Reovirus directly engages integrin to recruit clathrin for entry into host cells

Abstract

Reovirus infection requires the concerted action of viral and host factors to promote cell entry. After interaction of reovirus attachment protein σ1 with cell-surface carbohydrates and proteinaceous receptors, additional host factors mediate virus internalization. In particular, β1 integrin is required for endocytosis of reovirus virions following junctional adhesion molecule A (JAM-A) binding. While integrin-binding motifs in the surface-exposed region of reovirus capsid protein λ2 are thought to mediate integrin interaction, evidence for direct β1 integrin-reovirus interactions and knowledge of how integrins function to mediate reovirus entry is lacking. Here, we use single-virus force spectroscopy and confocal microscopy to discover a direct interaction between reovirus and β1 integrins. Comparison of interactions between reovirus disassembly intermediates as well as mutants and β1 integrin show that λ2 is the integrin ligand. Finally, using fluidic force microscopy, we demonstrate a functional role for β1 integrin interaction in promoting clathrin recruitment to cell-bound reovirus. Our study demonstrates a direct interaction between reovirus and β1 integrins and offers insights into the mechanism of reovirus cell entry. These results provide new perspectives for the development of efficacious antiviral therapeutics and the engineering of improved viral gene delivery and oncolytic vectors.

Fig. 1. Schematic of capsid composition of reovirus particles.

a–d Diagrams of cross-sections of reovirus virions, infectious subvirion particles (ISVPs), and cores. Schematics depict the arrangement and conformation of structural proteins in the double-layered shells of the virions in the absence (a) and presence (b) of Neu5Ac (triggering a conformational change of σ1); removal of σ3, cleavage of μ1, and rearrangement of σ1 in ISVPs (c); and removal of μ1 fragments, loss of σ1, and opening of the λ2 turret in cores (d).

Fig. 2. Reovirus directly engages β1 integrins in a cation-dependent manner.

Studies were conducted using  model surfaces (a–c) and living cells (d–g). a Experimental set up (left) to probe cation-dependent (right) virion-α2β1 integrin interactions using a model surface. b The adhesion map shows interaction forces (white pixels) between virions and integrins in the presence of Mn²⁺, which induces an extended integrin conformation. c Box plot of specific binding frequencies (BF) measured using AFM between virions and integrins in the presence of divalent cations and following injection of cRGD. d FD-based AFM setup to study T3SA+ binding to Lec2 cells, which express β1 integrins. Height (e) and corresponding adhesion maps (f) of the imaged area show specific binding of reovirus T3SA+ to living Lec2 cells in the presence of Mn²⁺ (white pixels). For better visibility, the pixel size in the adhesion image was enlarged two-fold. AFM images were acquired using an oscillation frequency of 0.25 kHz and amplitude of 750 nm under cell culture conditions. g Box plot shows BF of T3SA+ virions to β1 integrins on living Lec2 cells under conditions shown. The horizontal line within the box indicates the median, boundaries of the box indicate the 25th and 75th percentile, and whiskers indicate the highest and lowest values. Open square within each box indicates the mean. All data are representative of N = 5 independent experiments. P values were determined by two-sample t-test using Origin.

Fig. 3. Kinetic and thermodynamic insights into cation-dependent reovirus-integrin interactions.

Studies were conducted using AFM on model surfaces (a–e) and living cells (f–h) and using BLI (i). a Schematic of experimental set up to probe cation-dependent reovirus interactions with α2β1 integrin-coated model surface. b, c Dynamic force spectroscopy (DFS) plots show distribution of average rupture forces determined at eight distinct loading rate (LR) ranges for interactions between T3SA+ virions and β1 integrin-coated model surface in the presence of Mn²⁺ (for further details, see methods section) (b) or Mg²⁺ (c). Data corresponding to single interactions were fit with the Bell–Evans (BE) model (I, black curve), providing average k_off and x_u values. Dashed lines represent predicted binding forces for two (II) and three (III) simultaneous uncorrelated interactions (Williams-Evans [WE] prediction). d, e The binding probability is plotted as a function of the contact time. Least-squares fit of the data to a mono-exponential decay curve (line) provides average kinetic on-rate (k_on) of the probed interaction. Comparison of K_D (k_off/k_on) values shows that Mn²⁺ increases the affinity of T3SA+ virions for β1 integrins. f–h Assessment of cation-dependent reovirus interaction with integrins expressed on living cells. DFS plots of data obtained using model surfaces (gray circles and living cells (red dots) in the presence of either Mn²⁺ (g) or Mg²⁺ (h). Histogram of the force distribution observed on cells and a multi-peak Gaussian fit of data (n = 900 data points) are shown at the side. Error bars indicate s.d. of the mean value. All data are representative of N = 5 independent experiments. i Sensorgrams obtained using biolayer interferometry (BLI) show interaction of T3SA+ with β1 integrins immobilized on amine-reactive biosensors under conditions shown.

Fig. 4. Binding of the reovirus σ1 attachment protein to sialic acid alters β1 integrin interaction.

