- Head of research group
1. Use of high throughput techniques (next generation sequencing) in the detection of known and novel viral pathogens in acute and chronic diseases
Numerous infectious diseases emerged or re-emerged over the past decades. Pathogen identification/detection methods have significantly improved over time; however sample preparation (nucleic acid extraction), development of reliable diagnostic tools as well as proving causality (Hill's criteria) once a pathogen has been identified are still challenging.
Next generation sequencing together with PCR amplification techniques are used to hunt for new viruses in acute and chronic diseases with putative infectious etiology.
Improvements of sample preparation for successful detection of viral pathogens from diagnostic samples, development of reliable protocols suitable for detection of pathogens from clinical samples using next generation sequencing) are key elements in our research.
Being integrated together with the Department for Virus Genomics, Heinrich-Pette Institute (HPI) , Leibniz Institute for Experimental Virology, Hamburg in the German Center for Infectious Disease Research (DZIF) , thematic translational unit emerging infections we develop standardized NGS protocols and procedures, and together with our collaborators at the HPI optimize and adapt bioinformatic methods to detect and analyze pathogens in clinical samples of known or suspected infectious etiology. All protocols and methods are developed in close cooperation between clinical and research groups at the partner institutions.
2. The Merkel cell polyomavirus, a new human tumor virus: The function of the viral oncogene (LT-Antigen) in MCC pathogenesis
Merkel cell carcinoma (MCC) is a rare but highly aggressive form of skin cancer which mainly affects elderly and immunosuppressed patients.
In 2008, a novel human polyomavirus was identified using high throughput sequencing of RNA derived from MCC tissue (Feng et al., Science 2008). Due to a high association of the virus with this disease (80-97% of all MCC tumors are MCPyV positive), the monoclonal integration of its DNA in the host genome of the tumor cell and the expression of viral proteins (Large T-Antigen, LT, and small T-Antigen, sT) within the tumor cells this virus is classified as a human tumor virus.
Merkel cell polyomavirus (MCV) is one out of ten human polyomaviruses (hPyV) known to date, four of these are associated with diseases (Figure 1). Interestingly,
MCPyV is the only human PyV known associated with cancer.
Fig. 1: Phylogenetic tree of Large T-Antigen sequences of all human polyomaviruses known to date, mouse polyomavirus (MPyV) and monkey SV40 as well as Chimpanzee (ChPyV) polyomavirus. Human polyomaviruses associated with human diseases are labelled -*-.
Our research goals
Understanding the MCPyV life cycle:
To gain a better understanding of MCPyV biology, an in vitro MCPyV replication system is urgently needed. We have generated a synthetic MCPyV genomic clone (cMCPyV) based on the consensus sequence of MCC-derived sequences deposited in the NCBI database. Transfection of intramolecular recircularized cMCPyV DNA into some human cell lines recapitulates efficient replication of the viral genome, early and late gene expression together with moderate virus particle formation (Neumann, Borchert et al., PLoS one 2011). However, no serial transmission of infectious virus was achieved. This in vitro culturing system allows the molecular dissection of some aspects of the MCPyV viral life cycle which is important to study virus induced abrogation of the host cell.
Fig. 2: Electron micrographs showing SV40 particles in the nucleus of CV-1 cells (left side) and electron micrographs of PFSK-1 cells 4d past transfection with cMCPyV proviral DNA (right side). 40µm electron dense particles were observed in approximately 1 out of 50 cells with the particles localizing in the nucleus close to membrane structures (adapted from Neumann, Borchert et al., PLoS one 2011).
Applying multiple techniques, e.g. confocal microscopy, mass spectrometry, biochemical analysis (microscale thermophoresis), we study viral factors as well as host cell factors for their role in the MCPyV life cycle.
Fig. 3: Confocal laser scanning microscopy showing MCPyV LT-Ag localization (red) and PML-nuclear body localization (green) in our MCPyV replication system. Confocal micrographs of H1299 cells 4d past transfection with cMCPyV proviral DNA.
MCPyV contribution to MCC pathogenesis:
We comprehensively investigate MCPyV T-Ag function in order to elucidate their overall transforming potential, the contribution of individual T-Ag domains to the transformation process, the cell type dependency of T-Ag mediated transformation, the significance of the hallmark large T-Antigen (LT-Ag) truncations observed in MCC tissues, and the contribution of constitutive T-Ag expression to the sustained proliferation of MCC cells. We address these questions in heterologous cell systems as well as MCC-derived cell lines; as such cells represent the best available in vitro model system for MCC.Being integrated in the interdisciplinary graduate program - Hamburg School for Structure and dynamics in infection (SDI) - funded by the Landesexzellenzinitiative Hamburg, we investigate structural properties of MCPyV LT-Ag by applying structure biology methods (Dynamic Light Scattering, Small-angle X-ray scattering, X-ray crystallography).Projects:
- Identification and characterization of viral and host factors important for MCPyV replication
- Cell transformation by MCPyV
- The role of PML-NB components in MCPyV replication
- MudPIT analysis to identify MCPyV LT interacting factors
- Elucidating the function of MCPyV microRNA using HITSCLIP and NGS
- Structural characterization of tumor derived early gene products by X-ray crystallography
- Molecular biology methods (DNA/RNA/protein)
- Cell culture using established as well as primary cells
- Transfektion, transduction and infection of eukaryotic cells
- Confocal Laser Scanning Microscopy
- Protein co-localization studies using confocal microscopy and FACS-FRET analysis
- Real-time PCR
- Xenograft mouse models
- Methods to study protein protein interactions
- MALDI-ToF Mass spectroscopy
- Expression, purification of recombinant proteins using bacterial systems as well as baculovirus system
- Microscale thermophoresis, BIAcore
- X-ray crystallography
- Next generation sequencing