Research ArticleBIOPHYSICS

Multivalent binding of herpesvirus to living cells is tightly regulated during infection

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Science Advances  17 Aug 2018:
Vol. 4, no. 8, eaat1273
DOI: 10.1126/sciadv.aat1273
  • Fig. 1 Schematic of HV and AFM probing of HV binding to living animal cells.

    (A) Schematic model of MuHV-4 particle, showing a lipid bilayer–derived envelope (blue) carrying multiple glycoproteins, a subset of which are involved either in the attachment (gp70, gp150, and gL/gH) to cell surface molecules or in the entry mechanism into cells (gB and gH). (B) Schematic of HV binding to GAGs and virus release (either after attachment or after cellular infection). Before cell entry, the virus must bind to attachment factors (GAGs, HS, etc.) to concentrate virus on the cell surface before the specific binding to entry receptors. Interactions with GAG moieties will be monitored at the single-virus level using AFM, with tips derivatized with a single virion.

  • Fig. 2 Extraction of average rupture forces of WT MuHV-4 binding for discrete LRs.

    (A) Schematic representation of FD-based AFM allowing interactions between an AFM tip functionalized with a MuHV-4 viral particle and a heparin-coated model surface to be probed. Heparin molecules were immobilized, taking advantage of the oriented and strong biotin-streptavidin noncovalent attachment. Representative FD curve showing a specific adhesion event, as detected by the shape of the adhesion peak, where the extension of the PEG linker can be fitted with the WLC model for polymer extension (39). Representative retraction force-time (FT) curve from which the LR applied by the AFM tip can be extracted via the slope of the curve on the adhesive peak just before bond rupture. (B) Force and LR were extracted from FD and FT curves and sorted in narrow LR ranges (LR1, 300 to 1000 pN/s; LR2, 1000 to 3000 pN/s; LR3, 3000 to 104 pN/s; LR4, 104 to 3 × 104 pN/s; LR5, 3 × 104 to 105 pN/s; LR6, 105 to 3 × 105 pN/s). The rupture forces for each LR range were plotted as histograms. Histograms are fitted with multipeak Gaussian fits. n = 2400 from eight independent experiments.

  • Fig. 3 Extraction of kinetic parameters of WT and gp150-deficient viral particles binding toward purified heparin molecules.

    (A) AFM tip carrying a single WT MuHV-4 virion and DFS plot reporting the average values of the force distributions for WT MuHV-4–heparin interactions, enabling analysis using the Bell-Evans fit (23). Dashed lines represent the predicted binding forces for two, three, and four uncorrelated interactions rupturing simultaneously, according to the Williams-Evans model. (B) AFM tip carrying a single MuHV-4 virion from which the glycoprotein gp150 was deleted and DFS plot reporting the average values of the force distributions (fig. S8) for gp150 MuHV-4–heparin interactions, enabling analysis using the Bell-Evans fit. Dashed lines represent the Williams-Evans prediction for the simultaneous rupture of uncorrelated bonds. (C) Comparison of the multipeak Gaussian curves fitting the rupture force histograms of MuHV-4 WT and gp150 binding to heparin. Red-shaded areas correspond to higher binding probabilities for WT virions, while blue-shaded regions show more frequent gp150 virion binding. Single bonds (SBs) occur more frequently with the WT viral particles, while multiple bonds (MBs) are more probable for gp150-deficient virions. Thus, gp150 is thought to play a role in the regulation of GAG binding by limiting multivalent linkages between viral particles and long GAG chains. (D) Schematic representation of either single glycoprotein (gL/gH or gp70) interaction with heparin occurring more frequently with WT virions or multiple interactions involving a combination of the two glycoproteins more frequently observed upon gp150 virion binding.

  • Fig. 4 Probing the role of gp150 in virion-glycan binding on living cells.

