Capillary Electrophoresis of Liposome-Associated Virusses

Liposomes, lipid bilayer bounded droplets, are being frequently used as a model mimicking cellular membranes. In contrast to cells and membrane-delimited organelles, their lipid composition can be well defined, and membrane proteins can be inserted at predetermined concentration. Therefore, liposomes are considered a clean system allowing studying binding and transport phenomena occurring at the plasma membrane or at the surfaces of intracellular vesicles. We here propose to develop methods for the analysis and characterization of liposomes carrying proteins attached via various means by capillary electrophoresis. This system will be further employed to study the interaction of human rhinoviruses (HRVs) with membrane-anchored receptor molecules and the release of the viral genome, processes occurring at the plasma membrane and at endosomal membranes in vivo. HRVs that are responsible for the majority of mild infections of the upper airways, use two different receptors for cell attachment. Eighty-seven serotypes bind intercellular adhesion molecule 1 (ICAM-1), a member of the immunoglobulin superfamily and 12 HRVs bind members of the low-density lipoprotein receptor (LDLR) family, multidomain multimodular proteins. The ligand-binding domains of LDLRs consist of various numbers of imperfect direct repeats. A soluble, artificial concatemer of 5 copies of module 3 of very-LDLR attaches to HRV2 with high affinity indicating that more than one and most probably all five modules are involved. This suggests that 12 receptor molecules bind to the 12 vertices at the icosahedral fivefold axes of the virion. In contrast, 60 molecules of ICAM-1 attach simultaneously to their cognate serotypes. In any case, it is unclear how the virus binds to membrane anchored receptors and how the genomic RNA is subsequently injected through the membrane upon endocytosis. Whereas ICAM-1 aids in RNA release, it is not known whether LDL-receptors play any role in RNA penetration into the cytosol or are mere vehicles for viral entry. For the extensive characterization of these steps using liposomes carrying derivatives of viral receptors we propose to make use of the high sensitivity of detection, the low amount of material needed, and the speed of analysis of capillary electrophoresis. This will be achieved by combining capillary electrophoretic separation with fluorescence detection to follow virus, receptor, viral proteins, and genomic RNA during attachment to, and uncoating at or within liposomes.

Liposomes, lipid bilayer bounded droplets, are being frequently used as a model mimicking cellular membranes. In contrast to cells and membrane-delimited organelles, their lipid composition can be well defined, and membrane proteins can be inserted at predetermined concentration. Therefore, liposomes are considered a clean system allowing studying binding and transport phenomena occurring at the plasma membrane or at the surfaces of intracellular vesicles. We here propose to develop methods for the analysis and characterization of liposomes carrying proteins attached via various means by capillary electrophoresis. This system will be further employed to study the interaction of human rhinoviruses (HRVs) with membrane-anchored receptor molecules and the release of the viral genome, processes occurring at the plasma membrane and at endosomal membranes in vivo. HRVs that are responsible for the majority of mild infections of the upper airways, use two different receptors for cell attachment. Eighty-seven serotypes bind intercellular adhesion molecule 1 (ICAM-1), a member of the immunoglobulin superfamily and 12 HRVs bind members of the low-density lipoprotein receptor (LDLR) family, multidomain multimodular proteins. The ligand-binding domains of LDLRs consist of various numbers of imperfect direct repeats. A soluble, artificial concatemer of 5 copies of module 3 of very-LDLR attaches to HRV2 with high affinity indicating that more than one and most probably all five modules are involved. This suggests that 12 receptor molecules bind to the 12 vertices at the icosahedral fivefold axes of the virion. In contrast, 60 molecules of ICAM-1 attach simultaneously to their cognate serotypes. In any case, it is unclear how the virus binds to membrane anchored receptors and how the genomic RNA is subsequently injected through the membrane upon endocytosis. Whereas ICAM-1 aids in RNA release, it is not known whether LDL-receptors play any role in RNA penetration into the cytosol or are mere vehicles for viral entry. For the extensive characterization of these steps using liposomes carrying derivatives of viral receptors we propose to make use of the high sensitivity of detection, the low amount of material needed, and the speed of analysis of capillary electrophoresis. This will be achieved by combining capillary electrophoretic separation with fluorescence detection to follow virus, receptor, viral proteins, and genomic RNA during attachment to, and uncoating at or within liposomes.