Intracellular organization of Ebola virus RNA synthesis; formation, maturation and transport of viral RNP

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Research area: Molecular Virology

Replication of viral genomic RNA results in the formation of progeny genomes that need to be packaged and transported to the sites of viral budding. Replication of negative-strand Ebola virus genomes takes place in inclusion bodies located close to the nucleus. During synthesis, genomes are encapsidated to form ribonucleoprotein (RNP) complexes which mature to transport-competent nucleocapsids (NCs) inside the inclusions. Although the virus-induced inclusions play an important role in the synthesis of filovirus RNA, their organization is not well understood. In the proposed project we will investigate the spatial and temporal organization of filoviral RNA synthesis, its packaging into RNPs, the molecular basis for RNP maturation to become transport-competent NCs, and which cellular and viral factors are involved in the recruitment of the actin-polymerizing machinery that drives the transport of NCs. Results gained with surrogate systems under BSL-2 conditions will be validated using recombinant filoviruses and CRISPR/Cas9 knock out cell lines under BSL-4 conditions. We will use proteomics to identify cellular proteins associated with encapsidated genomes, quantitative live cell imaging to understand the transport dynamics, super resolution microscopy (dSTORM), and correlative light and electron microscopy (CLEM) to visualize actin and actin-binding proteins at the NCs.

Project-related publications of the investigator:

  • Takamatsu Y, Kolesnikova L, Schauflinger M, Noda T, Becker S. 2020. The Integrity of the YxxL Motif of Ebola Virus VP24 Is Important for the Transport of Nucleocapsid-Like Structures and for the Regulation of Viral RNA Synthesis. J Virol 94.
  • Grikscheit K, Dolnik O, Takamatsu Y, Pereira AR, Becker S. 2020. Ebola Virus Nucleocapsid-Like Structures Utilize Arp2/3 Signaling for Intracellular Long-Distance Transport. Cells 9.
  • Takamatsu Y, Dolnik O, Noda T, Becker S. 2019. A live-cell imaging system for visualizing the transport of Marburg virus nucleocapsid-like structures. Virol J 16:159.
  • Takamatsu Y, Kolesnikova L, Becker S. 2018. Ebola virus proteins NP, VP35, and VP24 are essential and sufficient to mediate nucleocapsid transport. Proc Natl Acad Sci U S A 115:1075-1080.
  • Mittler E, Schudt G, Halwe S, Rohde C, Becker S. 2018. A Fluorescently Labeled Marburg Virus Glycoprotein as a New Tool to Study Viral Transport and Assembly. J Infect Dis 218:S318-S326.
  • Wan W, Kolesnikova L, Clarke M, Koehler A, Noda T, Becker S, Briggs JAG. 2017. Structure and assembly of the Ebola virus nucleocapsid. Nature 551:394-397.
  • Schudt G, Dolnik O, Kolesnikova L, Biedenkopf N, Herwig A, Becker S. 2015. Transport of Ebolavirus Nucleocapsids Is Dependent on Actin Polymerization: Live-Cell Imaging Analysis of Ebolavirus-Infected Cells. J Infect Dis 212 Suppl 2:S160-6.
  • Dolnik O, Kolesnikova L, Welsch S, Strecker T, Schudt G, Becker S. 2014. Interaction with Tsg101 is necessary for the efficient transport and release of nucleocapsids in marburg virus-infected cells. PLoS Pathog 10:e1004463.
  • Schudt G, Kolesnikova L, Dolnik O, Sodeik B, Becker S. 2013. Live-cell imaging of Marburg virus-infected cells uncovers actin-dependent transport of nucleocapsids over long distances. Proc Natl Acad Sci U S A 110:14402-7.
  • Bharat TA, Noda T, Riches JD, Kraehling V, Kolesnikova L, Becker S, Kawaoka Y, Briggs JA. 2012. Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography. Proc Natl Acad Sci U S A 109:4275-80.

Function of lipid droplets in replication and pathogenesis of neurotropic flaviviruses

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Research area: Virus-host Interaction

Viruses rely on host lipid metabolic pathways for replication. Lipid droplets are the main cellular storage organelles of neutral lipids and central hubs of lipid trafficking. The Herker group investigates the function of lipid droplets in virus infection and pathogenesis and how viruses rewire cellular metabolic pathways for successful replication. Capsid proteins from hepatitis C virus (HCV) and different flavivirus family members localize to lipid droplets and, depending on cell types and viruses, lipid droplet accumulation and degradation have been observed in infected cells. However, it is currently unknown, how neurotropic flaviviruses interact with the intricate lipid metabolism of brain cells. This project aims to i) characterize host cell lipid droplets after expression of the capsid protein C or during West Nile virus (WNV), Zika virus (ZIKV), and tick-borne encephalitis virus (TBEV) infection in different cell types, such as neuron and astrocyte cell lines, human neurons and astrocytes differentiated from stem cells, and murine primary cells, ii) determine if WNV, ZIKV, and TBEV infection is inhibited by interference with lipid droplet formation and/or lipid mobilization, and iii) elucidate if virus-induced pathogenic effects are in part due to metabolic disturbance.

