Modulating the influenza A virus – target membrane fusion interface with synthetic DNA-lipid receptors

10.26434/chemrxiv-2021-cm9cl ◽

2021 ◽

Author(s):

Elizabeth Webster ◽

Katherine Liu ◽

Robert Rawle ◽

Steven Boxer

Keyword(s):

Cell Membrane ◽

Influenza A Virus ◽

Influenza A ◽

Viral Fusion ◽

Fusion Peptides ◽

Viral Receptor ◽

Synthetic Dna ◽

Rate Limiting Step ◽

Target Membrane ◽

Fusion Cell

Influenza A virus (IAV) binds to sialylated glycans on the cell membrane before endocytosis and fusion. Cell surface glycans are highly heterogenous in length and glycosylation density, which leads to variation in the distance and rigidity with which IAV is held away from the cell membrane. To gain mechanistic insight into how receptor length and rigidity impact the mechanism of IAV entry, we employed synthetic DNA-lipids as highly tunable surrogate receptors. We tethered IAV to target membranes with a panel of DNA-lipids to investigate the effects of the distance and tether flexibility between virions and target membranes on the kinetics of IAV binding and fusion. Tether length and the presence of a flexible linker led to higher rates of IAV binding, while the efficiencies of lipid and content mixing were typically lower for longer and more rigid DNA tethers. For all DNA tether modifications, we found that the rates of IAV lipid and content mixing were unchanged. These results suggest that variations in the interface between IAV and a target membrane do not significantly impact the rate-limiting step of fusion, or the low-pH triggered engagement of viral fusion peptides with the target membrane. However, our results imply that the flexibility of the viral receptor is important for ensuring that hemifusion events are able to successfully proceed to pore formation.

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New influenza A Virus Entry Inhibitors Derived from the Viral Fusion Peptides

PLoS ONE ◽

10.1371/journal.pone.0138426 ◽

2015 ◽

Vol 10 (9) ◽

pp. e0138426 ◽

Cited By ~ 11

Author(s):

Wenjiao Wu ◽

Dongguo Lin ◽

Xintian Shen ◽

Fangfang Li ◽

Yuxin Fang ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Virus Entry ◽

Viral Fusion ◽

Fusion Peptides ◽

Entry Inhibitors ◽

Virus Entry Inhibitors

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244. Synthetic DNA Vaccine Against Avian Influenza A Virus

Molecular Therapy ◽

10.1016/s1525-0016(16)36048-8 ◽

2012 ◽

Vol 20 ◽

pp. S96

Keyword(s):

Avian Influenza ◽

Influenza A Virus ◽

Dna Vaccine ◽

Influenza A ◽

Synthetic Dna ◽

Avian Influenza A Virus

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Rapalogs downmodulate intrinsic immunity and promote cell entry of SARS-CoV-2

10.1101/2021.04.15.440067 ◽

2021 ◽

Author(s):

Guoli Shi ◽

Abhilash I Chiramel ◽

Saliha Majdoul ◽

Kin Kui Lai ◽

Sudipto Das ◽

...

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Functional Divergence ◽

Severe Disease ◽

Drug Repurposing ◽

Mtor Inhibitors ◽

Cell Entry ◽

Viral Fusion ◽

Human Respiratory Tract ◽

The Impact

Infection by SARS-CoV-2 generally causes mild symptoms but can lead to severe disease and death in certain populations, including the immunocompromised. Drug repurposing efforts are underway to identify compounds that interfere with SARS-CoV-2 replication or the immunopathology it can elicit. Rapamycin is among those being currently tested in clinical trials for impacts on COVID-19 severity. While rapamycin and rapamycin analogs (rapalogs) are FDA-approved for use as mTOR inhibitors in multiple clinical settings, including cancer, we previously found that rapamycin can increase the susceptibility of cells to infection by Influenza A virus. In this study, we tested the impact of rapalogs on cellular susceptibility to SARS-CoV-2 infection. We report that rapamycin and rapalogs increased SARS-CoV-2 titers in human cervical epithelial and lung epithelial cell lines to different extents, and a similar pattern of enhancement was observed using pseudovirus incorporating viral fusion proteins from SARS-CoV-2, SARS-CoV, MERS, and Influenza A Virus. Rapalogs also promoted cell entry driven by SARS-CoV-2 Spike in nasal cells and primary small airway cells, representing proximal and distal ends of the human respiratory tract, respectively. Interestingly, cell entry enhancement by the rapalog ridaforolimus was cell type-dependent, revealing a previously unrecognized functional divergence between rapalogs. The differential activity of rapalogs was associated with their capacity to induce the degradation of interferon-inducible transmembrane (IFITM) proteins, restriction factors that broadly inhibit virus infection. Our findings will spur the development of mTOR inhibitors that do not suppress the first line of antiviral defense in cells.

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

10.1101/2021.04.26.441491 ◽

2021 ◽

Author(s):

Katherine N Liu ◽

Steven G. Boxer

Keyword(s):

Membrane Fusion ◽

Influenza A ◽

Pore Formation ◽

Fluorescent Labeling ◽

Fusion Pore ◽

Viral Fusion ◽

Enveloped Viruses ◽

Virus Assay ◽

Virus Content ◽

Target Membrane

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.

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

10.1101/2019.12.19.882738 ◽

2019 ◽

Author(s):

K. N. Liu ◽

S. G. Boxer

Keyword(s):

Membrane Fusion ◽

Influenza A Virus ◽

Receptor Binding ◽

Single Particle ◽

Influenza A ◽

Mixing Efficiency ◽

Membrane Cholesterol ◽

Host Membrane ◽

Target Membrane ◽

Viral Receptors

AbstractHost 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.

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