272150 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 (kH) 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.