Project-related publications of the investigator:

  • Herker E, Vieyres G, Beller M, Krahmer N, Bohnert M. 2021. Lipid Droplet Contact Sites in Health and Disease. Trends Cell Biol 31:345-358.
  • Bley H, Schobel A, Herker E. 2020. Whole Lotta Lipids-from HCV RNA Replication to the Mature Viral Particle. Int J Mol Sci 21.
  • Lassen S, Gruttner C, Nguyen-Dinh V, Herker E. 2019. Perilipin-2 is critical for efficient lipoprotein and hepatitis C virus particle production. J Cell Sci 132.
  • Schobel A, Rosch K, Herker E. 2018. Functional innate immunity restricts Hepatitis C Virus infection in induced pluripotent stem cell-derived hepatocytes. Sci Rep 8:3893.
  • Hofmann S, Krajewski M, Scherer C, Scholz V, Mordhorst V, Truschow P, Schobel A, Reimer R, Schwudke D, Herker E. 2018. Complex lipid metabolic remodeling is required for efficient hepatitis C virus replication. Biochim Biophys Acta Mol Cell Biol Lipids 1863:1041-1056.
  • Rosch K, Kwiatkowski M, Hofmann S, Schobel A, Gruttner C, Wurlitzer M, Schluter H, Herker E. 2016. Quantitative Lipid Droplet Proteome Analysis Identifies Annexin A3 as a Cofactor for HCV Particle Production. Cell Rep 16:3219-3231.
  • Eggert D, Rosch K, Reimer R, Herker E. 2014. Visualization and analysis of hepatitis C virus structural proteins at lipid droplets by super-resolution microscopy. PLoS One 9:e102511.
  • Camus G, Schweiger M, Herker E, Harris C, Kondratowicz AS, Tsou CL, Farese RV, Jr., Herath K, Previs SF, Roddy TP, Pinto S, Zechner R, Ott M. 2014. The hepatitis C virus core protein inhibits adipose triglyceride lipase (ATGL)-mediated lipid mobilization and enhances the ATGL interaction with comparative gene identification 58 (CGI-58) and lipid droplets. J Biol Chem 289:35770-80.
  • Vogt DA, Camus G, Herker E, Webster BR, Tsou CL, Greene WC, Yen TS, Ott M. 2013. Lipid droplet-binding protein TIP47 regulates hepatitis C Virus RNA replication through interaction with the viral NS5A protein. PLoS Pathog 9:e1003302.
  • Camus G, Herker E, Modi AA, Haas JT, Ramage HR, Farese RV, Jr., Ott M. 2013. Diacylglycerol acyltransferase-1 localizes hepatitis C virus NS5A protein to lipid droplets and enhances NS5A interaction with the viral capsid core. J Biol Chem 288:9915-9923.
  • Herker E, Ott M. 2012. Emerging role of lipid droplets in host/pathogen interactions. J Biol Chem 287:2280-7.
  • Herker E, Ott M. 2011. Unique ties between hepatitis C virus replication and intracellular lipids. Trends Endocrinol Metab 22:241-8.
  • Harris C, Herker E, Farese RV, Jr., Ott M. 2011. Hepatitis C virus core protein decreases lipid droplet turnover: a mechanism for core-induced steatosis. J Biol Chem 286:42615-42625.
  • Herker E, Harris C, Hernandez C, Carpentier A, Kaehlcke K, Rosenberg AR, Farese RV, Jr., Ott M. 2010. Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1. Nat Med 16:1295-8.

Mechanistic insights into morbillivirus-induced immunosuppression and antiviral responses

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Resarch areas: Molecular Virology, Innate Immunity, Viral Pathogenesis

Morbillivirus infections are a continuing threat to human and animal health. Measles virus still causes more than 140,000 fatalities annually. Canine distemper virus, the morbillivirus infecting carnivores like ferrets, is a potent surrogate model to study morbillivirus pathogenesis. Morbilliviruses cause immunosuppression, making infected individuals vulnerable to opportunistic secondary infections. They can cause loss of pre-acquired immunity, a phenomenon described as immune amnesia. Paradoxically, morbillivirus infections induce strong virus-specific humoral responses providing long-lasting immunity. Morbilliviruses infect and replicate in a subset of immune cells, including dendritic cells, macrophages, activated T cells, and naïve and activated B cells. We believe that immune amnesia is the result of depletion or functional reprogramming of infected cells, but detailed mechanistic insights remain elusive. Using the canine distemper virus/ferret-model, we are interested in investigating the interactions of morbilliviruses with immune cells to clarify how the virus changes immune organ environments and immune cell functions that consequently cause immune amnesia. We are also studying the duration and extent of immune amnesia induced by viruses exhibiting different degrees of viremia and attenuation. We try to answer important questions regarding morbillivirus biology: Which antiviral programs cause attenuation of morbilliviruses? How do they affect virus-induced immunosuppression and immune amnesia? By making use of mutant viruses expressing immunostimulatory defective-interfering RNAs we can assess their contributions to attenuation in ferrets and evaluate whether infection with immune-activating viruses can lead to productive antiviral responses that are able to protect from infection with lethal pathogenic virus. The identified mechanisms will be useful to develop novel strategies to alleviate morbillivirus-induced immunosuppression. Our findings will also allow rational design of novel, improved morbillivirus vaccines.

Project-related publications of the investigators:

