Single particle tracking (SPT) which allows for the visualization of key steps of the virus life cycle on a single virus particle level, promises to expand the knowledge in the field of virology drastically: it offers the possibility to reveal transient and dynamic processes that are otherwise masked in static or ensemble-averaged measurements. In the context of applying SPT to studying virus attachment and entry into host cells, virus pseudotypes, i.e. particles displaying the structural core of one virus and the functional envelope glycoprotein (GP) of a heterologous virus of interest, are promising candidates: they are compatible with biosafety level -2 conditions and can be easily labelled. However, heterogeneities in the GP distributions represent a significant hurdle for single particle applications, as they may affect the particle behaviour. Accordingly, it is important to characterize pseudotypes on an individual particle level, to optimize production, labelling and data acquisition strategies.
In this project, fluorescent pseudotypes of the deadly filovirus Ebola were produced, using a lentiviral pseudotyping system with a mCherry-tagged viral core. A workflow for their in-depth characterization on a single particle level is proposed. Additionally, the particles were used in single particle kinetic assays, to test the hypothesis that mucin-like domain of Ebola, a highly glycosylated region of the protein, is important in modulating the attachment and detachment of the virus from the cell surface.
A thorough characterization of pseudotype properties and the production of more homogenous samples will strengthen the interpretations of experimental results within virology and also contribute to the optimization of pseudotype-based vaccines. In addition, understanding the mechanisms by which virus interactions at the cell surface are modulated, can help developing therapeutics for future outbreaks.
