Rotavirus infection reduces sucrase-isomaltase expression in human intestinal epithelial cells by perturbing protein targeting and organization of microvillar cytoskeleton

Abstract

Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. These viruses infect mature enterocytes of the small intestine and cause structural and functional damage, including a reduction in disaccharidase activity. It was previously hypothesized that reduced disaccharidase activity resulted from the destruction of rotavirus-infected enterocytes at the villus tips. However, this pathophysiological model cannot explain situations in which low disaccharidase activity is observed when rotavirus-infected intestine exhibits few, if any, histopathologic changes. In a previous study, we demonstrated that the simian rotavirus strain RRV replicated in and was released from human enterocyte-like Caco-2 cells without cell destruction (N. Jourdan, M. Maurice, D. Delautier, A. M. Quero, A. L. Servin, and G. Trugnan, J. Virol. 71:8268-8278, 1997). In the present study, to reinvestigate disaccharidase expression during rotavirus infection, we studied sucrase-isomaltase (SI) in RRV-infected Caco-2 cells. We showed that SI activity and apical expression were specifically and selectively decreased by RRV infection without apparent cell destruction. Using pulse-chase experiments and cell surface biotinylation, we demonstrated that RRV infection did not affect SI biosynthesis, maturation, or stability but induced the blockade of SI transport to the brush border. Using confocal laser scanning microscopy, we showed that RRV infection induces important alterations of the cytoskeleton that correlate with decreased SI apical surface expression. These results lead us to propose an alternate model to explain the pathophysiology associated with rotavirus infection.

FIG. 1

Enzyme activity and RRV antigen expression in Caco-2 cells infected with RRV. (A) At 24 h p.i., total membrane fraction was prepared and assayed for SI, DPP IV, γ-GTP (γ-Glu), APN, and AP activities. Each bar represents the mean ± SD of six experiments. ∗, statistically significant difference (P < 0.01). Additional determination of enzymatic activity indicates that SI activity decreased from 16 to 24 h (not shown). , control; □, RRV infected. (B) At the same times p.i., infected cells were fixed with 3% paraformaldehyde and permeabilized with Triton X-100. RRV antigens were immunostained with a polyclonal anti-group A rotavirus and fluorescein-labeled anti-rabbit IgG secondary antibodies (left). Infected monolayers were also examined by phase-contrast microscopy (right). The bar indicates 25 μm.

FIG. 2

Alteration in the apical SI distribution in RRV-infected Caco-2 cells. At 24 h p.i., RRV-infected and mock-infected Caco-2 cells were fixed and permeabilized as described for Fig. 1. (A) SI was stained with a rat anti-SI MAb and fluorescein-coupled anti-rat IgG secondary antibodies. (B) DPP IV was immunostained with a rat anti-DPP IV MAb and fluorescein-coupled anti-rat IgG secondary antibodies. Horizontal (XY) sections at the apical level and vertical (XZ) sections were obtained by direct confocal analysis. Each bar indicates 10 μm.

FIG. 3

SI biosynthesis, maturation, and stability in RRV-infected Caco-2 cells. At 16 h p.i., RRV-infected (I) and mock-infected (C) Caco-2 cells were pulse-labeled for 1 h with [³⁵S]methionine-cysteine and chased for 0, 1, 3, and 6 h. Cells were then extracted, and SI was detected by immunoprecipitation with an anti-SI MAb followed by SDS-PAGE (7.5% gel) and fluorography (A). (B) Densitometric quantification of the fluorogram shown in panel A. Values are means ± SD of three independent experiments.

FIG. 4

DPP IV biosynthesis, maturation, and stability in RRV-infected cells. After the immunoprecipitation with an anti-SI MAb (Fig. 3), the supernatants depleted of SI were immunoprecipitated with an anti-DPP IV MAb. The immunoprecipitates were then analyzed by SDS-PAGE on a 6 to 15% (A) or 7.5% (B) polyacrylamide gel without endo H treatment (panel B, lanes 1, 3, 5, 7, 9, and 11) or after endo H treatment (panel B, lanes 2, 4, 6, 8, 10, and 12) in order to differentiate the high-mannose DPP IV form from viral protein VP4 as described in Materials and Methods. After endo H treatment, the immature form of DPP IV became deglycosylated and its molecular mass decreased to 85 kDa, whereas the molecular mass of VP4 did not change. Labeled proteins were visualized by fluorography (black square, immature 100-kDa DPP IV; white square, deglycosylated 85-kDa DPP IV; black arrowhead, mature 110-kDa DPP IV; white arrowhead, viral protein VP4). I, RRV-infected Caco-2 cells; C, mock-infected Caco-2 cells.

FIG. 5

Delivery of SI to the apical surface is blocked by RRV infection. At 16 h p.i., RRV-infected (I) and mock-infected (C [control]) Caco-2 cells were pulse-labeled for 1 h with [³⁵S]methionine-cysteine and chased for 2, 3, or 6 h. The monolayers were then biotinylated on the apical surfaces and extracted, and SI reaching the apical surface was determined by immunoprecipitation, streptavidin precipitation, SDS-PAGE (7.5% gel), and fluorography. (A) SI apical arrival; (B) densitometric quantification of the fluorogram in panel A. Values are means ± SD of three independent experiments.

FIG. 6

Apical and basolateral delivery of DPP IV is delayed by RRV infection. After immunoprecipitation with an anti-SI MAb (Fig. 5), the supernatants depleted of SI were immunoprecipitated with an anti-DPP IV MAb. Apical (Ap) and basolateral (Bl) biotinylated DPP IV were subjected to streptavidin precipitation, SDS-PAGE (15% gel), and fluorography. After a 3-h chase, DPP IV was entirely processed to the 110-kDa complex glycosylated form and represented only by the upper 110-kDa band. The 100-kDa band represents the viral protein VP 4 (A). (B) Densitometric quantification of the DPP IV bands shown in panel A. Values are means ± SD of three independent experiments. C, control; I, infected.

FIG. 7

Three-dimensional F-actin alteration induced by RRV infection. At 24 h p.i., RRV-infected and mock-infected Caco-2 cells were fixed, permeabilized, and stained with fluorescein-phalloidin, which binds to F-actin. Horizontal sections were generated by CLSM along an axis perpendicular to the monolayer, at the apex (A), the middle (B), and the base (C) of the cell. Bars, 10 μm.

FIG. 8

Alteration of villin organization induced by RRV infection. At 24 h p.i., RRV-infected and mock-infected Caco-2 cells were fixed, permeabilized, and immunostained with polyclonal antivillin antibodies and fluorescein-labeled anti-rabbit IgG antibodies. Apical (A) and subcortical (B) horizontal sections were generated by CLSM. Bars, 10 μm.