  • Pfaller CK, Bloyet LM, Donohue RC, Huff AL, Bartemes WP, Yousaf I, Urzua E, Claviere M, Zachary M, de Masson d’Autume V, Carson S, Schieferecke AJ, Meyer AJ, Gerlier D, Cattaneo R. 2020. The C Protein Is Recruited to Measles Virus Ribonucleocapsids by the Phosphoprotein. J Virol 94.
  • Ayasoufi K, Pfaller CK. 2020. Seek and hide: the manipulating interplay of measles virus with the innate immune system. Curr Opin Virol 41:18-30.
  • Petrova VN, Sawatsky B, Han AX, Laksono BM, Walz L, Parker E, Pieper K, Anderson CA, de Vries RD, Lanzavecchia A, Kellam P, von Messling V, de Swart RL, Russell CA. 2019. Incomplete genetic reconstitution of B cell pools contributes to prolonged immunosuppression after measles. Sci Immunol 4.
  • Donohue RC, Pfaller CK, Cattaneo R. 2019. Cyclical adaptation of measles virus quasispecies to epithelial and lymphocytic cells: To V, or not to V. PLoS Pathog 15:e1007605.
  • Thakkar VD, Cox RM, Sawatsky B, da Fontoura Budaszewski R, Sourimant J, Wabbel K, Makhsous N, Greninger AL, von Messling V, Plemper RK. 2018. The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity. J Virol 92.
  • Sawatsky B, Cattaneo R, von Messling V. 2018. Canine Distemper Virus Spread and Transmission to Naive Ferrets: Selective Pressure on Signaling Lymphocyte Activation Molecule-Dependent Entry. J Virol 92.
  • Pfaller CK, Donohue RC, Nersisyan S, Brodsky L, Cattaneo R. 2018. Extensive editing of cellular and viral double-stranded RNA structures accounts for innate immunity suppression and the proviral activity of ADAR1p150. PLoS Biol 16:e2006577.
  • Pfaller CK, Mastorakos GM, Matchett WE, Ma X, Samuel CE, Cattaneo R. 2015. Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations. J Virol 89:7735-47.
  • Pfaller CK, Radeke MJ, Cattaneo R, Samuel CE. 2014. Measles virus C protein impairs production of defective copyback double-stranded viral RNA and activation of protein kinase R. J Virol 88:456-68.
  • Sawatsky B, Wong XX, Hinkelmann S, Cattaneo R, von Messling V. 2012. Canine distemper virus epithelial cell infection is required for clinical disease but not for immunosuppression. J Virol 86:3658-66.

Molecular basis for the virulence of deformed wing virus infections in honey bees

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Research area: Molecular Virology

The Deformed wing virus (DWV; order Picornavirales) is a major pathogen of honey bees (Apis mellifera) and responsible for colony collapses. It is hypothesized that virulent emerging DWV strains have evolved from less virulent precursors. In this funding period, we want to decipher the molecular determinants of DWV virulence using combined molecular biological and protein biochemical approaches. Molecular markers of DWV virulence will be elucidated by studying (I) inter-strain recombination, (II) in vivo passaging and adaptation of DWV-B, and (III) the role of the viral leader protein as potential RNAi escape factor. An avirulent DWV-B will be used as a platform to localize the genomic regions responsible for virulence. (I) The series of genetically engineered DWV recombinants will be systematically studied for replication, viral titers, host cell range and virulence in honey bee pupae and primary bee cells. (II) We will passage a DWV-B clone in vivo to study virulence emergence. Responsible mutations will be characterized using reverse genetics. (III) We hypothesize that the leader protein of DWV is an antagonist of the innate immune defense of honey bees and hence a central virulence factor. We will characterize the leader protein of DWV using reporter assays, recombinant cell culture expression, and pull-down experiments to identify the interacting host cell factors. Hopefully, our results will contribute to the establishment of new concepts against the devastating colony losses in beekeeping.

Project-related publications of the investigator:

  • Seitz K, Buczolich K, Dikunova A, Plevka P, Power K, Rumenapf T, Lamp B. 2019. A molecular clone of Chronic Bee Paralysis Virus (CBPV) causes mortality in honey bee pupae (Apis mellifera). Sci Rep 9:16274.
  • Riedel C, Lamp B, Chen HW, Heimann M, Rumenapf T. 2019. Fluorophore labelled BVDV: a novel tool for the analysis of infection dynamics. Sci Rep 9:5972.
  • Kiesler A, Seitz K, Schwarz L, Buczolich K, Petznek H, Sassu E, Durlinger S, Hogler S, Klang A, Riedel C, Chen HW, Motz M, Kirkland P, Weissenbock H, Ladinig A, Rumenapf T, Lamp B. 2019. Clinical and Serological Evaluation of LINDA Virus Infections in Post-Weaning Piglets. Viruses 11.
  • Schwarz L, Riedel C, Hogler S, Sinn LJ, Voglmayr T, Wochtl B, Dinhopl N, Rebel-Bauder B, Weissenbock H, Ladinig A, Rumenapf T, Lamp B. 2017. Congenital infection with atypical porcine pestivirus (APPV) is associated with disease and viral persistence. Vet Res 48:1.
  • Lamp B, Schwarz L, Hogler S, Riedel C, Sinn L, Rebel-Bauder B, Weissenbock H, Ladinig A, Rumenapf T. 2017. Novel Pestivirus Species in Pigs, Austria, 2015. Emerg Infect Dis 23:1176-1179.
  • Schurischuster S., S. Zambanini, M. Kampel, and B. Lamp*. 2016. Sensor Study for Monitoring Varroa Mites on Honey Bees (Apis mellifera). Proceedings of the Visual observation and analysis of Vertebrate And Insect Behavior, VAIB 5:11, http://homepages.inf.ed.ac.uk/rbf/VAIB16PAPERS/vaibchurischuster.pdf
  • Lamp B, Url A, Seitz K, Eichhorn J, Riedel C, Sinn LJ, Indik S, Koglberger H, Rumenapf T. 2016. Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV). PLoS One 11:e0164639.
  • Lamp B, Riedel C, Wentz E, Tortorici MA, Rumenapf T. 2013. Autocatalytic cleavage within classical swine fever virus NS3 leads to a functional separation of protease and helicase. J Virol 87:11872-83.
  • Riedel C, Lamp B, Heimann M, Konig M, Blome S, Moennig V, Schuttler C, Thiel HJ, Rumenapf T. 2012. The core protein of classical Swine Fever virus is dispensable for virus propagation in vitro. PLoS Pathog 8:e1002598.
  • Lamp B, Riedel C, Roman-Sosa G, Heimann M, Jacobi S, Becher P, Thiel HJ, Rumenapf T. 2011. Biosynthesis of classical swine fever virus nonstructural proteins. J Virol 85:3607-20.

