- Lyssaviren (Rabies Virus, Europäische Fledermaus Lyssavirus Typ I und II)
- Vesicular Stomatitis Virus (VSV)
The Rhabdoviridae virus family includes numerous animal pathogens. Within rhabdoviruses infecting mammals, Rabies virus (RV) and vector transmitted viruses such as Vesicular Stomatitis Virus (VSV) are the best investigated members and are considered as model viruses for rhabdovirus pathogens. Compared to other members of the Mononegavirales order, rhabdoviruses are regarded as minimal viruses because of their rather simple genome organisation, making rhabdoviruses a suitable model system to investigate viral replication mechanisms.
In spite of the broad host range of rhabdoviruses, host species specific differences regarding the replication potential of rhabdoviruses are observed. Beside basic, cell independent mechanisms of the rhabdovirus replication a species specific and/or cell type dependent replication of the pathogens is a decisive factor for the zoonotic potential of these viruses. Knowledge about molecular principles of host cell dependent and independent virus replication is therefore of significant interest for the assessment of risk potentials of known and emerging rhabdoviruses as well as for the development or advancement of vaccines for prevention.
A key technology in the lab is the use of genetically modified recombinant rhabdoviruses that allow the functional characterization of virus proteins and regulatory nucleotide sequences. Besides the consistent advancement of already existing reverse genetics systems, new systems are developed for rhabdoviruses that are of special interest for the investigation of molecular replication mechanisms of rhabdoviruses or that have to be characterized on the molecular level and functionally investigated because of current or conceivable emergence.
A further focus in the lab is the use of modern imaging techniques for the visualization of viral replication steps. For this purpose, a technology platform has been established that allows real time tracking of fluorescence-labelled proteins and viruses in living cells (live cell/virus imaging).
Membrane Budding by Rabies Virus Matrix Protein
DFG Schwerpunktprogramm 1175 „Dynamics of Cellular Membranes and their Exploitation by Viruses“
This project aims at the identification of specific viral and cellular functions required for the formation of enveloped rhabdoviruses. By integrating virological, biochemical, and imaging approaches, tagged M proteins will be used to identify cellular interaction partners of the M protein. Since M is the major driving force in rhabdovirus budding, the identification of interacting cell proteins that support virion release is crucial for the basic understanding the molecular mechanisms of rhabdovirus assembly. Besides the unravelling of cellular mechanisms abused for pathogen release, knowledge about the molecular mechanisms may support target structures for virus inhibition and rational attenuation of recombinant rhabdovirus vaccines.
Molecular Determinants of Lyssavirus Replication (DFG)
Bats act as reservoirs for many zoonotic viruses. Reasons for this preferential reservoir function and molecular requirements for trans-species transmission and establishment of bat-derived viruses in new hosts are unknown. Among the bat-associated viruses are Lyssaviruses. Lyssaviruses comprise rabies virus (RV) circulating in carnivores and bat associated viruses, making this a suitable model to investigate virus adaptation to different hosts. In the proposed project we plan to assay for virus-host protein interactions that explain host specific replication of different lyssaviruses. Murine and human cells are infected with bat and non-bat lyssaviruses and protein interactions required for efficient replication in non-bat systems are identified in a comparative mass spectrometry screen, in which RV and bat virus proteomes of purified intracellular subviral complexes are compared. Differences between RV and bat virus complexes in their cell protein content are supposed to reflect host specific variations. Influences of identified proteins on virus replication are evaluated and domains/motifs involved in host specific interactions are mapped. The relevant sequences are exchanged between recombinant RV and bat viruses and their influence on host specific virus replication of the recombinant viruses are investigated in cell culture and in vivo. Recombinant bat viruses with proteins or protein-domains adopted from RV will for the first time allow systematic characterization of molecular processes in host specific lyssavirus replication. Identification of host-related differences will contribute to the risk assessment related to infection of new hosts by bat-lyssaviruses and will provide a basis for further investigation of molecular principles of virus replication in bat reservoirs.
Live-Imaging of viral structures
Viral replication is a dynamic process. For the understanding of the mechanisms involved, aside mere qualitative aspects, the spatial and temporal resolution of the individual steps is required. Whereas classical imaging and biochemical techniques so far supported rather static informations about the investigated processes, modern fluorescence microscopy techniques for live cell imaging allow the analysis of intracellular processes with a very high temporal and spatial resolution. Investigations are targeted to direct interactions of viral and cellular proteins as well as intracellular transport mechanisms of subviral or viral particles. The Establishment of a “live imaging”-platform at the FLI allows the corresponding investigations with selected viral pathogens. In this regard, Rabies viruses play an important role as a model system for infectious pathogens of the central nervous system (CNS). Aside investigations to the “retrograde axonal transport” of virus paticles to the CNS, fluorescence labelled particles or virus proteins allow the analysis of virus entry, cytoplasmic replication and virus assembly/budding in real time and in the three-dimensional cellular space.