Research

Our lab is interested in the interaction of viral structures (proteins and nucleic acids) with host factors and the relevance for antiviral immunity. We aim to gain functional and mechanistic insights in the interplay between viruses and the organism by studying virus-host interactions and protein expression profiles that are elicited by viral infections. Through this approach we identify yet unstudied proteins and pathways that we are further testing in focused hypothesis-driven approaches that include testing of interactions on molecular basis, in vitro cell culture assays and in vivomodels using genetically modified animals.

(1) Interaction of viral nucleic acids with host proteins

We identified viral triphosphorylated RNA as specific ligand for the virus sensor RIG-I (Pichlmair et al., Science 2006). Using affinity proteomics followed by mass spectrometry we identified additional proteins binding specifically to this type of RNA. Interferon induced proteins with tetratricopeptide repeats (IFIT), for instance, bind PPP-RNA and perturb virus growth (Pichlmair et al, Nature Immunology, 2011). IFIT proteins bind PPP-RNA using a uniqe mechanism ensuring high specificity and affinity (Abbas et al., Nature 2013). IFIT1 depletion in vitro and in mice are specifically susceptible to infection with viruses including orthomyxo- (e.g. influenza A virus) and paramyxoviruses (e.g. vesicular stomatitis virus). Functionally, IFITs specifically target translation of viral RNA (Habjan et al., Plos Pathogens, 2013). Using similar approaches we identified a yet unstudied protein, NCBP3, as cap-binding protein (Gebhardt et al., Nature Communications 2015). NCBP3 binds NCBP1 to from an alternative Cap-RNA complex (CBC) that binds to mRNA and is important for RNA processing and export. Lack of NCBP3 is increasing vulnerability to virus infections, suggesting an important role of the alternative CBC during antiviral responses.

(2) Systematic analysis of changes in the proteome after viral infection

Though comprehensive knowledge on changes in the transcriptome, comparable little is known on the global changes of the proteome after infection with individual viruses. We are assessing virally-induced changes in the global composition of the proteome as well as specific post-translational modifications.

(3) Interactions viral and host proteins and functional consequences

Viruses require the host cellular machinery to replicate. We use viruses go guide us to cellular proteins and pathways that are determining virus pathogenicity. To this aim we are using mass spectrometry to study cellular binding partners of viral proteins using systems biology approaches (Pichlmair et al., Nature 2012). We identified 600 cellular proteins that are binding to of viral immune modulators (iVIMs). We are now complementing this survey to assess the functional consequences for antiviral immunity. This survey so led to identification of novel modes of transcriptional regulation by an orthomyxovirus that specifically affects genes required for host defense (Haas et al., Plos Pathogens, 2018). Furthermore, we identified a novel cell death pathway named Oxeiptosis that is targeted by viral proteins derived from diverse viruses. Intracellular reactive oxygen species (ROS) that are commonly generated during virus infections, engagement of toxic substances or teratogenic transformation of cells (Holze et al., Nature Immunology, 2018). The cellular protein KEAP1 senses increased ROS levels and activates a cell death cascades that involves the mitochondrial enzyme PGAM5 that dephosphorylates the protein AIFM1. This leads to cell death. Lack of oxeiptosis in mice induces hyperinflammation after virus infection, which is associated to increase immunopathology.

Infrastructure

Our laboratory offers the opportunity to use top-notch technologies to study virus host interactions and that allow us high flexibility and independence:

(1) Molecular biology laboratory

Our laboratories allow to work with genetically modified organisms at all different levels – from recombinant proteins to ex vivo material. Access to local mouse facilities and a BSL3 laboratory with licenses to work with human pathogens is also available.

(2) Mass spectrometry

We are running our own mass spectrometry platform (Q-Exactive plus HF mass analyzer coupled to a NanoLC 1200 UHPLC system), which gives us the opportunity to perform large scale screening experiments including testing protein-protein interactions, protein expression profiling, studying protein dynamics as well as post-translational modifications. Labmembers are preparing and analyzing their own samples after a short training period.

(3) Compute server/storage

We are operating our own server (HPE ProLiant RackServer with 36 physical Cores/72 threats, 256 gByte RAM, 20TB HDD) and storage system (HPE StoreEasy, 70 TB), which we use for analysis ofmass spectrometry and other high content data. An own dedicated Bioinformatics subgroup is scripting all sort of algorithms that are essential to analyse mass spectrometry data and to generate high quality datasets.

(4) Incucyte S3 light microscopy screening platform

The most recent addition to our laboratory. This system allows phenotypic screens and bridges the gap between mass spectrometry analysis (mostly discovering hundreds of candidates) to detailed functional and mechanistic studies on a very limited number of proteins. The incucyte S3 system allows fluorescence based screens of up to 584 conditions in a time-resolved manner. Combination with CRISPR/Cas9 and/or Drung screens will allow us to specifically perturb proteins identified by mass spectrometry analysis. A great tool that will transform the way we perform experiments in the lab.

(5) BD Cytoflex FACS analyser

Top modern FACS machine with a 96-well multisampler and the ability to test up to 13 colors in parallel.

(6) BioRAD NGC Quest10 FPLC

A reliable and easy to use FPLC system with sample pump, the ability to mout 5 collumns in parallel, coupled to a fraction collector. This FPLC can be used to generate recombinant proteins, to fractionate cells or nuleic acids using various size exclusion or affinity collumns.

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