Mechanisms and regulation of Ebola virus RNA synthesis

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Research areas: Molecular Virology, RNA Biochemistry

Ebola virus (EBOV) RNA synthesis is a highly regulated process that involves the interplay of cis-acting elements in the viral genome with viral RNA binding proteins. While replication of the EBOV genome is driven by the viral polymerase L, its cofactor VP35 and the nucleoprotein NP, viral transcription additionally requires VP30, an EBOV-specific transcription factor whose activity is regulated via phosphorylation. In the second funding period, we were able to unveil the mechanism of VP30’s dynamic phosphorylation, identifying the serine/arginine-rich protein kinase 1 (SRPK1), and demonstrating that NP acts as recruitment factor for subunit B56 of the protein phosphatase 2A to dephosphorylate VP30 if simultaneously bound to NP. Based on the study of mutant minigenomes (MGs), we further demonstrated that hexamer phasing in the 3’-leader promoter is not only key to replication, but also to efficient transcription initiation. The genomic EBOV replication promotor is bipartite, consisting of promoter elements 1 (PE1) and 2 (PE2), that are separated by the transcription start sequence/site (TSS) for the first gene (NP) and a spacer sequence. We found that spacer extensions of up to ~54 nt are tolerated, while minor incremental stabilization of hairpin (HP) structures at the TSS rapidly abolished viral polymerase activity. Balanced viral transcription and replication can still occur when any potential RNA structure formation at the TSS is eliminated, and transcription remains VP30-dependent also in this case. In addition, the HP structure at the TSS of the native 3’-leader was demonstrated to be optimized for tight regulation by VP30 and to enable the switch from transcription to replication when VP30 is not part of the polymerase complex. Increasing stability of the HP impaired viral transcription. Short leader RNAs of 60-80 nt length are synthesized from the EBOV genome. Like replicative antigenomic RNA, leader RNAs are initiated opposite to the penultimate C residue at the genomic 3’-end and their amount is reduced in the presence of VP30, suggesting that leader RNAs are products of abortive antigenome synthesis.

For the next funding period, we would like to intensify our efforts regarding the interplay between cis-acting regulatory elements in the EBOV genome and viral proteins that contribute to RNA synthesis. We will address the following work packages: (i) Advanced investigations on the mechanistic role of structural elements and hexamer phasing in the 3’-leader promotor and at internal TSS by mutational analysis in the context of mono-, bi-, or tetracistronic MGs. Selected mutations will be introduced into the EBOV genome and recombinant viruses will be generated and characterized. (ii) Characterization of constraints for leader RNA synthesis by engineering the 3’-leader in terms of length, sequence and structure. (iii) To investigate the accessibility of the encapsidated RNA genome by the polymerase complex, we want to analyze protein-RNA interactions by iCLIP or PAR-CLIP, either using reconstituted complexes of recombinantly expressed polymerase complex and leader/trailer RNAs or nucleocapsids derived from tetracistronic MGs. (iv) We aim at establishing an in vitro transcription/replication system based on the expression of the viral polymerase complex in insect cells. Moreover, we will investigate (v) the functional role of VP35 helicase activity and (vi) will search for host factors interacting with the viral polymerase complex.

The herein proposed work packages will contribute to a deeper understanding of regulatory mechanisms involved in EBOV RNA synthesis.

Project-related publications of the investigators:

  • Bach S, Demper JC, Biedenkopf N, Becker S, Hartmann RK. 2021. RNA secondary structure at the transcription start site influences EBOV transcription initiation and replication in a length- and stability-dependent manner. RNA Biol 18:523-536.
  • Takamatsu Y, Krahling V, Kolesnikova L, Halwe S, Lier C, Baumeister S, Noda T, Biedenkopf N, Becker S. 2020. Serine-Arginine Protein Kinase 1 Regulates Ebola Virus Transcription. mBio 11.
  • Bach S, Demper JC, Grunweller A, Becker S, Biedenkopf N, Hartmann RK. 2020. Regulation of VP30-Dependent Transcription by RNA Sequence and Structure in the Genomic Ebola Virus Promoter. J Virol doi:10.1128/JVI.02215-20.
  • Bach S, Biedenkopf N, Grunweller A, Becker S, Hartmann RK. 2020. Hexamer phasing governs transcription initiation in the 3′-leader of Ebola virus. RNA 26:439-453.
  • Kruse T, Biedenkopf N, Hertz EPT, Dietzel E, Stalmann G, Lopez-Mendez B, Davey NE, Nilsson J, Becker S. 2018. The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30. Mol Cell 69:136-145 e6.
  • Lier C, Becker S, Biedenkopf N. 2017. Dynamic phosphorylation of Ebola virus VP30 in NP-induced inclusion bodies. Virology 512:39-47.
  • Biedenkopf N, Lange-Grunweller K, Schulte FW, Weisser A, Muller C, Becker D, Becker S, Hartmann RK, Grunweller A. 2017. The natural compound silvestrol is a potent inhibitor of Ebola virus replication. Antiviral Res 137:76-81.
  • Biedenkopf N, Hoenen T. 2017. Modeling the Ebolavirus Life Cycle with Transcription and Replication-Competent Viruslike Particle Assays. Methods Mol Biol 1628:119-131.
  • Schlereth J, Grunweller A, Biedenkopf N, Becker S, Hartmann RK. 2016. RNA binding specificity of Ebola virus transcription factor VP30. RNA Biol 13:783-98.
  • Biedenkopf N, Schlereth J, Grunweller A, Becker S, Hartmann RK. 2016. RNA Binding of Ebola Virus VP30 Is Essential for Activating Viral Transcription. J Virol 90:7481-7496.

