Pseudotyping systems and their use in coronavirus entry research
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The research of virus host cell entry is essential for anti-viral drug development. However, for coronaviruses, such as the highly pathogenic SARS virus, the entry mechanisms are not yet fully discovered. Therefore, many research groups are currently studying the activation of the coronavirus spike protein, which mediates fusion between the viral envelope and plasma membrane. In many fundamental studies, artificial assays are used instead of genuine virus to circumvent the requirement for a high Biosafety level. In this thesis, we discuss systems as pseudotyping assays, cell-cell fusion assays, virus-like particles and some reconstituted systems as well. Some of these systems also have purposes outside viral entry research, for example in gene therapy, targeted drug-delivery and anti-cancer treatment by specifically altering the tropism of the vector to the target cells. Pseudotyping and other artificial assays have the advantages of being allowed to be performed less stringent biosafety conditions and making reverse genetics easier to perform. However, all experimental systems have limitations which we evaluated. While VSV∆G and retrovirus based pseudotyping assays gave comparable results as authentic virus, real virus has still to be taken along as well. Cell-cell fusion assays are simplistic systems to study the basic functionality of a fusion protein. However, these assays gave contradictory results in comparison to virus entry in some studies, which can be explained by the difference in the interaction context. Therefore, cell-cell fusion assays are not suitable to study entry mediation by fusion proteins. The experimenter has to choose the systems carefully to approach his questions, because of the limitations of the experimental set ups. Finally, we try to experimentally set up a new pseudotyping system based on the coronavirus Murine Hepatitis Virus (MHV). We also make a stable cell line expressing recombinant MHV spike protein which could be used for both retrovirus and this new pseudotyping assay.