Periodic Reporting for period 1 - REMIND EBOV (Role of the mucin-like domain of the Ebola virus in modulating virus-glycosaminoglycan interactions)
Reporting period: 2021-08-01 to 2023-07-31
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.
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.
For the characterisation of the pseudotypes cell culture, cloning, immunostaining, flow cytometry, fluorescence microscopy, western blots, and negative staining transmission electron microscopy were used. The findings reveal that standard particle production protocols based on transient transfection and sucrose-gradient-purification result in samples containing significant fractions of particles lacking either GP or the viral core, with large batch-to-batch variability. The project results show that these aspects can be significantly improved through the creation of stable cell lines for pseudotyping. The distribution and abundance of GPs on the pseudotype surface was investigated and it was shown that pseudotypes obtained from different cell lines do not differ in their size nor in the number of GPs. Nor did the addition of a fluorescent marker into the pseudotype affect those properties.
To assess the influence of the mucin-like domain of Ebola on the virus binding dynamics, a pseudotype virus containing a GP protein with this domain deleted was produced. A platform mimicking the carbohydrate layer covering the cells (glycocalyx) produced by end-immobilized heparan sulfate chains, was used to investigate the initial attachment behaviour of the virus. The results indicate that the deletion of the mucin-like domain, does not impact the attachment or detachment behaviour of the pseudotypes to heparan sulfate, in comparison to the wildtype GP.
The findings from this project were presented at several scientific conferences and seminars. A manuscript is currently under preparation and will be published in an open-access journal. In addition, the results and scripts generated in this project will be made available.
To assess the influence of the mucin-like domain of Ebola on the virus binding dynamics, a pseudotype virus containing a GP protein with this domain deleted was produced. A platform mimicking the carbohydrate layer covering the cells (glycocalyx) produced by end-immobilized heparan sulfate chains, was used to investigate the initial attachment behaviour of the virus. The results indicate that the deletion of the mucin-like domain, does not impact the attachment or detachment behaviour of the pseudotypes to heparan sulfate, in comparison to the wildtype GP.
The findings from this project were presented at several scientific conferences and seminars. A manuscript is currently under preparation and will be published in an open-access journal. In addition, the results and scripts generated in this project will be made available.
The work performed in this project will contribute substantially to the field of virology. Pseudotyping is widely used and with more and more advanced imaging methods, more and more groups are employing single-particle approaches. Thus, it is crucial to have well-characterised and homogenous samples. The knowledge uncovered with this project will improve the interpretation of experimental results obtained with viral pseudotypes. As viral pseudotypes are also widely used in the development of vaccines and medications, it could have a more direct impact on the society and health care.