Central resource facility for (quantitative) proteomics applications of RNA-virus infected cells

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Research areas: 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:

  • 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. 2020. Distinct IL-1alpha-responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner. EMBO J 39:e101533.
  • Aznaourova M, Janga H, Sefried S, Kaufmann A, Dorna J, Volkers SM, Georg P, Lechner M, Hoppe J, Dokel S, Schmerer N, Gruber AD, Linne U, Bauer S, Sander LE, Schmeck B, Schulte LN. 2020. Noncoding RNA MaIL1 is an integral component of the TLR4-TRIF pathway. Proc Natl Acad Sci U S A 117:9042-9053.
  • 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.
  • Franz-Badur S, Penner A, Strass S, von Horsten S, Linne U, Essen LO. 2019. Structural changes within the bifunctional cryptochrome/photolyase CraCRY upon blue light excitation. Sci Rep 9:9896.
  • Bruhl J, Trautwein J, Schafer A, Linne U, Bouazoune K. 2019. The DNA repair protein SHPRH is a nucleosome-stimulated ATPase and a nucleosome-E3 ubiquitin ligase. Epigenetics Chromatin 12:52.
  • Robledo M, Schluter JP, Loehr LO, Linne U, Albaum SP, Jimenez-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.
  • 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:e1006286.
  • Tenekeci U, Poppe M, Beuerlein K, Buro C, Muller H, Weiser H, Kettner-Buhrow D, Porada K, Newel D, Xu M, Chen ZJ, Busch J, Schmitz ML, Kracht M. 2016. K63-Ubiquitylation and TRAF6 Pathways Regulate Mammalian P-Body Formation and mRNA Decapping. Mol Cell 62:943-957.

Central resource facility for virus genomics and host transcriptomics

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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:

  • Vazquez-Armendariz AI, Heiner M, El Agha E, Salwig I, Hoek A, Hessler MC, Shalashova I, Shrestha A, Carraro G, Mengel JP, Gunther A, Morty RE, Vadasz I, Schwemmle M, Kummer W, Hain T, Goesmann A, Bellusci S, Seeger W, Braun T, Herold S. 2020. Multilineage murine stem cells generate complex organoids to model distal lung development and disease. EMBO J 39:e103476.
  • Shaban MS, Müller C, Mayr-Buro C, Weiser H, Albert B, Weber A, Linne U, Hain T, Babayev I, Karl N, Hofmann N, Becker S, Herold S, Lienhard Schmitz M, Ziebuhr J, Kracht M. 2020. Inhibiting coronavirus replication in cultured cells by chemical ER stress. bioRxiv doi:10.1101/ 2020.08.26.266304.
  • Schwengers O, Hoek A, Fritzenwanker M, Falgenhauer L, Hain T, Chakraborty T, Goesmann A. 2020. ASA3P: An automatic and scalable pipeline for the assembly, annotation and higher-level analysis of closely related bacterial isolates. PLoS Comput Biol 16:e1007134.
  • Schwengers O, Hain T, Chakraborty T, Goesmann A. 2020. ReferenceSeeker: rapid determination of appropriate reference genomes. Journal of Open Source Software 5:1994.
  • 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. 2019. A critical role for miR-142 in alveolar epithelial lineage formation in mouse lung development. Cell Mol Life Sci 76:2817-2832.
  • Hu P, Wilhelm J, Gerresheim GK, Shalamova LA, Niepmann M. 2019. Lnc-ITM2C-1 and GPR55 Are Proviral Host Factors for Hepatitis C Virus. Viruses 11.
  • 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, Stroher U, Gunther S. 2018. New Lineage of Lassa Virus, Togo, 2016. Emerg Infect Dis 24:599-602.
  • Ehmann R, Kristen-Burmann C, Bank-Wolf B, Konig M, Herden C, Hain T, Thiel HJ, Ziebuhr J, Tekes G. 2018. Reverse Genetics for Type I Feline Coronavirus Field Isolate To Study the Molecular Pathogenesis of Feline Infectious Peritonitis. mBio 9.
  • Rodriguez-Gil A, Ritter O, Saul VV, Wilhelm J, Yang CY, Grosschedl R, Imai Y, Kuba K, Kracht M, Schmitz ML. 2017. The CCR4-NOT complex contributes to repression of Major Histocompatibility Complex class II transcription. Sci Rep 7:3547.
  • 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:e1006286.

Impact of virus and host factors on lung epithelial cell injury and repair in severe influenza virus pneumonia

/in /von

Research areas: Pneumology, Infectious Diseases, Cell Biology, Molecular Virology

Viral pneumonia is characterized by extensive lung injury, usually followed by stem cell-mediated epithelial repair. We recently defined a pool of bronchoalveolar stem cells (BASC) that repair both bronchial and alveolar tissue after IAV-induced injury. Using transgenic in vivo mouse and bronchoalveolar lung organoid (BALO) infection models, as well as first-in-man approaches such as inhalative granulocyte/macrophage colony-stimulating factor (GM-CSF) in patients with severe virus-induced lung injury, we revealed that BASC´s and alveolar epithelial cells type II´s regenerative responses depend on cooperation with district mesenchymal cells of the stem cell niche and on GM‑CSF release upon IAV infection.

Therefore, our main aims are to identify the host signaling pathways at the virus-host interface that drive alveolar epithelial cell injury and foster repair from distinct lung stem cell niches, and to elucidate how viral pathogenicity factors impact on these mechanisms, resulting in aggravated damage of the distal lung epithelium and in impaired or aberrant repair after virus-induced pneumonia.

