In vitro assembly of a viral envelope.

Viruses such as influenza and Ebola are enveloped in lipid bilayers annexed from host cells and containing glycoproteins essential for the infection process. At the molecular level little is known about the assembly process in terms of physical interactions between the lipids and glycoproteins. In this paper we assemble HIV glycoproteins in lipid vesicles in order to examine envelope assembly, a process that is usually only executed under control of a host cell. Using atomic force microscopy it was possible to observe fusion of individual envelope like particles, and contrast this with the behaviour of lipid vesicles without envelope glycoproteins. It was found that the inclusion of glycoproteins caused the vesicles to distort and that the subsequent fusion "footprint" with a lipid bilayer was related to the envelopes' unique morphology. This non-spherical morphology suggests that the presence of a viral capsid may be essential for the stability of an enveloped virus. Interactions between trans-membrane gp41 and gp120, the spikes protruding from a virion, were examined using supported lipid bilayers. Interactions between the gp120 and membrane-located gp41 resulted in the assembly of unusual molecular wires, one molecule in height and with a zigzag arrangement of gp120 molecules. In this work we have shown that purely physical/chemical interactions have dramatic effects on glycoprotein/lipid assembly and should be considered in the development of virus based technologies such as virosomes.

Lipid directed assembly of the HIV capsid protein.

Experimental evidence for in vivo capsid assembly suggests that capsid formation initiates from interactions between capsid (CA) proteins and lipids in the viral envelope. Various in vitro studies aiming to elucidate the detailed mechanisms of capsid self-assembly products have been carried out in conditions far removed from those, which would be encountered in a physiological environment. In this work we used lipid bilayers as a platform for studying the assembly of the CA protein with the rationale that the lipid-CA interactions play an important role in the nucleation of these structures. Observations using atomic force microscopy (AFM) have allowed a 'curling tadpole' mechanism to be suggested for the capsid self-assembly process. Stable dimeric CA proteins are able to move across the lipid bilayer to associate into trimers-of-dimers. These trimers form distinctly curved chains, which coil up to form larger features. As the feature grows additional trimers associate with the feature, giving a tadpole-like appearance. By comparing capsid assembly on mica, on single component lipid bilayers, and phase separated lipid bilayers, it was possible to determine the effect of lipid-protein interactions on capsid assembly.