Detailseite
Projekt
Investigating the relevance of lipid microdomains for (HI) virus protein assembly and virus budding in an artifical cell environment
Antragstellerin
Professorin Dr. Petra Schwille
Fachliche Zuordnung
Virologie
Förderung
Förderung von 2005 bis 2013
Projektkennung
Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 13204897
During the HIV replication cycle, the immature viral core is assembled at the plasma membrane and subsequently released from the host cell via a budding process. This process is primarily driven by its cytoplasmic core-precursor protein Gag. In addition, constituents of the plasma membrane become incorporated into the viral envelope. These are, e.g., GPI (glycosylphosphatidyl inositol)-anchored proteins, but also the ganglioside GM1, cholesterol and Sphingomyelin, which are supposed to be enriched in so-called lipid rafts , putative platforms for protein sorting and signaling events in the cell membrane which exhibit a condensed lipid order. Consequently, lipid rafts seem to play a crucial role in virus infection of a cell, and their control could be an essential step in antiviral therapies. However, to date very little is known about the dynamics of the protein-lipid interactions during virus assembly and budding, due to the complexity of the cellular system being infected by viruses. To investigate the role of lipid microdomains or rafts on virus proliferation independent from other factors such as membrane proteins or the cellular secretion machinery, we propose to study the assembly, membrane-association and budding of viral particles, after in vitro expression of Gag protein, in an artificial cell system of defined membrane composition. To this end, a generally applicable scheme of cell-free protein expression in Giant Unilamellar Vesicles (GUVs) will be established. Of particular relevance will further be the control of cholesterol levels in the GUV, i.e., artificial cell membrane. Using fluorescent lipids and Gag protein chimeras fused to various fluorescent proteins, the different steps such as Gag protein aggregation, membrane association and budding of viral particles will be investigated by various kinds of single molecule-based fluorescence correlation spectroscopy and imaging, techniques specifically developed and tailored in our laboratory.
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