Wege und Kinetik der Foamyvirus Aufnahme und des Glykoprotein-vermittelten Fusionsprozesses
Biochemie
Biophysik
Zusammenfassung der Projektergebnisse
Understanding the entry pathway of viruses is of eminent importance as 1) viruses represent an everincreasing pathogenic threat, 2) the delivery of nucleic acids and macromolecules to cells is becoming a potent tool in gene therapy and drug delivery and 3) viruses serve as important model systems for investigating molecular and cell biology. For these reasons, we have focused on investigating the uptake and fusion pathway of foamy virus (FV). Within the project, we had three main aims: 1) the development of tools for visualizing and quantifying lipidmixing, content mixing and fusion, and using the developed tools 2) to characterize the uptake pathways and fusion processes using enzymatic assays and single virus tracing (SVT), as well as 3) to investigate how the kinetics of entry and fusion are affected by different components of the virus by use of chimeric viruses assembled from components of different types of retroviruses. With respect to the first aim, we have made extensive progress. We generated and characterized a myriad of different foamy virus constructs with different fluorescent tags attached at different positions within the virus. These constructs included labeling of the Gag protein and Env proteins with combinations of GFP and mCherry, Cerulean and YFP, and RFP670 and Cerulean/mTurquoise2. To utilize the new viral constructs for live-cell imaging, the spinning disc confocal microscope was upgraded with an additional channel for monitoring content mixing, lipid mixing or visualization of cellular components. We could show that mTurquoise2 is a better choice as Cerulean for the imaging experiments. RFP670 appears to be suitable as a fluorescent marker is not yet measurable on the microscope for technical reasons. We successfully developed an enzymatic content mixing assay that was used to investigate virus uptake and fusion kinetics, temperature requirements and cellular trafficking pathways involved. This revealed, that the majority of fusion events in a human fibroblast cell line occurred between 10 to 40 minutes post attachment, that fusion activity was reduced 2-fold at 26°C and 5 to 10 fold at 20°C, and that inhibitors preventing endosomal acidification or micropinocytosis strongly interfered with viral fusion. A variant that will allow visualization of content mixing optically by SVT in real time is still under development. As the fusion process is a rare event, we invested time in improving the analysis routines to increase the throughput of the data analysis. The workflow of the analysis was streamlined and the ability to read in previously extracted trajectories was implemented. As the quality of the analysis and the quantity of trajectories depends on tracking algorithm used, we also spent great efforts in automating the SVT software. The wavelet approach adapted here is powerful and general enough that it could be applied to other imaging modalities. With the developed software, we could track single atoms diffusing on a surface measured using scanning tunneling microscopy, even when the imaging artifacts were severe. With the tools we have successfully developed and those that are almost completed in aim 1, we are now in the position to complete aim 2 of the project and thus makes it possible to pursue aim 3.
Projektbezogene Publikationen (Auswahl)
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2019. Density fluctuations as door-opener for diffusion on crowded surfaces. Science. 363: 715–718
Henß, A-K, Sakong S, Messer PK, Wiechers J, Schuster R, Lamb DC, Groß A, and Wintterlin J.
