(473d) Stochastic Studies of Influenza Virus Fusion to Biomimetic Membranes

Influenza
viruses are membrane-enveloped, negative-strand RNA viruses that employ
membrane fusion to release its RNA into host cells and initiate replication.
Influenza enters the cell via clathrin-mediated endocytosis. Fusion of the
viral and endosomal membrane is facilitated by the conformational change of the
viral protein hemagglutinin (HA) at low pH. Traditional bulk fusion assays rely
on the fusion fluorescently labeled viruses to synthetic lipid vesicles to
obtain kinetic data about the fusion pathway. However, fusion is a stochastic
event and only ensemble averages of fusion kinetics are obtained from bulk
measurements. To obtain more detail, we use fluorescence dequenching and total
internal reflection microscopy (TIRFM) to track and quantify fusion of
individual viruses to supported lipid membranes. Imaging individual virus
fusion events enables hemifusion kinetics to be differentiated from pore
formation kinetics. Our assays are carried out in high-throughput microfluidic devices where fusion is initiated by reducing
the pH in the device. Hemifusion lag times are determined for each individual
fusing virus and from this data we can determine hemifusion rate constants (k_H) and the number of steps in the hemifusion
pathway (N). By using a distinct two-fluorophore labeling approach, we can also
measure the time to pore formation of individual viruses following the
hemifusion event. In this study we compare the fusion characteristics of strains
of influenza H3N2 that have undergone varying levels of laboratory adaptations.
Adaptations occur when strains of influenza, such as the commonly studied X:31, undergo multiple passages in eggs to improve virus
yield. Multiple passages in eggs result in strains that exhibit significant
morphological changes and altered receptor-specificity compared to circulating
strains. Our result shows that some less adapted strains exhibit a significant
shift in the optimal pH for endosomal fusion compared to the highly adapted X:31.