Glycoprotein S on the surface of the SARS-CoV-2 virus is a key factor in infection. In addition to the viral attachment and entry into target cells, protein S also enables syncytia formation. Syncytia are larger multinucleated cells that result from the fusion of infected cells with neighboring uninfected cells. Their formation increases the spread of the virus and the complexity of the disease COVID-19. Cell fusion depends mainly on the fusogenicity of the S protein on the surface of infected cells. In addition, syncytium formation requires the same receptor as viral entry, ACE2. The presence of the TMPRSS2 protease on the surface of target cells further promotes cell fusion, as it enables the proteolytic cleavage of protein S. Like other RNA viruses, SARS-CoV-2 mutates rapidly. Since its emergence, the wild type has been replaced by variants containing many mutations, especially in protein S. Mutations in protein S affect the virus's transmissibility, pathogenicity, and ability to form syncytia. In this master's thesis, we examined the ability of cells expressing variants of the S protein of SARS-CoV-2 to form syncytia. We analyzed the wild type of protein S that appeared in Wuhan, as well as the delta and XBB.1.5 variants. In addition, we introduced point mutations N501Y in the receptor binding domain, P681H in the FCS, and T716I in the S2 subunit of protein S. In the scope of the master's thesis, we studied the formation of syncytia in two ways. We compared the fusogenicity of protein S variants using a fusion assay with split luciferase, and examined the influence of the presence of the ACE2 receptor and TMPRSS2 protease on syncytia formation. All selected natural variants of SARS-CoV-2 protein S showed a comparable rate of syncytia formation. Mutations N501Y and P681H increased cell fusion, while T716I decreased it. In addition, syncytia were formed even in the absence of the ACE2 receptor, if the TMPRSS2 protease was present on the surface of the acceptor cells. In the second part of this master's thesis, we also studied the kinetics of syncytia formation. For this purpose, we prepared the Simplicon vector carrying sequences for the studied variants of protein S and performed in vitro transcription. We then confirmed the expression of all recombinant proteins in transfected cells and the presence of all variants of protein S except delta on the surface of transfected cells. The kinetics of the formation of syncytia was monitored with an automatic microscope, but the experiments were not successful, and we did not obtain reproducible results. Therefore, the process should be improved at the level of transfection by using another cell line, another method of introducing RNA replicons, or by adding the Vaccinia virus protein B18R. Additionally, it would be necessary to optimize the analysis of the results and test the use of other programs. The results of the master's thesis confirm that the variants of the SARS-CoV- 2 S protein have different fusion potentials and contribute to a better understanding of the influence of other factors, such as ACE2 and TMPRSS2, on cell fusion.
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