Project-related publications of the investigator:

  • Vazquez-Armendariz AI, Herold S. 2021. From Clones to Buds and Branches: The Use of Lung Organoids to Model Branching Morphogenesis Ex Vivo. Front Cell Dev Biol 9:631579.
  • Vazquez-Armendariz AI, Heiner M, El Agha E, Salwig I, Hoek A, Hessler MC, Shalashova I, Shrestha A, Carraro G, Mengel JP, Gunther A, Morty RE, Vadasz I, Schwemmle M, Kummer W, Hain T, Goesmann A, Bellusci S, Seeger W, Braun T, Herold S. 2020. Multilineage murine stem cells generate complex organoids to model distal lung development and disease. EMBO J 39:e103476.
  • Schmoldt C, Vazquez-Armendariz AI, Shalashova I, Selvakumar B, Bremer CM, Peteranderl C, Wasnick R, Witte B, Gattenlohner S, Fink L, Vadasz I, Morty RE, Pleschka S, Seeger W, Gunther A, Herold S. 2019. IRE1 Signaling As a Putative Therapeutic Target in Influenza Virus-induced Pneumonia. Am J Respir Cell Mol Biol 61:537-540.
  • Salwig I, Spitznagel B, Vazquez-Armendariz AI, Khalooghi K, Guenther S, Herold S, Szibor M, Braun T. 2019. Bronchioalveolar stem cells are a main source for regeneration of distal lung epithelia in vivo. EMBO J 38.
  • El Agha E, Moiseenko A, Kheirollahi V, De Langhe S, Crnkovic S, Kwapiszewska G, Szibor M, Kosanovic D, Schwind F, Schermuly RT, Henneke I, MacKenzie B, Quantius J, Herold S, Ntokou A, Ahlbrecht K, Braun T, Morty RE, Gunther A, Seeger W, Bellusci S. 2017. Two-Way Conversion between Lipogenic and Myogenic Fibroblastic Phenotypes Marks the Progression and Resolution of Lung Fibrosis. Cell Stem Cell 20:261-273 e3.
  • Quantius J, Schmoldt C, Vazquez-Armendariz AI, Becker C, El Agha E, Wilhelm J, Morty RE, Vadasz I, Mayer K, Gattenloehner S, Fink L, Matrosovich M, Li X, Seeger W, Lohmeyer J, Bellusci S, Herold S. 2016. Influenza Virus Infects Epithelial Stem/Progenitor Cells of the Distal Lung: Impact on Fgfr2b-Driven Epithelial Repair. PLoS Pathog 12:e1005544.
  • Peteranderl C, Morales-Nebreda L, Selvakumar B, Lecuona E, Vadasz I, Morty RE, Schmoldt C, Bespalowa J, Wolff T, Pleschka S, Mayer K, Gattenloehner S, Fink L, Lohmeyer J, Seeger W, Sznajder JI, Mutlu GM, Budinger GR, Herold S. 2016. Macrophage-epithelial paracrine crosstalk inhibits lung edema clearance during influenza infection. J Clin Invest 126:1566-80.
  • Herold S, Hoegner K, Vadasz I, Gessler T, Wilhelm J, Mayer K, Morty RE, Walmrath HD, Seeger W, Lohmeyer J. 2014. Inhaled granulocyte/macrophage colony-stimulating factor as treatment of pneumonia-associated acute respiratory distress syndrome. Am J Respir Crit Care Med 189:609-11.
  • Unkel B, Hoegner K, Clausen BE, Lewe-Schlosser P, Bodner J, Gattenloehner S, Janssen H, Seeger W, Lohmeyer J, Herold S. 2012. Alveolar epithelial cells orchestrate DC function in murine viral pneumonia. J Clin Invest 122:3652-64.
  • Herold S, Tabar TS, Janssen H, Hoegner K, Cabanski M, Lewe-Schlosser P, Albrecht J, Driever F, Vadasz I, Seeger W, Steinmueller M, Lohmeyer J. 2011. Exudate macrophages attenuate lung injury by the release of IL-1 receptor antagonist in gram-negative pneumonia. Am J Respir Crit Care Med 183:1380-90.
  • Cakarova L, Marsh LM, Wilhelm J, Mayer K, Grimminger F, Seeger W, Lohmeyer J, Herold S. 2009. Macrophage tumor necrosis factor-alpha induces epithelial expression of granulocyte-macrophage colony-stimulating factor: impact on alveolar epithelial repair. Am J Respir Crit Care Med 180:521-32.
  • Herold S, Steinmueller M, von Wulffen W, Cakarova L, Pinto R, Pleschka S, Mack M, Kuziel WA, Corazza N, Brunner T, Seeger W, Lohmeyer J. 2008. Lung epithelial apoptosis in influenza virus pneumonia: the role of macrophage-expressed TNF-related apoptosis-inducing ligand. J Exp Med 205:3065-77.

