weitnauer M, Linne U, Görlach A. 2015. NOX1 supports the metabolic remodeling of HepG2 cells. PLoS One. 10(3):e0122002. doi:10.1371.