Modulating the Influenza A Virus–Target Membrane Fusion Interface With Synthetic DNA–Lipid Receptors

Abstract

Influenza A virus (IAV) binds to sialylated glycans on the cell membrane before endocytosis and fusion. Cell-surface glycans are highly heterogeneous in length and glycosylation density, which leads to variations 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.

Lipid Membrane Interface Viewpoint: From Viral Entry to Antiviral and Vaccine Development

“…By tuning the amount of cholesterol in vesicles, the study found that cholesterol does enhance the (hemi)­fusion efficiency by presumably stabilizing the hemifusion state but does not affect the fusion rate. To ensure the fusion event, the target vesicles can be loaded with a self-quenching concentration to monitor the pore formation, and the tether length between the target vesicles and viral particles can be modulated to study the effects of receptor length and flexibility …”

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

“…To observe content mixing between virus and vesicle compartments, typically, a water-soluble dye is encapsulated at self-quenching concentration within the virus or target vesicle interior which turns on in fluorescence due to dilution upon fusion (14)(15)(16)(17). These assays have been popular because content label dyes like calcein and sulforhodamine B (SRB) are relatively inexpensive (an important practical consideration since only a small fraction of dye gets encapsulated during preparation of the target vesicles).…”

DNA-Tethered Lipid Membrane Formation via Solvent-Assisted Self-Assembly

“…Tethered lipid bilayers − have been developed to host membrane proteins in a fluid–lipid bilayer configuration while maintaining the stability, mobility, and controllability of planar supported lipid bilayers (SLBs). − Despite these advantages, SLBs interact strongly with their substrates and are therefore often incompatible with integral membrane proteins that can bind to their substrates. − Tethered lipid bilayers take advantage of chemical tethering to separate the model lipid membrane from solid supports, so membrane-associated proteins can function without interference. − Various methods have been applied, including polymer tethers, − protein tethers, tethered bilayer lipid membranes (tBLMs) with diphytanyl ethylene glycol anchors, − and DNA–lipid conjugates, − which have been used in a variety of self-assembled membrane structures by exploiting the well-defined DNA specificity and length. This superior controllability was demonstrated in a model system using the segregation pattern of mobile tethers, and in the study of membrane fusion .…”

“…This superior controllability was demonstrated in a model system using the segregation pattern of mobile tethers, and in the study of membrane fusion . The DNA-tethered lipid bilayers have been further developed to enable patterning, and to mimic the viral membrane fusion via synthetic DNA–lipid receptors . A wide range of analyses investigating ligand-gated ion channels, pore-forming peptides, and membrane pores can be conducted if the DNA-tethered lipid membrane fully covers an electrode surface.…”