A fluorogenic pseudo-infection assay to probe transfer and distribution of influenza viral contents to target vesicles

Fusion of enveloped viruses with endosomal membranes and subsequent release of viral genome into the cytoplasm is crucial to the viral infection cycle. It is often modelled by carrying out fusion between virus particles and target lipid vesicles. We utilized fluorescence microscopy to characterize the kinetic and spatial aspects of the transfer of influenza viral ribonucleoprotein (vRNP) complexes to target vesicles and their distribution within the fused volumes to gain deeper insight into the mechanistic aspects of endosomal escape. The fluorogenic RNA-binding dye QuantiFluor® (Promega) was found to be well-suited for direct and sensitive microscopic observation of vRNPs which facilitated background-free detection and kinetic analysis of fusion events on a single particle level. To determine the extent to which the viral contents are transferred to the target vesicles through the fusion pore, we carried out virus-vesicle fusion in a side-by-side fashion. Measurement of the Euclidean distances between the centroids of super-localized membrane and content dye signals within the fused volumes allowed determination of any symmetry (or the lack thereof) between them as expected in the event of transfer (or the lack thereof) of vRNPs, respectively. We found that in case of fusion between viruses and 100 nm target vesicles, ~39% of the events led to transfer of viral contents to the target vesicles. This methodology provides a rapid, generic and cell-free method to assess the inhibitory effects of anti-viral drugs and therapeutics on the endosomal escape behavior of enveloped viruses.