Fusion of Epstein-Barr virus with epithelial cells can be triggered by αvβ5 in addition to αvβ6 and αvβ8, and integrin binding triggers a conformational change in glycoproteins gHgL

Abstract

Fusion of herpesviruses with their target cells requires a minimum of three glycoproteins, namely, gB and a complex of gH and gL. Epstein-Barr virus (EBV) fusion with an epithelial cell requires no additional virus glycoproteins, and we have shown previously that it can be initiated by an interaction between integrin αvβ6 or αvβ8 and gHgL. We now report that integrin αvβ5 can also bind to gHgL and trigger fusion. Binding of gHgL to integrins is a two-step reaction. The first step, analyzed by surface plasmon resonance, was fast, with high association and low dissociation rate constants. The second step, detected by fluorescence spectroscopy of gHgL labeled at cysteine 153 at the domain I-domain II interface with the environmentally sensitive probes acrylodan and IANBD, involved a slower conformational change. Interaction of gHgL with neutralizing monoclonal antibodies or Fab' fragments was also consistent with a two-step reaction involving fast high-affinity binding and a subsequent slower conformational change. None of the antibodies bound to the same epitope, and none completely inhibited integrin binding. However, binding of each decreased the rate of conformational change induced by integrin binding, suggesting that neutralization might involve a conformational change that precludes fusion. Overall, the data are consistent with the interaction of gHgL with an integrin inducing a functionally important rearrangement at the domain I-domain II interface.

Fig. 1.

Cartoon of the structure of gHgL. The four domains of gHgL are indicated, as are the positions of the flap structure, the CL59 epitope, and the region of the protein thought to contain the E1D1 epitope. The position of C153 is shown, and gL is depicted in gray.

Fig. 2.

Integrin αvβ5 in addition to integrins αvβ6 and αvβ8 binds to gHtgL with single-exponential kinetics and triggers cell-to-cell fusion mediated by gHgL and gB. SPR analysis was performed to evaluate binding of different concentrations of gHtgL to integrin αvβ5 (A), integrin αvβ6 (B), integrin αvβ8 (C), and integrin αvβ3 (D) immobilized to sensor chips by antibody. (E) Differences between the theoretical fit to a single-exponential function and experimental data (residuals) for the formation portion of each SPR trace at different concentrations of gHtgL added to αvβ5. (F) Fusion of CHO-K1 cells transfected with plasmids expressing gB and gHgL and overlaid for 20 h with integrins. Cells were fixed and stained with MAb to gB, and the percentage of cells expressing gB that contained 4 or more nuclei (% fusion) was calculated.

Fig. 3.

Linear plot of k_obs^on versus gHtgL concentration for the gHtgL interaction with αvβ5, αvβ6, or αvβ8. Cognate but not scrambled peptides inhibited binding. Sensorgrams show gHtgL binding to αvβ5 (A and D), αvβ6 (B and E), and αvβ8 (C and F) submitted to global fit analysis (12 to 24 traces at a time), with each point representing a result from one analysis by the global fit function of BIAevaluation. Closed circles in upper panels represent binding of gHtgL alone to integrins. Open circles in lower panels represent binding in the presence of scrambled peptide, and squares represent binding in the presence of cognate peptide. Correlation coefficients for the least-squares fits are 0.97 (A), 0.99 (B), and 0.96 (C). In panels D to F, the correlation coefficient for the scrambled peptide is 0.99, and those for the cognate peptide are 0.99, 0.93, and 0.99, respectively.

Fig. 4.

