Project Details
Projekt
Lateral sorting of proteins into lipid-rafts and protein-protein interactions as prerequisite for assembly of influenza virus: A complementary biophysical approach on model membranes and living cells
Subject Area
Virology
Term
from 2005 to 2015
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 12714198
Assembly and budding of enveloped viruses requires local enrichment of viral components at the budding site of the host membranes. Two non-excluding mechanisms can account for this: inclusion of membrane proteins into and association of other viral components with subdomains of the plasma membrane (lipid-rafts), and specific interactions between viral proteins. The lipid-raft concept for virus budding is based on two observations: (i) membrane proteins of many viruses are present in rafts and (ii) disintegration of rafts inhibits assembly of virus particles. However, published results demonstrating the occurrence of viral proteins in rafts were obtained with the Triton-extraction method and it is highly controversial whether partitioning of proteins into these detergent-resistant membranes reflects their association with rafts inside living cells. Furthermore, the molecular basis, i.e. the signal for sorting a viral protein into a lipid domain is an open question. Likewise, interactions between glycoproteins and internal components of virus particles were only indirectly deduced from genetic experiments, but have not been directly demonstrated inside cells. In this project we will address partitioning of proteins into rafts and interactions between viral proteins using essentially biophysical approaches: ¿ Fluorescence-energy transfer (FRET) between established raft-markers and viral proteins as well as between two viral proteins inside cells. ¿ Reconstitution of viral membrane proteins into giant unilameHar vesicles (GUVs) harbouring a liquid-disordered and a liquid-ordered, raft-like phase. ¿ Specific interaction between transmembrane domain sequences and lipids. We will analyze the well-characterized influenza virus membrane proteins hemagglutinin (HA), neuraminidase (NA) and the matrix-protein (Ml). Based on Triton-extraction experiments NA and HA are constituents of lipid-rafts, whereas Ml binds to rafts only when either NA or HA are present. We will also address whether artificial systems as GUVs can be used to model membrane bending processes associated with virus budding, and to identify the viral components necessary to trigger membrane bending. However, the proposed methods are suitable to analyze the raft-affinities and protein-protein-interactions for any viral protein, and efforts will be undertaken in the Schwerpunkt to apply our approach to other enveloped viruses.
DFG Programme
Priority Programmes
