A fluorogenic method to directly observe transfer and distribution of influenza viral contents to target vesicles.

Abstract

There is a long-standing interest in studying viral infection processes using bottom-up biophysical models to dissect various underlying molecular determinants. Fusion of enveloped viruses with cellular membranes and subsequent release of viral contents into the cytoplasm is a key event in the infection cycle. In case of influenza virus, fusion of the viral membrane is mediated by conformational changes of its surface protein hemagglutinin triggered by acidic pH within endosomes. Previously, content transfer assays have relied on fluorescence enhancement of an encapsulated self-quenched water-soluble dye upon fusion of the viral compartment with a target membrane. However these assays do not track the transfer of actual viral macromolecular contents, and suffer from drawbacks such as the need to use high concentration of the content dye which may have perturbative effects on the membranes. To overcome these limitations, we developed a method utilizing nucleic acid-binding dyes to directly observe the transfer of viral ribonucleoprotein complexes to target vesicles by total internal reflection fluorescence (TIRF) microscopy. The dyes were encapsulated within surface-tethered vesicles and the unlabeled virus particles were bound via sialic acid receptors. The fluorogenic nature of the dyes allowed background-free detection of fusion events when pH was dropped. We determined the kinetics of viral content transfer and found that it is slower than that of lipid mixing. We further employed a super-localization strategy to investigate to what extent the viral contents are transferred to the target vesicle through the fusion pore. Overall, our method allows observation of viral fusion events on a single-particle level and will provide the groundwork for development of biosensors for rapid quantification of fusogenic virus particles.