The SARS coronavirus E protein interacts with PALS1 and alters tight junction formation and epithelial morphogenesis

Abstract

Intercellular tight junctions define epithelial apicobasal polarity and form a physical fence which protects underlying tissues from pathogen invasions. PALS1, a tight junction-associated protein, is a member of the CRUMBS3-PALS1-PATJ polarity complex, which is crucial for the establishment and maintenance of epithelial polarity in mammals. Here we report that the carboxy-terminal domain of the SARS-CoV E small envelope protein (E) binds to human PALS1. Using coimmunoprecipitation and pull-down assays, we show that E interacts with PALS1 in mammalian cells and further demonstrate that the last four carboxy-terminal amino acids of E form a novel PDZ-binding motif that binds to PALS1 PDZ domain. PALS1 redistributes to the ERGIC/Golgi region, where E accumulates, in SARS-CoV-infected Vero E6 cells. Ectopic expression of E in MDCKII epithelial cells significantly alters cyst morphogenesis and, furthermore, delays formation of tight junctions, affects polarity, and modifies the subcellular distribution of PALS1, in a PDZ-binding motif-dependent manner. We speculate that hijacking of PALS1 by SARS-CoV E plays a determinant role in the disruption of the lung epithelium in SARS patients.

Figure 1.

Interaction of SARS-E and PALS1 proteins. (A) Sequence of the cytoplasmic tail (CT) of SARS-E protein that was used as the bait for yeast-two-hybrid screening. NT, amino-terminus; TM1–2, transmembrane domains. (B) Schematic representation of clones 67, 131, and full-length PALS1. Functional domains are listed below and identified by different shapes. Among 28 PALS1 cDNA clones isolated, #67 (panel a) and #131 (panel b) encoded the smallest and largest PALS1 cDNA fragments, respectively. Numbers indicate amino acid position. (C) SARS-E binds to PALS1 in mammalian epithelial cells. Vero E6 cells were either mock transfected or transfected with plasmids expressing Flag-PALS1 and E protein, alone or in combination. Forty-eight hours post-transfection, cells were lysed and Flag-PALS1 was immunoprecipitated with anti-Flag M2 antibodies conjugated to agarose resin (lanes 2–5). Samples were separated by gel electrophoresis (4–12% acrylamide) and proteins revealed by immunoblotting (IB) using either mouse monoclonal anti-Flag M2 antibody (panel a) or rabbit anti-E serum (panel b). SARS-E protein was coimmunoprecipitated with Flag-PALS1 from cotransfected cells (lane 5) whose input is shown in lane 1. The molecular mass (in kDa) and migration of protein standards are indicated on the left edge of each gel. Results shown are representative of two independent experiments.

Figure 2.

Characterization of PALS1 functional domains that interact with SARS-E protein. (A) Schematic representation of PALS1 clone 131 and truncation mutants fused to glutathione-S-transferase (GST) at the N-terminus. Functional domains are depicted by different shapes as in Figure 1B. (B) The PDZ domain of PALS1 is necessary and sufficient to bind to SARS-E in vitro. Purified GST-PALS1 (clone 131) and its truncation mutants linked to sepharose beads were incubated overnight at 4°C with cell lysates of Vero E6 cells transiently expressing E protein. Two assays were performed in parallel for each GST-PALS1 construct with increasing amounts (0.5 μg or 1.0 μg) of fusion protein, as indicated by the triangles above each blot. Beads were washed five times with cell lysis buffer and E protein was analyzed by SDS-PAGE and immunoblotting (IB) using a rabbit anti-E serum. The molecular mass (in kDa) and migration of protein standards are indicated on the left edge of each gel. SARS-E was pulled down only by beads linked to constructs containing the PDZ domain of PALS1, whereas no signal was seen in mock-transfected cells or when control beads were used (a). Results shown are representative of two independent experiments.

Figure 3.