RNA viruses induce heterologous immune protection from atopy and asthma

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Research areas: Molecular Virology, Immunology, Allergy

We showed that a preceding influenza A H1N1 HH/05/2009 virus infection protects from experimental asthma in a murine model. The protective effect was dependent on cross-reactivity between virus- and allergen-derived epitopes at the level of CD44+ CD62L– T effector memory cells (Tem). Virus-induced T1 immune responses to allergens conferred a long-lasting protection. We were the first to demonstrate influenza-induced heterologous immune responses against allergens and aimed to investigate whether such responses were broadly applicable for several other RNA viruses and allergens. We have established and applied a robust bioinformatics pipeline (collaboration with Z02) for the identification of potentially cross-reactive epitope pairs between Respiratory Syncytial Virus (RSV), Rhinovirus (RV), SARS-CoV-2 and influenza virus strains of the 2019/2020 quadrivalent influenza vaccine (QIV) vs >2500 food and inhalant allergen protein sequences. In silico results from our extensive systematic approach generated a priority list of peptides, which were subsequently screened for MHC binding in vitro. Potentially cross-reactive RSV A2- and allergen-derived peptides which showed strong MHC binding, induced T cell activation and IFNγ production upon ex vivo stimulation of RSV-polarized lymphocytes. Importantly, combinatorial pentamer staining revealed truly cross-reactive T cells in the lungs of animals previously infected with RSV A2. Moreover, we provided proof-of-principle evidence for RV-mediated attenuation of allergen-induced experimental asthma. The remaining ultimate question is the clinical relevance of our findings for people with atopic asthma. We hypothesize that virus peptide immunization induces allergen-specific protective immune responses via attenuation of T2 responses to cross-reactive peptides. We thus aim to investigate whether immunization with RV- or RSV-derived cross-reactive peptides protect from experimental asthma either directly or via transfer of Tem from immunized mice. Second, we shall decipher airway immune signatures by means of mass cytometry (CyTOF) among animals developing allergen-induced experimental asthma vs. those that have been previously infected with an RNA virus or immunized with virus peptides and thereby protected from asthma development. Furthermore, we will examine the impact of RNA virus (RSV, RV) infection and of virus peptide immunization on the pulmonary virome composition and diversity, and thus its contribution to RNA virus-induced heterologous immune protection from asthma. Vaccination with the seasonal QIV is currently recommended for asthmatics based on most national immunization guidelines. We shall thus vaccinate wild type and Class I HLA transgenic mice before subjecting to experimental asthma, induced by allergens cross-reactive to the vaccine. Cross-reactive T cells will be deeply phenotyped by means of mass cytometry, tetramer staining, and transcriptomics. Finally, we shall investigate T cell cross-reactivity in the human context, i.e. among healthy controls and atopic individuals previously vaccinated with the seasonal influenza vaccine. Frequencies of cross-reactive T cells will be correlated with natural infection- and immunization-history. Results from this line of experimentation are expected to advise public health policies in regards to influenza vaccination and pave the way for clinical trials involving peptide immunization of atopic asthmatic individuals.

Project-related publications of the investigator:

  • Fragkou PC, De Angelis G, Menchinelli G, Can F, Garcia F, Morfin-Sherpa F, Dimopoulou D, Mack E, de Salazar A, Grossi A, Lytras T, Skevaki C. 2022. ESCMID COVID-19 guidelines: diagnostic testing for SARS-CoV-2. Clin Microbiol Infect doi:10.1016/j.cmi.2022.02.011.
  • Feng A, Yang E, Moore A, Dhingra S, Chang S, Yin X, Pi R, Mack E, Volkel S, Gessner R, Gundisch M, Neubauer A, Renz H, Tsiodras S, Fragkou P, Asuni A, Levitt J, Wilson J, Leong M, Lumb J, Mao R, Pinedo K, Roque J, Richards C, Stabile M, Swaminathan G, Salagianni M, Triantafyllia V, Bertrams W, Blish C, Carette J, Frankovich J, Meffre E, Nadeau KC, Singh U, Wang T, Prak EL, Herold S, Andreakos E, Schmeck B, Skevaki C, Rogers A, Utz P. 2022. Autoantibodies targeting cytokines and connective tissue disease autoantigens are common in acute non-SARS-CoV-2 infections. Res Sq doi:10.21203/rs.3.rs-1233038/v1.
  • Skevaki C, Ngocho JS, Amour C, Schmid-Grendelmeier P, Mmbaga BT, Renz H. 2021. Epidemiology and management of asthma and atopic dermatitis in Sub-Saharan Africa. J Allergy Clin Immunol 148:1378-1386.
  • Skevaki C, Karsonova A, Karaulov A, Fomina D, Xie M, Chinthrajah S, Nadeau KC, Renz H. 2021. SARS-CoV-2 infection and COVID-19 in asthmatics: a complex relationship. Nat Rev Immunol 21:202-203.
  • Renz H, Skevaki C. 2021. Early life microbial exposures and allergy risks: opportunities for prevention. Nat Rev Immunol 21:177-191.
  • Chang SE, Feng A, Meng W, Apostolidis SA, Mack E, Artandi M, Barman L, Bennett K, Chakraborty S, Chang I, Cheung P, Chinthrajah S, Dhingra S, Do E, Finck A, Gaano A, Gessner R, Giannini HM, Gonzalez J, Greib S, Gundisch M, Hsu AR, Kuo A, Manohar M, Mao R, Neeli I, Neubauer A, Oniyide O, Powell AE, Puri R, Renz H, Schapiro J, Weidenbacher PA, Wittman R, Ahuja N, Chung HR, Jagannathan P, James JA, Kim PS, Meyer NJ, Nadeau KC, Radic M, Robinson WH, Singh U, Wang TT, Wherry EJ, Skevaki C, Luning Prak ET, Utz PJ. 2021. New-onset IgG autoantibodies in hospitalized patients with COVID-19. Nat Commun 12:5417.
  • Balz K, Kaushik A, Chen M, Cemic F, Heger V, Renz H, Nadeau K, Skevaki C. 2021. Homologies between SARS-CoV-2 and allergen proteins may direct T cell-mediated heterologous immune responses. Sci Rep 11:4792.
  • Balz K, Trassl L, Hartel V, Nelson PP, Skevaki C. 2020. Virus-Induced T Cell-Mediated Heterologous Immunity and Vaccine Development. Front Immunol 11:513.
  • Skevaki C, Hudemann C, Matrosovich M, Mobs C, Paul S, Wachtendorf A, Alashkar Alhamwe B, Potaczek DP, Hagner S, Gemsa D, Garn H, Sette A, Renz H. 2018. Influenza-derived peptides cross-react with allergens and provide asthma protection. J Allergy Clin Immunol 142:804-814.
  • Skevaki C, Van den Berg J, Jones N, Garssen J, Vuillermin P, Levin M, Landay A, Renz H, Calder PC, Thornton CA. 2016. Immune biomarkers in the spectrum of childhood noncommunicable diseases. J Allergy Clin Immunol 137:1302-16.
  • Skevaki CL, Psarras S, Volonaki E, Pratsinis H, Spyridaki IS, Gaga M, Georgiou V, Vittorakis S, Telcian AG, Maggina P, Kletsas D, Gourgiotis D, Johnston SL, Papadopoulos NG. 2012. Rhinovirus-induced basic fibroblast growth factor release mediates airway remodeling features. Clin Transl Allergy 2:14.
  • Skevaki CL, Christodoulou I, Spyridaki IS, Tiniakou I, Georgiou V, Xepapadaki P, Kafetzis DA, Papadopoulos NG. 2009. Budesonide and formoterol inhibit inflammatory mediator production by bronchial epithelial cells infected with rhinovirus. Clin Exp Allergy 39:1700-10.

