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

Abstract

<jats:title>Abstract</jats:title><jats:p>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.</jats:p><jats:sec><jats:title>Statement of Significance</jats:title><jats:p>Influenza 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.</jats:p></jats:sec>

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

“…Virus was pelleted in HB buffer by centrifugation at 21130 rcf for 50 min and resuspended in fresh HB buffer. DNA-lipids were incorporated into the IAV envelope by incubating virus sample at 4˚C on ice overnight as previously described (22,26). IAV is a BSL-2 agent and was handled following an approved biosafety protocol at Stanford University.…”

“…1. In a microfluidic flow cell (see SI Methods for details), glass slides were functionalized as previously described (26). Next, 5 µL of IAV in HB buffer (roughly 5.4 nM) displaying DNA sequence A' (antisense to A) were introduced to the flow cell and tethered to the substrate.…”

“…To avoid the process of adding a self-quenched concentration of fluorescently-tagged lipids to the viral envelope, we previously created a new version of the single-virus lipid mixing assay where the experimental readout for viral fusion is fluorescence dequenching of lipid-labeled target vesicles. Using this assay, we found that that target membrane cholesterol enhances the efficiency of influenza A virus (IAV) lipid mixing but has no effect on the rate (26). Here, we present a single-virus content mixing assay that utilizes content-labeled vesicles to eliminate the difficult process of content-labeling virions.…”

Kinetic Modeling of West Nile Virus Fusion Indicates an Off-Pathway State

“…As with other virus families (Floyd et al, 2008;Ivanovic et al, 2013;Kim et al, 2017;Liu and Boxer, 2020;Otterstrom et al, 2014;Rawle et al, 2016;Yang et al, 2015Yang et al, , 2017, single virus fusion experiments have been applied fruitfully to study fusion of flaviviruses (Chao et al, 2014(Chao et al, , 2018Rawle et al, 2018). In these studies, single virus fusion events are collected by fluorescence microscopy, typically observing pH-triggered lipid exchange between fluorescently labeled virions or virus-like particles (VLPs) and model lipid membranes as surrogates for the host cell membrane.…”

Enhanced Cholesterol-dependent Hemifusion by Internal Fusion Peptide 1 of SARS Coronavirus-2 Compared to its N-terminal Counterpart

“…16 The role of cholesterol in membrane fusion is firmly established from the results obtained from viral and model membrane fusion. [17][18] Cho-lesterol is also known to promote oligomerization of the SARS-CoV FP. 19 The lipid stalk hypothesis assumes that the sequential evolution of the intermediates toward the opening of fusion pore.…”