Binding of integrin to acrylodan-labeled gHtgL or IANBD-labeled gHtgL induces an increase in fluorescence and a shift in the fluorescence emission peak. (A) Flow cytometric analysis of binding to AGS cells of acrylodan-labeled gHtgL (dashed line) and unlabeled gHtgL (solid line). The dotted line represents the isotype control. (B) Corrected fluorescence emission spectra at an excitation wavelength of 359 nm and an emission wavelength of 380 to 600 nm for acrylodan-labeled gHtgL alone (1) and 5 min (2), 60 min (3), and 120 min (4) after addition of αvβ8. (C) Corrected time-based emission of acrylodan-gHtgL after addition of αvβ6, measured at an excitation wavelength of 359 nm and an emission wavelength of 504 nm (1) or 540 nm (2). (D) Corrected time-based emission of acrylodan-gHtgL after addition of αvβ5, measured at an excitation wavelength of 359 nm and an emission wavelength of 504 nm (1) or 540 nm (2). (E) Fluorescence emission at an excitation wavelength of 505 nm and an emission wavelength of 515 to 707 nm for IANBD-labeled gHtgL alone (1) and 5 min (2), 30 min (3), and 60 min (4) after addition of αvβ8.

Fig. 5.

Increase in fluorescence of acrylodan-labeled gHtgL after addition of integrin is not a result of protein aggregation. (A) Overlay of emission spectra of unlabeled gHtgL alone and 5 min and 60 min after addition of αvβ8. (B) Overlay of emission spectra of acrylodan-labeled gHtgL measured immediately (1) or after 1 h (2). (C) Time-based emission fluorescence of acrylodan-labeled gHtgL after addition of αvβ8. At the time point indicated by the arrow, the reading was stopped, the sample was centrifuged at 16,000 × g for 15 min, and the sample was returned to the cuvette for resumption of the reading. (D) Overlay of emission spectra of acrylodan-labeled gHtgL alone and 5 min, 60 min, and 120 min after addition of αvβ3. Fluorescence was recorded at an excitation wavelength of 359 nm and an emission wavelength of 380 to 600 nm (A, B, and D) or an excitation wavelength of 359 nm and an emission wavelength of 540 nm (C).

Fig. 6.

Binding of MAb against gH or gHgL to gHtgL fits a single-exponential function. SPR analysis was performed to determine the interaction between gHgL and immobilized E1D1 (A), CL40 (B), or CL59 (C). (D) The differences between the theoretical and experimental data (residuals) for the formation portion of each SPR trace of binding to E1D1 were fitted to a single-exponential equation.

Fig. 7.

Binding of gHtgL to immobilized E1D1 or CL59, but not to CL40, is affected by prebinding of gHtgL with MAbs to gHtgL, and only E1D1 has an effect on binding to an integrin. (A) Binding to immobilized E1D1 of gHtgL alone (4) or gHtgL complexed with E1D1 (1), CL40 (2), or CL59 (3). (B) Binding to immobilized CL59 of gHtgL alone (4) or gHtgL complexed with CL59 (1), E1D1 (2), or CL40 (3). (C) Binding to immobilized CL40 of gHtgL alone (4) or gHtgL complexed with CL40 (1), E1D1 (2), or CL59 (3). (D) Binding to immobilized αvβ8 of gHtgL alone (4) or gHtgL complexed with E1D1 (1), CL40 (2), or CL59 (3).

Fig. 8.

Binding of E1D1, CL40, CL59, or Fab′ fragments of antibodies induces an increase in fluorescence of acrylodan-labeled gHtgL. Emission spectra are shown for an excitation wavelength of 359 nm and an emission wavelength of 380 to 600 nm for acrylodan-labeled gHtgL alone (1) and 3 min (2) and 30 min (3) after addition of CL40 (A), CL59 (B), E1D1 (C), or Fab′ fragments of CL40 (D) or CL59 (E).

Fig. 9.

Preincubation of acrylodan-labeled gHtgL with whole antibody or Fab′ fragments of CL40 or CL59 alters the subsequent ability of an integrin to increase fluorescence. Time-based emission is shown for acrylodan-labeled gHtgL preincubated for 100 min at 25°C with CL40 (1), CL59 (2), or no antibody (3) (A) or with Fab′ fragments of CL40 (1) or CL59 (2), no antibody (3), or antipeptide antibody to gH (4) before addition of αvβ8 (B).