Integrin activation decreased EV1 binding to cellular α2β1. CHO-α2 and CHO-α2E318W cells were allowed to adhere to immobilized EV1 or collagen I for 15 min. Adhered cells were detected using WST-1 reagent. Integrin activation significantly (P<0.001) increased cell adhesion to collagen I. At the same time, EV1 seemed to favour CHO-α2WT cells (P<0.05). Mean values±s.d. of eight parallel measurements are shown. Statistical significances were determined by two-tailed Student's t-test.

Activation of p38 after α2β1 clustering by antibodies or natural ligands requires E336-dependent conformational changes in α2 subunit. (A) CHO-α2 and CHO-α2E336A cells were sub-cultured onto collagen I (Col I) or fibronectin-coated cell culture plates. After o/n incubation, cells were lysed and p38 phosphorylation (P-p38) was detected by immunoblotting. CHO-α2 cells showed higher p38 activation on collagen I than on fibronectin. Fibronectin seemed to activate p38 even in CHO-α2E336A cells; however, the E336A mutation inhibited α2β1 integrin-mediated p38 activation induced by collagen I. (B, C) Similarly, E336A caused a dramatic decrease in p38 activation after antibody-mediated α2 clustering both in (B) CHO-α2E336A and (C) Saos-α2E336A cells, whereas the p38 phosphorylation was induced in CHO and Saos cells expressing α2WT. Immunoblots and quantified data from representative experiments are shown. The mean level of p38 activation is shown relative to (A) total p38 or (B, C) β-actin levels.

α2β1 clustering, not the activation of p38 or E336-mediated conformational activation of the receptor, is required for the EV1 entry. (A) In Saos-α2 cells EV 1 induced cluster formation of α2 integrins in 15 min as indicated by the co-localization image (scale bar, 10 μm). Manders' co-localization coefficients are 0.52 for integrin and 0.51 for EV1 and P=1.0. (B) A 15-min EV1 clustering does not, however, induce p38 activation similar to that seen in antibody-mediated clustering in Saos-α2 cells (C). In both Saos-α2 and Saos-α2E336A cells, 30-min EV1 clustering induced a clear PKCα activation. (B, C) An immunoblot and quantified data (mean±s.e.) of five or three representative experiments are shown. Untreated cells or cells treated with α2 primary antibody (16B4) only were used as controls. (D) When the ability of EV1 to infect Saos-α2 and Saos-α2E336A was studied, both EV1 and α2β1 were labelled, and EV1-positive cells were manually calculated from confocal microscopy images. The E336A mutation introduced into α2 subunit seemed not to prevent EV1 entry into Saos cells. Data shown are mean values±s.d. of EV1-positive cells (%).

Clustering of α2β1 integrins induces a rapid transient phosphorylation of p38. (A, bottom) Volume renderings of confocal image data show that secondary antibodies (goat anti-mouse IgG) were able to induce integrin clustering in Saos-α2 cells treated with α2 primary antibody (Alexa Fluor 555-conjugated 16B4). (A, top) Without secondary antibodies, no clustering occurs. In both cases, the same living cell was imaged at 0- and 15-min time points. (B) When p38 phosphorylation (P-p38) induced by the antibody (16B4 and anti-mouse IgG)-mediated α2β1 clustering was analysed in Saos-α2 cells at successive time points, a rapid and transient p38 phosphorylation, peaking at 15 min, was obvious. (C) Similarly, antibody-mediated clustering caused p38 activation even in CHO-α2 cells in 15 min. Representative immunoblots of one experiment (B, C) and statistical analyses of scanned blot images of one (C) or five (B) independent experiments are shown. Mean levels of phosphorylated p38 (P-p38)±s.d. relative to total p38 or β-actin levels are shown. (D) In addition to immunoblotting, flow cytometric analysis of Saos-α2 cells showed that neither α2-specific primary nor secondary antibody alone caused p38 activation, whereas the combination of the antibodies elicited p38 phosphorylation. For analysis, cells were treated with clustering antibodies for 15 min, fixed with isopropanol, permeabilized with methanol and stained with Alexa Fluor 488-conjugated phospho-p38 antibody.

