We combine structure-based mutagenesis with imaging of influenza-virus membrane fusion in real time at the resolution of individual virions to reach previously inaccessible level of mechanistic understanding. We are also adapting these approaches to study of nonenveloped (reovirus) virus membrane penetration.
Home-built Total Internal Reflection Fluorescence (TIRF) microscope.
Experimental toolkit. (A) We form planar membrane bilayers on the surface of a glass microscope coverslip. The membranes incorporate glycolipids or glycoproteins displaying sialic-acid receptors that recruit virions. Virions on membranes are imaged under TIR. (B) Virons incorporate fluorescent dyes serving as reporters for fusion intermediates. Hundreds of individual virions are observed in a single imaging experiment. Single-particle detection and analyses permit extraction of lag times to fusion intermediates for individual virions over the course of membrane fusion.
Membrane Fusion of WT Influenza Virions at 20X the Actual Rate. White dots represent individual influenza virions incorporating fluorescent membrane-dye at a quenching concentration. Fusion reaction is triggered by low-pH buffer introduced using a flow-cell system. Virions initially move in the direction of the buffer flow and invariably stop (arrest) before fusion. Initial mixing of outer membrane leaflets (hemifusion) releases the dye from the viral into the target membrane and is observed as a spike in localized fluorescence followed by radial spreading of the dye 'cloud' from the site of membrane merger.
STOCHASTIC COMPUTER SIMULATIONS
Individual HAs within the target-membrane interface refold at random as a result of proton binding. They extend with a rate determined by the stability of the fusion peptides in their pre-fusion pockets. Productive HAs insert into into the target bilayer but cannot exert enough force to bring membranes together. They remain stretched out between the two membranes as ‘extended intermediates’. Membrane fusion ensues only after several successful insertion attempts happen by chance next to each other, so that HAs pulling on the two membranes can combine forces.
We employ reverse genetics systems for influenza (Neumann et al. 1999; Hoffmann et al., 2000) to generate viruses with site directed mutations in HA. We have previously generated a small panel of HA mutants, which was instrumental in deriving the new model of influenza membrane fusion. A key enabling step we have recently achieved is develop a method to generate non replicating viruses.
Viruses harboring deleterious
mutations in HA. MDCK or MDCK-HA (stably expressing
WT HA) cells were infected with a non replicating mutant virus. In
the absence of the ‘helper’ HA, the recovered
infectivity was below the detection limit (<100
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