Integrins alpha1beta1 and alpha2beta1 are receptors for the rotavirus enterotoxin

Abstract

Rotavirus NSP4 is a viral enterotoxin capable of causing diarrhea in neonatal mice. This process is initiated by the binding of extracellular NSP4 to target molecule(s) on the cell surface that triggers a signaling cascade leading to diarrhea. We now report that the integrins alpha1beta1 and alpha2beta1 are receptors for NSP4. NSP4 specifically binds to the alpha1 and alpha2 I domains with apparent K(d) = 1-2.7 muM. Binding is mediated by the I domain metal ion-dependent adhesion site motif, requires Mg(2+) or Mn(2+), is abolished with EDTA, and an NSP4 point mutant, E(120)A, fails to bind alpha2 integrin I domain. NSP4 has two distinct integrin interaction domains. NSP4 amino acids 114-130 are essential for binding to the I domain, and NSP4 peptide 114-135 blocks binding of the natural ligand, collagen I, to integrin alpha2. NSP4 amino acids 131-140 are not associated with the initial binding to the I domain, but elicit signaling that leads to the spreading of attached C2C12-alpha2 cells, mouse myoblast cells stably expressing the human alpha2 integrin. NSP4 colocalizes with integrin alpha2 on the basolateral surface of rotavirus-infected polarized intestinal epithelial (Caco-2) cells as well as surrounding noninfected cells. NSP4 mutants that fail to bind or signal through integrin alpha2 were attenuated in diarrhea induction in neonatal mice. These results indicate that NSP4 interaction with integrin alpha1 and alpha2 is an important component of enterotoxin function and rotavirus pathogenesis, further distinguishing this viral virulence factor from other microbial enterotoxins.

Fig. 1.

Dose-dependent binding of NSP4 to immobilized α2 I as measured by ELISA and SPR. (A) Increasing concentrations of SA11 NSP4_FL were incubated with immobilized α2 I or BSA with either 1 mM Mg²⁺ or 1 mM EDTA. NSP4_FL binds to α2 I with 1 mM Mg²⁺ (filled circles), whereas it does not with 1 mM EDTA (open circles). NSP4_FL does not react with BSA in either 1 mM Mg²⁺ (filled triangles) or 1 mM EDTA (open triangles). (B) Representative profiles of the relative SPR response of the binding of increasing concentrations of SA11 NSP4_FL to immobilized α2 I. y axis values are resonance units (RU) normalized to the maximum resonance units of NSP4_FL to α2 I. “Resp. Diff.” refers to “Response Difference.” This is a quantitative calculation in RU that is made by subtraction of the baseline response from the absolute response of the binding.

Fig. 2.

Localization of the binding region on NSP4 to α2 I determined by SPR. Representative profiles of the relative SPR response of the binding of various NSP4 proteins and peptides to immobilized α2 I. (A) Increasing concentrations of SA11 NSP4_112–175 were applied to immobilized α2 I. (B) Similar concentrations (4.2–4.6 μM) of SA11 NSP_FL, OSU-v NSP4_FL, OSU-a NSP4_FL, OSU-v NSP4_Δ131–140, and SA11 NSP4_E120A were applied to immobilized α2 I. (C) Increasing concentrations of NSP4_pep114–135 were applied to immobilized α2 I. y axis values are resonance units (RU).

Fig. 3.

Cell adhesion and spreading on NSP4_FL is mediated by α2β1 integrin. (A) Twenty-four-well plates were coated with increasing concentrations of collagen (open circles), SA11 NSP4_FL (filled circles), or BSA (open triangles). Then 1.5 × 10⁴ C2C12-α2 cells per well were added, and bound C2C12-α2 cells were fixed, stained with crystal violet, and quantitated by measuring OD₅₉₀. Specificity of the binding was shown by using soluble α2 I to block the cell attachment (filled squares). (B) Twenty-four-well plates were coated with collagen, SA11 NSP4_FL, NSP4_112–175, and BSA. Then 1.5 × 10⁴ C2C12-α2 or C2C12 cells per well were seeded and allowed to spread. C2C12-α2 cells were fixed and stained, and the morphology of spread cells was analyzed by inverted light microscopy. The morphology of C2C12-α2 cells seeded onto BSA-coated wells and C2C12 cells seeded onto all substrate-coated wells was directly observed by inverted light microscopy without washing, fixation, or staining. (C) A 96-well plate was coated with collagen. Then 5 × 10³ C2C12-α2 cells per well were first incubated with 2.1 nmol of NSP4_pep114–135 or NSP4_pep150–175 for 60 min and then added into collagen-coated wells. The attached cell numbers were counted after fixation and staining.

Fig. 4.

C2C12-α2 adhesion and spreading on different NSP4 proteins. Ninety-six-well plates were coated with collagen, BSA, or various NSP4 and NSP4 mutants. C2C12-α2 cells (5 × 10³ cells per well) were applied and allowed to attach and spread. Attached and spread cells were fixed, stained, and counted by using an inverted microscope. (A and B) Attached and spread C2C12-α2 cells on protein-coated wells, respectively.

Fig. 5.

Effect of anti-NSP4 peptide antibodies to C2C12-α2 attachment and spreading on NSP4. (A) Ninety-six-well plate was coated with NSP4_FL alone, NSP4_FL with preimmune IgG, NSP4_FL with anti NSP4_pep114–135 antibody, or NSP4_FL with anti- NSP4_pep120–147 antibody. Then 5 × 10³ C2C12-α2 cells per well were applied and incubated. Attached cells were fixed, stained, and counted under a light microscope. Collagen and BSA were used as controls. The y axis indicates the number of attached cells. (B) The percentage of spread cells versus attached cells on NSP4_FL with preimmune IgG (filled triangles) and anti-NSP4_pep120–147 antibody (filled circles). + and ++ represent protein amounts of 10 and 20 μg per well, respectively.

Fig. 6.

Colocalization of NSP4 with integrin α2 in RV-infected polarized Caco2. (A–F) Polarized Caco2 cells grown on Transwell membranes were mock infected (A–C) or RV-infected (D–F), fixed, permeabilized, and stained as described in SI Materials and Methods, Method 4. Integrin α2 is detected in A and D with anti-integrin α2 and Alexa Fluor 488-conjugated antibodies (green). NSP4 is analyzed in B and E with anti-NSP4_114–135 and Alexa Fluor 594-conjugated antibodies (red). (C and F) Merged images of A and B or D and E, respectively. (F) Colocalization of NSP4 (red) with integrin α2 (green) on the lateral membrane of noninfected cells is indicated by a yellow arrow in the XY section, which corresponds to the yellow arrow in the Z section. Lack of NSP4 detection on the lateral membrane of distal noninfected cells is indicated by a white arrow in the XY section, which corresponds to the white arrow in the Z section. (a) Apical surface. (b) Basolateral surface.

Fig. 7.

PLC-specific inhibitor U-73122 and PI3K inhibitor wortmannin do not affect C2C12-α2 cell adhesion, but disrupt C2C12-α2 cell spreading. Ninety-six-well microtiter plates were coated with NSP4_FL. C2C12-α2 cells (5 × 10³ cells per well) were mixed with various amounts of U-73122, its analog U-73343 as control, or wortmannin and then added to the wells and allowed to attach and spread for 120 min. Attached and spread cells were fixed, stained, and counted by using an inverted microscope. (A) Attached cell numbers. (B) Percentages of spread C2C12-α2 among the attached cells in the various concentrations of U-73122 or U-073343, respectively. (C) Percentage of spread C2C12-α2 in the presence of 3 μM U-73343 or 1 μM wortmannin.