Regulation of RNA metabolism, translation and protein degradation pathways in the host response to coronavirus infection

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Research areas: Signal Transduction, Molecular Virology

In this project, we aim to develop an integrative (and comprehensive) molecular view on the human coronavirus (CoV) host response and to achieve deep mechanistic insights into the (coordinated) changes affecting host cell gene expression during viral replication.  A specific overall goal is to define protein kinase modules that control transcriptional and post-transcriptional / translational levels of gene regulation. In the last funding period, by focusing on HCoV-229E, we systematically studied the host response to CoV at the signaling, chromatin and mRNA levels. We found that HCoV-229E causes an unconventional, attenuated activation of the NF-κB system, profoundly reshapes the genome-wide enhancer landscape in the nucleus of infected cells and strongly induces mRNAs encoding a set of transcription factors (TFs), inflammatory mediators and genes downstream of ER stress signaling involved in the unfolded protein response. However, genes upregulated at the mRNA level were often differentially expressed at the protein level. A combination of genetic and pharmacological loss of function experiments revealed a prominent activation of the PERK / IRE1α protein kinase systems by CoV and has led to the discovery of a potent antiviral effect of the ER stress trigger thapsigargin. Thapsigargin inhibits replication of HCoV-229E, MERS-CoV and SARS-CoV-2 in the lower nanomolar range and proteomic data suggest the involvement of ER-associated degradation (ERAD), autophagy and specific parts of the ubiquitin proteasome system in this effect. Together, these findings have resulted in multiple new insights into the CoV – host interactions, both at the level of individual molecules but also globally, that will be exploited in the third funding period. Part (A) of the work program defines four specific goals to identify (i) the chromatin-based mechanisms and genomic targets of the TFs ATF3, KLF6, ANKRD1 and c-JUN, (ii) the CoV-specific activation mechanisms, interactomes and functions of PERK and IRE1α, (iii) their roles in selective translation and (iv) the host cell factors required for CoV replication by unbiased genome-wide loss of function screens. Part (B) aims at identifying the molecular basis for the thapsigargin antiviral effects by focussing on (i) core ERAD components, (ii) factors involved in selective autophagy and (iii) candidate E1 and E2 enzymes, proteasome targeting factors (e.g. ubiquilins) and the role of the ubiquitin proteasome system in degrading viral components. In part (C) a final goal is to integrate the different levels of mechanistic, genome- and proteome-wide data into a (deep) holistic molecular view of the CoV host response also taking into account publically available data sets with the long-term aim to identify key nodes of signaling that distinguish the highly pathogenic from adapted human CoV and that are amenable to therapeutic intervention.

Project-related publications of the investigator:

  • Shaban MS, Muller C, Mayr-Buro C, Weiser H, Meier-Soelch J, Albert BV, Weber A, Linne U, Hain T, Babayev I, Karl N, Hofmann N, Becker S, Herold S, Schmitz ML, Ziebuhr J, Kracht M. 2021. Multi-level inhibition of coronavirus replication by chemical ER stress. Nat Commun 12:5536.
  • Meier-Soelch J, Mayr-Buro C, Juli J, Leib L, Linne U, Dreute J, Papantonis A, Schmitz ML, Kracht M. 2021. Monitoring the Levels of Cellular NF-kappaB Activation States. Cancers (Basel) 13.
  • Zarnack K, Balasubramanian S, Gantier MP, Kunetsky V, Kracht M, Schmitz ML, Strasser K. 2020. Dynamic mRNP Remodeling in Response to Internal and External Stimuli. Biomolecules 10.
  • Kracht M, Muller-Ladner U, Schmitz ML. 2020. Mutual regulation of metabolic processes and proinflammatory NF-kappaB signaling. J Allergy Clin Immunol 146:694-705.
  • 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.
  • Mayr-Buro C, Schlereth E, Beuerlein K, Tenekeci U, Meier-Soelch J, Schmitz ML, Kracht M. 2019. Single-Cell Analysis of Multiple Steps of Dynamic NF-kappaB Regulation in Interleukin-1alpha-Triggered Tumor Cells Using Proximity Ligation Assays. Cancers (Basel) 11.
  • 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.
  • 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. Front Immunol 9:775.
  • 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:e1006286.