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First published online 19 October 2004
doi: 10.1242/jcs.01425

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The rotavirus surface protein VP8 modulates the gate and fence function of tight junctions in epithelial cells

¹ Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (CINVESTAV), Mexico D.F. 07000, Mexico
² Department of Developmental Genetics and Molecular Physiology, Institute of Biotechnology, National University of Mexico (UNAM), Cuernavaca, Morelos 62250, Mexico

View larger version (14K): [in a new window]   Fig. 1. Viral protein VP8 alters the transepithelial electrical resistance (TER) of epithelial monolayers (MDCK) in a reversible and dose dependent manner. (A) TER was determined in MDCK monolayers receiving 4 µg/ml of GST-VP5 (open squares) or GST-VP8 (closed triangles). In all the panels of this figure the closed squares correspond to the TER values obtained with MDCK monolayers treated with 4 µg/ml of GST. Monolayers that had been cultured for 30 minutes with 4 µg/ml of GST-VP8 (closed triangles) were washed (arrow) and transferred to media without GST-VP8 (open triangles). (B) MDCK monolayers incubated with 0.4 µg/ml (open circles), 4 µg/ml (closed triangles) or 10 µg/ml (open triangles) of GST-VP8. (C) At different times (1-5 days) after plating at confluence, the epithelial monolayers were treated with control media (gray bars) or 4 µg/ml of GST-VP8 added for 30 minutes (black bars) and 2 hours (white bars). Statistical significance was determined using a one-way ANOVA test. *P<0.05; **P<0.001; ***P<0.0001. (D) Confluent MDCK monolayers cultured for 20 hours in LC (1-5 µM Ca2+) were transferred to NC (1.8 mM Ca2+) (closed squares). Some monolayers were instead transferred to NC media containing either 4 µg/ml of VP5 (open squares) or of VP8 (closed triangles). In all these Figures, the values of TER reported as media±s.e.m. were normalized as explained in the Materials and Methods section. At each experimental point the number of monolayers on which the TER was measured is indicated.

View larger version (10K): [in a new window]   Fig. 2. VP8 disturbs the gate function of the tight junction (TJ) as determined by the paracellular passage of non-ionic tracers. MDCK cells were plated at confluency on Transwells and grown for 3 days. Paracellular flux of 4 kDa FITC-Dextran (FD4), and 70 kDa FITC-Dextran (FD70) was measured in the apical to basolateral direction. The amount of tracer diffusion was normalized to control MDCK cells. The data of six monolayers are reported as the mean±s.e.m. Open columns=control monolayers, closed columns=monolayers treated for 1 hour with 4 µg/ml GST-VP8, broken columns=monolayers treated for 1 hour with 4 µg/ml GST-VP5. Statistical significance was determined using the ANOVA test. *P<0.05; **P<0.001; ***P<0.0001.

View larger version (105K): [in a new window]   Fig. 3. VP8 perturbs the fence function of the tight junction (TJ). In all the experiments presented here, cells were plated at confluency on Transwell inserts for 3 days. Some monolayers remained in control media, whereas others were treated for 1 hour with 4 µg/ml of GST or GST-VP8, or for 10 minutes with 1.8 mM EGTA. The membrane distribution of the fluorescent markers was determined by z-sectioning on a confocal microscope. Bar, 8 µm. (A) Bodipy®-sphingomyelin-BSA complex diffusion assay. Fluorescent lipid/BSA was loaded to the apical surface. Arrows indicate lateral membrane staining. (B) Movement of the apical membrane protein GP135 (green) to the lateral membrane. In this and the following images arrows indicate occludin (blue) staining in the TJ region of control and GST-treated monolayers. The arrowheads in GST-VP8 and EGTA-treated monolayers denote the protein that has moved to the opposite plasma membrane. (C) Movement of the basolateral membrane protein Na+K+-ATPase (green) to the apical surface. (D) Redistribution of ß3 integrin (green) from the basolateral to the apical surface. (E) Another set of monolayers treated in the same manner was employed for selective surface membrane labeling (AP, apical; BL, basolateral) with activated biotin. Densitometric values of ß1 integrin labeling are expressed as mean percentages of the total (apical plus basolateral)±s.e.m. Immunoblots are representative of those from three separate experiments.

View larger version (123K): [in a new window]   Fig. 4. VP8 induces changes in the tight junction (TJ) strand organization observed by freeze-fracture. The upper panel shows representative freeze-fracture images of control (A) and VP8 (4 µg/ml) treated monolayers (B). In the latter several loose ends (arrowheads) are found between regions where the network is profound and complex (asterisks). P=protoplasmic face; E=exoplasmic face; Bar, 200 nm. The lower panel corresponds to the morphometric results obtained from 1239 and 1124 TJ sites analyzed for the control (white bars) and VP8-treated monolayers (black bars), respectively. The distribution of the number of TJ strands and the total amount of junction were studied as described in the Materials and Methods section.

View larger version (82K): [in a new window]   Fig. 5. VP8 treatment does not alter the protein content of the tight junction (TJ) proteins ZO-1, occludin and claudin-3, but modifies the distribution pattern observed by immunofluorescence. (A) Confluent MDCK monolayers were incubated in media with 4 µg/ml of GST or GST-VP8 or kept in control media. After one hour, the monolayers were fixed and processed for immunofluorescence. Bar, 40 µm. (B) Western blot detection of ZO-1, claudin-3 and occludin in the Triton X-100 soluble and insoluble fractions, obtained from monolayers cultured in control media, treated for 1 hour with 4 µg/ml of GST or GST-VP8.

View larger version (20K): [in a new window]   Fig. 6. VP8 facilitates the passage of molecules through the intestinal epithelium in an animal model. (A) Experiments were performed either in rats that had previously received an intraperitoneal injection of streptozotocin (75 mg/kg of body weight) to induce them to become diabetic (closed squares) or a mock injection (closed triangles). (B) Rats with streptozotocin-induced diabetes received insulin (Humulin, intermediate action) parenterally (6 IU; continuous lines) or orally (30 IU; broken lines). (C) Rats with streptozotocin-induced diabetes orally received VP8 (100 µg) (broken lines) or VP8 (100 µg) and insulin (30 IU) (continuous lines). Blood samples were taken after overnight fasting (time=0) or at different times after the animals received their diet. Each line corresponds to the results obtained with one animal.

View larger version (25K): [in a new window]   Fig. 7. The sequence of VP8 contains several regions similar to segments present in the external loops of occludin and claudins. The shadowed segments of the VP8 sequence have 50% identity to regions of the extracellular loops of occludin or claudin. The name of the similar protein is indicated next to the brackets (e.g. claudin 2, cl 2; occludin, occl, etc.).

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