Structural models of the binding of α2β1 integrin clusters to EV1. (A) Five α2β1 heterodimers (different shades of orange and red) in the extended conformation are shown bound to the five EV1 capsid (light blue) protomers around one fivefold symmetry axis. α2I domains are drawn in green and the transmembrane helices in dark green. The position of the membrane is marked with a grey bar. The position and orientation of the unoccupied α2I domain-binding sites on the virus surface (one in each of the 60 protomers) is marked with green L-shaped lines. (B) Five α2β1 heterodimers in the bent conformation are shown bound to five protomers (forming a pentamer) around a fivefold symmetry axis. This arrangement results in the five integrins packing very closely together and against the membrane, but it may still be sterically feasible. The α2I domain and the transmembrane helices are connected to the rest of the integrin structure by highly flexible linkers that may allow a bent conformation that can fit in the neighbouring binding sites of one pentamer. (C) Five α2β1 heterodimers in the bent conformation, each bound to one protomer in five adjacent pentamers around the EV1 capsid. A grey line marks the position of the plasma membrane. The transmembrane helices of the five integrins are positioned such that the membrane would be required to be substantially curved around the virus. The same arrangement, looking down an axis perpendicular to the membrane, is shown in (D). In this pentagonal arrangement, the distance from the intracellular end of one transmembrane helix to corresponding part in the proximal, neighbouring integrin is approximately 320 Å and to a distal integrin is approximately 500 Å.

EV1 favours inactive α2β1 integrin. (A) A structural model of the human α2β1 integrin head region was built based on the crystal structures of the αVβ3 integrin and the α2I domain. (A; bottom, left) Modelling indicates that the closed conformation of the α2I domain does not form specific contacts with the β1I domain (blue). (A; bottom, right) However, when the α2I domain adopts an open conformation, E336 at the C-terminal end of helix α7 is positioned in such a way that it could coordinate to the metal ion (yellow) of the MIDAS in the β1I domain and act as an intrinsic ligand. E309 does not seem to participate in the process. (B) In a cell spreading assay, CHO-α2 and CHO-α2E309A cells attached and spread on a collagen I (Col I) matrix in 120 min, whereas α2E336A mutation caused a dramatic decrease in the spreading. Cells were not able to attach to or spread on BSA, which was used as a negative control. (C) When CHO-α2 and CHO-α2E336A cells were allowed to adhere to immobilized EV1 or collagen I for 15 min, the E336A mutation seemed to decrease α2-mediated cell adhesion to collagen I. Interestingly, EV1 seemed to favour CHO-α2E336A cells. Mean values±s.d. of four parallel measurements are shown (B, C). (D) However, at 15 min, CHO-α2 and CHO-α2E336A cell adhesion to both collagen I and EV1 was significantly increased in the presence of integrin cluster-inducing TPA (100 nM; black columns). Mean values±s.d. of four parallel measurements are shown.

EV1 prefers the closed conformation of the α2I domain. (A) The crystal structure of the α2I domain in the closed (left) and the open conformations (right). Both conformations are also shown docked onto the surface of EV1 on the basis of the structure of the α2I–EV1 complex determined by cryo-EM (bottom). The surfaces of two protomers of the EV1 capsid are shown, one coloured light blue and the other light grey. The fivefold symmetry axis of the icosahedral capsid is labelled ‘5'. The regions in α2I that undergo the most extensive conformational changes (grey arrows) are the α7 helix (green), the βE-α6 loop (orange; includes the αC helix in the closed conformation) and the MIDAS (the metal ion in yellow). The position of amino-acid E318 is indicated. Residues that have been shown by mutagenesis to affect EV1 binding are coloured red (residues 199–201, 212–216 and 289), and the residues that are within 4.0 Å of the virus structure in the model of the complex are coloured blue. Most of the regions of the α2I involved in the conformational changes are not in close contact with the virus. Microtitre plates were coated with (B) collagen I (Col I) or (C) EV1. Delfia^® Diluent II (BSA; Δ) was used as a background control. The GST fusion proteins, containing wild-type α2I (▪) or high affinity mutant α2I E318W (O) domains, were allowed to react with the ligand for 1 h in the presence of 2 mM MgCl₂. Bound αI domains were detected with Eu³⁺-labelled GST antibody, and the signal was measured using time-resolved fluorescence. Data are presented as mean values±s.d. of triplicate measurements. Approximate K_d values for αI domain binding were obtained by fitting the binding data for αI domain concentrations series to a Michaelis–Menten form equation. (D) BIAcore analyses of the interactions between 1 μM α2I WT (solid line) or α2I E318W (dashed line) with immobilized EV1 are presented as overlaid sensograms. After a short (120 s) association phase, the measurement of the dissociation was continued for 25 min. (E, F) Binding of α2I WT (▪) and α2I E318W (O) to immobilized EV1 was competed with increasing concentrations of soluble (E) EV1 (0.04–0.7 nM) or (F) triple helical GFOGER peptide (0.01–1000 μM) that mimic a high-affinity integrin-binding site on collagen by using Eu³⁺-based solid phase binding assay described above (B, C). Results were fitted to the model representing a dose–response curve with variable Hill slope.