    (A) AFM height image of two adjacent CHO cells: a GAG+ (fluorescently tagged) cell and an unlabeled GAG CHO cell. Inset: 30 μm × 30 μm fluorescent image of both cells. (B) Corresponding adhesion map, showing interactions between WT viral particles and CHO cells. The adhesion intensity is lower in GAG+ cells than in GAG cells. (C) Zoom on the adhesion map recorded on the GAG+ cell [see dashed square in (B)]. The upper map displayed the low forces (range, 100 to 200 pN), and the lower map showed the higher forces (range, 200–300 pN). (D) Representative FD curves extracted from the adhesion map shown in (B), showing either nonadhesive FD curves on both cell surfaces (black pixels) or adhesion events (white pixels) on GAG+ and GAG cells. Data are representative of 26 measurements from seven independent experiments. (E) Box plot of the relative binding frequency (GAG+/GAG) observed for WT and gp150 virions. (F) AFM height image, with fluorescence image in the inset showing two adjacent GAG+ and GAG cells. (G) Corresponding adhesion map recorded with gp150 virions. gp150 virions bind strongly to GAG+ cells in comparison to WT virions. Data are representative of 15 measurements from six independent experiments. (H) Zoom on the adhesion map recorded on the GAG+ cell [see dashed square in (G)]. The upper map displayed the low forces (range, 100 to 200 pN), and the lower map displayed the higher forces (range, 200–300 pN). (I) DFS plot with data obtained on purified heparin (Fig. 3A), where force distributions between WT virions and GAG+ cells are overlaid. Data in gray and black were obtained at tip oscillation frequencies of 0.125 and 0.250 kHz.

  • Fig. 5 Investigation of virus binding and release.

    (A) TEM images recorded 48 hours after infection at a multiplicity of infection of 2 using WT and gp150 virions on GAG+ and GAG cells. (B) ELISA tests monitoring virus binding to heparin-coated surfaces using anti-gN signal and titration using increasing concentration of soluble heparin. (C) Flow cytometry assay of virus binding to cell surfaces in the presence of increasing heparin concentration. MFI, mean fluorescence intensity. (D) Confocal microscopy images of fluorescent-tagged virions binding to cell surfaces in the presence of heparin.

  • Fig. 6 Free-energy landscape describing the first binding steps of MuHV-4 and gp150 to cell surfaces.

    (Left) After landing on the cell surface, the HV particle binds to GAGs. gp150 acts as a binding regulator by maintaining the number of foothold low, enabling the virus to diffuse laterally to seek for specific receptors. (Right) gp150 virion lacking this regulatory element increases its adhesion to cellular surfaces, preventing the virus from lateral diffusion. The multiples bonds, preventing the virus from lateral diffusion and release, kinetically and thermodynamically stabilize the virus.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/8/eaat1273/DC1

    Fig. S1. Principle of FD-based AFM to probe HV binding to living animal cells.

    Fig. S2. Characterization of MuHV-4 viral particles and validation of tip and surface functionalization.

    Fig. S3. Attachment and infection of GAG+ and GAG CHO cells.

    Fig. S4. Exploring a wide range of LRs with FD-based AFM.

    Fig. S5. Control experiments showing the specificity of MuHV-4 virus binding toward immobilized heparin.

    Fig. S6. Binding force distribution and multipeak Gaussian fit of different LR sets.

    Fig. S7. Multipeak Gaussian fit of average rupture forces of gp150 MuHV-4 binding extracted for discrete LRs.

    Fig. S8. Principle of combined optical and FD-based AFM imaging of animal cells to extract topography images and quantitative multiparametric maps.

    Fig. S9. Consecutive mapping of WT virus binding to CHO cells shows similar results.

    Fig. S10. Investigation of virus binding to cells using flow cytometry.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Principle of FD-based AFM to probe HV binding to living animal cells.
    • Fig. S2. Characterization of MuHV-4 viral particles and validation of tip and surface functionalization.
    • Fig. S3. Attachment and infection of GAG+ and GAG CHO cells.
    • Fig. S4. Exploring a wide range of LRs with FD-based AFM.
    • Fig. S5. Control experiments showing the specificity of MuHV-4 virus binding toward immobilized heparin.
    • Fig. S6. Binding force distribution and multipeak Gaussian fit of different LR sets.
    • Fig. S7. Multipeak Gaussian fit of average rupture forces of gp150 MuHV-4 binding extracted for discrete LRs.
    • Fig. S8. Principle of combined optical and FD-based AFM imaging of animal cells to extract topography images and quantitative multiparametric maps.
    • Fig. S9. Consecutive mapping of WT virus binding to CHO cells shows similar results.
    • Fig. S10. Investigation of virus binding to cells using flow cytometry.

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