The effect of a point mutation in the SA-binding site of σ1 protein (c–e) or addition of exogenous α-SA (f–h) on reovirus-α2β1 integrin interactions was determined using model surfaces. a Schematic depicts the experimental set up. All experiments were conducted in the presence of Mn²⁺. b Box plot shows BF between T3SA+ or T3SA- virions and integrins quantified using AFM under the conditions shown. The horizontal line within the box indicates the median, boundaries of the box indicate the 25th and 75th percentile, and whiskers indicate the highest and lowest values. An open square within each box indicates the mean. c DFS plot shows interaction forces between T3SA- and integrins. d Comparison of forces required to rupture bonds between integrins and T3SA+ (green) or T3SA- (yellow). e BF plotted as a function of contact time shows comparable k_on and K_D values for T3SA+ (green) and T3SA- (yellow) interactions with integrins. f–h DFS plot (f) and kinetic on-rate measurements (h) show differences in T3SA+ interactions with β1 integrins in the presence (red) and absence (green) of α-SA (1 mM Neu5Ac) (g). Error bars indicate s.d. of the mean. All data are representative of N = 5 independent experiments. P values were determined by two-sample t-test using Origin.

Fig. 5. Analysis of β1 integrin interactions with reovirus disassembly intermediates suggests λ2 as the viral integrin ligand.

a Schematic depicts the experimental set up. All experiments were conducted in the presence of Mn²⁺. b Box plot of BF between β1 integrins and T3SA+ virions, ISVPs, cores, or IBM-1/-2 (integrin binding motif mutant) quantified using AFM under the conditions shown. The horizontal line within the box indicates the median, boundaries of the box indicate the 25th and 75th percentile, and whiskers indicate the highest and lowest values. An open square within each box indicates the mean. c–e Binding of T3SA+ ISVPs to β1 integrins (colored in blue) in comparison with virions before (colored in green) and after (colored in red) incubation with Neu5Ac. c DFS plot of the interaction forces between integrins and T3SA+ ISVPs show that ISVPs display a different kinetic bond rupture behavior compared with T3SA+ virions in the absence of Neu5Ac but the same behavior as T3SA+ virions in the presence of Neu5Ac (as shown in d). e k_on and K_D determined from measuring BF as a function of contact time indicates that the affinity of ISVPs for integrin is comparable to that of virions in the presence of Neu5Ac. f–h Binding of T3SA+ cores (colored in gray) to β1 integrins differs from that of T3SA+ virions (colored in green). Plots display DFS data (f), comparison of T3SA+ cores (gray) with virions (green) (g), and kinetic on-rate measurements (h). Control experiments are shown in Supplementary Fig. 5. Error bars indicate s.d. of the mean. All data are representative of N = 3 independent experiments. Ten AFM tips were analyzed per condition. P values were determined by two-sample t-test using Origin .

Fig. 6. Integrins function in clathrin recruitment by reovirus bound to cells.

Integrin-induced recruitment of clathrin (labelled with blue fluorescent protein [BFP]) at the plasma membrane following reovirus binding was quantified using Fluid-FM coupled with confocal microscopy. a Fluid-FM coupled to a confocal imaging setup. Fluorescent T3SA+ reovirus particles covalently coupled to gold-coated nanoparticles were trapped using a micro-channeled cantilever and brought in contact with clathrin-BFP-expressing CHO-JAM-A cells. Fast-scanning confocal microscopy was used to simultaneously visualize reovirus and clathrin fluorescence signals. b Representative time-lapse images from fluid-FM/confocal imaging show recruitment of clathrin (BFP, for better visibility shown in green instead of blue) to a reovirus (Alexa 647, red) contact site. c Recruitment of clathrin observed over time using blue fluorescence intensity around beads coated with T3SA+ virions in the absence (green) or presence of integrin-blocking peptides KGE and cRGD (black) or Neu5Ac (red). d Recruitment of clathrin observed over time using blue fluorescence intensity around beads coated with T3SA+ virions (green) and control particles ISVP (blue), IBM-1 (black), IBM-2 (gray), or Alexa 700 dye (purple). Representative images from control experiments are displayed in Supplementary Figs. 6 and 7. e Box plot shows the rate of clathrin recruitment calculated from the quantification of fluorescence over time (slopes of intensity vs. time curves fit to linear regressions). The horizontal line within the box indicates the median, boundaries of the box indicate the 25th and 75th percentile, and whiskers indicate the highest and lowest values. An open square within each box indicates the mean. Error bars indicate s.d. of the mean value. All data are representative of N = 10 independent experiments. P values were determined by two-sample t-test using Origin.

Fig. 7. Integrin interaction with λ2 reovirus outer capsid protein mediates clathrin-recruitment for entry into host cells.

a Crystal structure of the λ2 turret (pentamer, about 120 Å diameter and 80 Å tall), viewed from the top. Cryo-electron microscopy reconstruction of the virion is shown on the side^,. The five elongated λ2 monomers, four shown in green and one in gray, wrap around the other surface, with their long axes about 45° to the radial direction. b The λ2 monomer, viewed from the side (the gray monomer in a). β1 integrin binding sequences, RGD and KGE, are highlighted as red sticks. c Zoom-in on β1 integrin binding domain in λ2. Images are based on related crystal structure 1EJ6 [10.1038/35010041]. d Conclusive model: Virions efficiently bind β1 integrins (“high-avidity” binding), which mediates recruitment of clathrin for virus internalization. Virions in the presence of abundant free α-SA or ISVPs bind poorly to integrins (“low-avidity” binding), potentially because of steric hindrance from conformational changes in σ1. Consequently, they use alternate mechanisms for entry such as caveolin-mediated endocytosis, similar to virions lacking the β1 integrin domain (IBM).