Liu, K. N. | bioRxiv Science

bioRxiv2021

Single-virus content mixing assay reveals cholesterol-enhanced influenza membrane fusion efficiency

In order to infect a cell, enveloped viruses must first undergo membrane fusion, which proceeds through a hemifusion intermediate, followed by the formation of a fusion pore through which the viral genome is transferred to a target cell. Single-virus fusion studies to elucidate the dynamics of content mixing typically require extensive fluorescent labeling of viral contents. The labeling process must be optimized depending on the virus identity and strain and can potentially be perturbative to viral fusion behavior. Here, we introduce a single-virus assay where content-labeled vesicles are bound to unlabeled influenza A virus (IAV) to eliminate the problematic step of content-labeling virions. We use fluorescence microscopy to observe individual, pH-triggered content mixing and content loss events between IAV and target vesicles of varying cholesterol compositions. We show that target membrane cholesterol increases the efficiency of IAV content mixing and decreases the fraction of content mixing events that result in content loss. These results are consistent with previous findings that cholesterol stabilizes pore formation in IAV entry and limits leakage following pore formation. We also show that content loss due to hemagglutinin fusion peptide engagement with the target membrane is independent of composition. This approach is a promising strategy for studying the single-virus content mixing kinetics of other enveloped viruses. Statement of SignificanceTo replicate, enveloped viruses, like influenza A virus, must successfully deliver their contents to a host cell through viral membrane fusion. Most single-virus fusion assays require extensive fluorescent labeling of virions which can be perturbative to fusion kinetics. Here, we utilize content-labeled vesicles in a single-virus content mixing assay, which eliminates the need to fluorescently label virus contents. We use this assay to show that target membrane cholesterol increases the fraction of stable influenza virus content mixing events. This assay also enables the study of target membrane destabilization due to viral fusion peptide engagement.

biophysics↗

bioRxiv2019

Target membrane cholesterol modulates single influenza virus membrane fusion efficiency but not rate

Host lipid composition influences many stages of the influenza A virus (IAV) entry process, including: initial binding of IAV to sialylated glycans, fusion between the viral envelope and the host membrane, and the formation of a fusion pore through which the viral genome is transferred into a target cell. In particular, target membrane cholesterol has been shown to preferentially associate with virus receptors and alter physical properties of the membrane like fluidity and curvature. These properties affect both IAV binding and fusion, which makes it difficult to isolate the role of cholesterol in IAV fusion from receptor binding effects. Here, we develop a new fusion assay that uses synthetic DNA-lipid conjugates as surrogate viral receptors to tether virions to target vesicles. To avoid the possibly perturbative effect of adding a self-quenched concentration of dye-labeled lipids to the viral membrane, we tether virions to lipid-labeled target vesicles, and use fluorescence microscopy to detect individual, pH-triggered IAV membrane fusion events. Through this approach, we find that cholesterol in the target membrane enhances the efficiency of single-particle IAV lipid mixing, while the rate of lipid mixing is independent of cholesterol composition. We also find that the single-particle kinetics of influenza lipid mixing to target membranes with different cholesterol compositions is independent of receptor binding, suggesting that cholesterol-mediated spatial clustering of viral receptors within the target membrane does not significantly affect IAV hemifusion. These results are consistent with the hypothesis that target membrane cholesterol increases lipid mixing efficiency by altering host membrane curvature. Statement of SignificanceInfluenza A virus is responsible for millions of cases of flu each year. In order to replicate, influenza must enter a host cell through virus membrane fusion, and cholesterol in the target membrane is vital to the dynamics of this process. We report a receptor-free, single virus fusion assay that requires no fluorescent labeling of virus particles. We use this assay to show that cholesterol increases the fraction of fusion events in a manner that is correlated with the spontaneous curvature of the target membrane but is independent of receptor binding. This assay represents a promising strategy for studying viral fusion processes of other enveloped viruses.

biophysics↗