Virus binding and fusion to cell membranes





Introduction

             The time is ripe for the next pandemic influenza strain to emerge. Our ability to provide vaccine protection against flu is challenged because flu mutates so quickly and vaccines must be developed based on anticipated strain mutations from the prior season. Thus new vaccines must be developed and distributed each year. Young children and seniors are usually most susceptible to infection and are the likely recipients of flu shots because vaccines are always in short supply. However, population susceptibility to infection is not predictable, but varies by strain. Take for example the case of the 1918 pandemic, healthy young adults were most susceptible to infection and had higher fatality rates. Given the variability in infectivity, vaccine cannot be produced or distributed during an inevitable epidemic until the strain is identified and its infectivity fully characterized. Furthermore, if the wrong vaccine is developed in the prior year or an unanticipated strain suddenly emerges, it would provide limited or no protection from infection. Antiviral medicines offer another defense strategy except that the flu virus quickly develops resistances to these drugs. Therefore, in a pandemic it would be essential to have readily available tools for rapid identification of the virulent factors of the strain. These factors include optimal viral binding, proteolytic activation, rapid fusion of viral membrane with host cell, and rapid delivery of genetic material to the cytosol. It is important to consider that these factors can vary among various cell types, environmental conditions, and populations.

The Daniel group is creating tools to rapidly assess these factors using cell membrane mimics and single particle microscopy techniques.

A key factor in optimal virus binding to the cell is multivalency. This means that the virus has multiple ligand-receptor interactions, which greatly strengthens its adhesion to the cell membrane. The virus can make these connections because the receptors within the cell membranes diffuse in two dimensions and can reorganize to make proper attachments. In order to develop assays that truely mimic virus-cell adhesion, this important property of the cell membrane must be preserved.

            


We can create artificial planar analogs of the cell membrane on solid supports that can be used to study various biophysical phenomena, ranging from protein binding to membrane elasticity. These so-called "solid-supported bilayers" can be created on flat glass slides as well as inside microfluidic channels of intricate architecture. The SLBs self-assemble during vesicle fusion, creating contiguous bilayer films, as depicted in the diagram below. These SLBs preserve the very important property of membrane fluidity, so that membrane reactions and organizations can proceed like they do in real cells.


            


We use total internal reflection fluorescence microscopy to study virus binding and fusion. This technique is surface specific because only a thin (~100 nm) layer is illuminated by the evanescent wave that leaks into the flow cell during total internal reflection of the laser beam inside the prism. Therefore, only fluorophores within this thin region can be excited and and monitored. Since the lipid bilayer is coated on the walls of the flow cell, it is well within the evanescent wave and binding to it can easily be quantified using fluorescently-labeled virus.


            



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