Physicochemical tools for studying virus interactions with targeted cell membranes in a molecular and spatiotemporally resolved context

Figures

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  Fig. 1. Virus binding to cell membranes is a complex and highly dynamic process. (A) Viruses are known to present multiple copies of proteins (e.g., VP1 in the polyomavirus SV40) that are able to bind to receptors or attachment factors (AFs) presented by the cell’s plasma membrane. In order to achieve firm attachment to cell membranes, viruses typically have to bind many receptors/attachment factors in parallel, which leads to a very dynamic and complex binding process. The understanding of this process requires precise knowledge on the properties of the constituents (e.g., the mobility D of receptors/attachment factors in the membrane, the rate constants kon and koff of the molecular interaction between single viral proteins and their receptors/attachment factors) and the underlying biophysical laws, which can be obtained using biochemical and biophysical techniques (some of which are discussed in this review), but often requires to approximate the native situation by simplified model systems (e.g., by approximating virus-cell interactions by the interaction of virus-like particles with less complex lipid bilayers). Such investigations have, for example, enabled to generate a numerical…

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  Fig. 2. Bioanalytical methods to probe virus-membrane interactions. (A) A surface-based biosensor monitors in real time the change of an acoustic or optical signal upon adsorption of a virus particle or viral protein to surface-deposited receptors or cell surface mimics. In such an experiment, one of the binding partners is immobilized at the surface of a sensor chip while the other binding partner is added using a flow chamber. The acoustic or optical signal generated upon binding at the biosensing interface can be related to the amount of material deposited at the sensor surface. Analysis of the signal upon virus or protein adsorption together with analysis of the detachment behavior upon rinsing yield information on the ensemble-averaged interaction kinetics and affinity. (B) Binding of single viruses to membranes can be efficiently probed using total internal reflection fluorescence (TIRF) microscopy, which generates a very short-ranged evanescent excitation field at the interface, making it possible to visualize surface-bound fluorescently labeled viruses while discriminating them from the ones in solution. Analysis of the rate of arrival under equilibrium conditions yields…

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  Fig. 3. Probing the binding of norovirus virus-like particles (VLPs) to membranes using a quartz crystal microbalance with dissipation monitoring (QCM-D; A, B) and total internal reflection fluorescence (TIRF; C, D) microscopy. (A) QCM-D allows to probe the binding of viruses or lectins to receptor/attachment factor-presenting membranes by providing information on the adsorbed mass (measured in terms of a resonance frequency f, the value of which decreases with increasing mass) and the interfacial hydrodynamic friction (measured in terms of a dissipation D, which increases with increasing friction). As the friction strongly depends on the size of the adsorbed object, it is possible to distinguish binding events caused by viruses and proteins and thus to probe the release of membrane-bound viruses upon addition of a binding competitor having a higher affinity to the receptor/attachment factor (e.g., a lectin). This provides information on the binding strength of the virus-membrane interaction and (B) on the accumulation of receptors/attachment factors by the virus, which generates deviations between the relative virus surface coverage, determined during the initial binding process (B,…

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  Fig. 4. Probing the interaction of influenza A viruses (IAVs) with cells and attachment factors using dynamic force spectroscopy (DFS; A, B) and TIRF microscopy (C, D). (A) AFM-based DFS applied to the interaction of IAVs with cells showed mostly single rupture events, which are attributed to the unbinding of single viral proteins from sialylated structures on the cell’s plasma membrane. (B) The loading rate–dependent unbinding force showed only little variation across the different IAV strains and cell lines used, which is in line with previous studies showing only minor differences in the affinity to IAV’s protein hemagglutinin to α2,3- and α2,6-linked sialic acids [84]. Adapted figure with permission from (Sieben 2012); Copyright by the National Academy of Sciences. (C) TIRF microscopy applied to dye-labeled IAVs interacting with sialic acid–presenting supported lipid bilayers (SLBs) showed that due to the fluid nature of the membrane, IAVs can move laterally while being bound to the membrane (C, left). The speed of this process (quantified in terms of a diffusion coefficient) contains information on the number of attachment factors engaged by the IAV, which can be used to determine…

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  Fig. 5. Probing HSV-glycosaminoglycan (GAG) interactions using surface-immobilized GAGs. (A) A biomimetic GAG-platform based on the end-on immobilization of GAGs via biotin-streptavidin is used to probe the interaction between GAGs and viral glycoproteins or viruses. (B) Dynamic force spectroscopy (DFS) experiments are used to probe the virus-GAG binding strength. The histograms, showing the rupture force frequency recorded between CS and HSV-1-WT, are fitted with multiple Gaussian curves for different ligand-receptor interactions (top) and then compared for HSV-1-WT and HSV-1-gC-Δmuc (bottom). Black-shaded areas show higher binding frequencies for HSV-1-WT, while the gray-shaded areas correspond to higher binding frequencies for HSV-1-gC-Δmuc. The overall higher binding force for the mutant virus translates into a smaller koff (reproduced with permission from [98]). (C) Single particle tracking (SPT) with total internal reflection fluorescence (TIRF) microscopy is used to determine the diffusion coefficient of individual GAG-binding viruses. (i) The distribution of the diffusion coefficient is broad, spanning over several orders of magnitudes. (ii) Studies of the diffusion behavior…

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  Fig. 6. HIV-1 binding and fusion events observed using confocal fluorescence and TIRF microscopy. (A) Scheme showing cell membrane derived giant plasma membrane vesicle (GPMV) formation with lipid ordered (Lo=red) and disordered (Ld=blue) phases and HIV-1 binding preferentially to the Lo/Ld boundaries. (Ai) Number of HIV-1 particles bound to Lo, Ld, and Lo/Ld. Corresponding representative confocal images are shown and described in panels below. (Aii) The bright-field image shows GPMVs formed and attached to cells. (B) Confocal micrographs of GPMVs depict how, at higher resolution of selected rectangle, CD4 (immunolabeled) distributes into Lo phase, separated from Dil staining (lipid anchoring dye, known to distribute into the Ld phase). (C) Fluorescent micrographs showing the distribution of HIV-1 (labeled green with mKO-Gag) at the single particle level binding to the Lo phase and possibly to Lo/Ld boundaries but less to the Ld phase (labeled with DiO). (D) Scheme of formation of a supported planar plasma membrane (SPPM) and its application for dynamic imaging of HIV-1 binding and fusion using TIRF microscopy. (E) TIRF micrographs showing how differently CD4 distributes into the Lo…