Dreidimensionale Einzelmolekülverfolgung von Herpesviren zur Untersuchung ihrer Zellausschleusung und des Zusammenbaus ihrer Tegumentschicht
Virologie
Zusammenfassung der Projektergebnisse
The objective of the proposed work was to study the details of herpesvirus assembly and egress dynamics inside infected living cells. In order to achieve these goals, I chose together with the host lab an interdisciplinary approach which combined advanced fluorescence microscopy, virus biology and quantitative single particle tracking. 2D single particle tracking of the fluorescently labelled capsids - the DNA packaging core structure of herpesvirus - in the cytoplasm of infected cells where the assembly compartments are located during late stages in infection revealed two areas with different capsid mobility. Slow movement was related to areas rich in viral glycoproteins - membrane proteins in the viral envelope - whereas fast movement was observed during transport along microtubules. However, the original goal of the project was to follow single virus capsids through the cell in 3D using light sheet microscopy. But the light sheet microscope that existed at the time of the start of my project in the host lab was not suited for subcellular imaging. A new setup had to be designed and built for the project including troubleshooting of sample mounting and installation of environmental control. In the meantime, I followed another interesting route of research related to herpesvirus assembly which evolved from the three-colour single particle tracking experiments that I performed. The assembly process is a highly complex process during which viruses take advantage of cellular mechanisms inside their hosts. That means that the virus coordinates big changes to the host cell organisation. These changes also greatly influence cell dynamics and directionality of transport processes which made the study of them very relevant for the DFG project. We first established a fluorescent reporter virus which provided for the first time a direct visual readout for the stage of infection at the individual cell level. I identified four stages in infection. This classification was used to map the morphological changes of various cellular structures and cell organelles involved in virus transport and assembly over the time course of the infection cycle. Most structures undergo severe changes, and we can distinguish two major concerted rearrangement periods, one presumably related to virus assembly and the other to cell stress and death. The results of this work were presented at international conferences for microbiology and microscopy, respectively. Towards the end of the funding, the host lab had to shut down for four months due to a broken pipe which caused a flood, but I am now able to resume work on studying single virus dynamics using the newly built light sheet microscope which is now set up for the proposed experiments. In addition to the funded project, a master student established under my supervision expansion microscopy in the host lab and optimised the protocol for herpesvirus infected cells. We applied this technology for imaging cellular structures in infected cells with fine detail, and it is now used in the host lab within different collaborations and PhD projects. Also, I led a collaboration with Prof. Andrew Lever from the Department of Medicine, University of Cambridge, to verify that ESCRT-II is involved in HIV budding. In support of biochemical data from the Lever group, we used two-colour TIRF imaging to visualize the colocalisation between HIV Gag and ESCRT- II EAP45 protein. This project became to form a big part of a PhD project in the host lab.