Infection with SARS-CoV causes redistribution of PALS1 proteins to the ERGIC/Golgi region in Vero E6 cells. (A) SARS-CoV–infected Vero E6 cells were fixed, permeabilized, and labeled with antibodies against the SARS-S (S), SARS-E (E), PALS1, and ERGIC-53 (ERGIC, a protein marker of this compartment), as indicated. Images shown were single focal plane images and were acquired with a Zeiss LSM 510 Axiovert 200M confocal microscope. SARS-S and SARS-E colocalized in the ERGIC (panels a–c, white arrows). PALS1 was mainly present at cell-cell contacts (panels d–f, red arrows) and at the ERGIC (panel f, white arrowhead), where it colocalized with S protein (arrowheads in panels d and e) in infected cells. This cytoplasmic distribution of PALS1 was conspicuously absent from cells expressing little or no SARS-S (asterisks in panels d and e). (B) SARS-CoV-infected Vero E6 cells were fixed, permeabilized, and labeled with antibodies against the PALS1, Calnexin (ER), Golgin-97 (trans Golgi), and ZO-1 (tight junctions), as indicated. Images were acquired with a ZEISS Axio Observer Z1 fluorescent microscope. PALS1 did not redistribute to the ER (panel a). However, it colocalized with Golgin-97 (panel b, white arrows) whereas ZO-1 remained at cell to cell contacts (panel c, white arrowhead) and did not colocalize with the cytoplasmic accumulated PALS1 (panel c, white arrow). Bar, 10 μm.

Figure 4.

SARS-E protein possesses a PDZ domain-binding motif (PBM) at its carboxy-terminus. (A) Pull-down assay. Purified GST-PALS1 fusion proteins (clone 131, PDZ and SH3 domains; see Figure 2A) linked to sepharose beads were incubated overnight at 4°C with lysates of HEK 293T human epithelial cells that transiently expressed either full-length (wt) or a truncated (ΔPBM) SARS-E protein containing a hemagglutinin (HA) tag at the N-terminal position. Two assays were performed in parallel for each construct with increasing amounts (0.5 μg or 1.0 μg) of fusion protein, as indicated by the triangles above each blot. Beads were washed five times with cell lysis buffer and E protein was analyzed by SDS-PAGE and immunoblotting (IB) using a mouse anti-HA serum. Deletion of PBM abolished the interaction with PALS1 (cf. #131 and PDZ in panels a and b). The SH3 construct was used as the negative control. (B) Coimmunoprecipitation assay. Cells were transfected with the combination of plasmids as indicated on top. Forty-eight hours posttransfection, cells were lysed and proteins immunoprecipitated with anti-Flag M2 antibodies conjugated to agarose resin (panels c and d). Samples were separated by gel electrophoresis (4–12% acrylamide), and proteins were revealed by immunoblotting (IB) using either anti-Flag M2 or anti-HA mouse monoclonal antibodies. Deletion of PBM resulted in an almost complete disruption of the interaction between SARS-E and PALS1 (cf. lanes 5–6 in panel d). (C) Competition assay. 1 μg of purified GST-PDZ fusion protein linked to sepharose beads was preincubated for 6 h at 4°C with DMSO, E, or CRB3 CT peptides in DMSO (200 μM and 1 mM concentrations, indicated by a triangle above the blots). Pull-down of Myc-CRB3 and HA-E (wt) from cell lysate of transfected HEK293T human epithelial cells was analyzed subsequently using a rabbit anti-CRB3 serum (panel a) and a mAb anti-HA (panel b), respectively. E and CRB3 CT peptides could interfere with CRB3 protein interaction with GST-PDZ (cf. lanes 4–7 in panel a). Conversely, CRB3 CT peptide, but not E CT peptide, competed with HA-E (wt) interaction with GST-PDZ (cf. lanes 4–7 in panel b). In all panels, the molecular mass (in kDa) and migration of protein standards are indicated on the left edge of each gel. Results shown are representative of two independent experiments.

Figure 5.

MDCKII cyst morphogenesis is altered by expression of SARS-E protein. Single MDCKII, eGFP-PALS1 cells were embedded into cell culture medium supplemented with 4% GelTrex matrix and incubated at 37°C for five days until cysts developed. Mature cysts were fixed, permeabilized, and stained with antibodies against ZO-1 (a marker of tight junctions), the apical protein GP-135 and hemagglutinin (HA) tag at the amino terminus of HA-E (wt/ΔPBM) proteins, as indicated. Images were acquired with an inverted confocal microscope (Zeiss LSM 510 Axiovert 200M). (A) Control cells stably expressing eGFP-PALS1 formed cysts with a single lumen. (B) In contrast, transfection with SARS-E (wt; upper panels) or ΔPBM (lower panels) resulted in the formation of cysts with multiple lumens, as shown in these examples. Occasionally, a low fraction of E (wt) could be detected with eGFP-PALS1 at the apical site of the cell (white arrow). Bar, 10 μm. (C) Qualitative analysis of cyst formation by MDCKII cells ectopically expressing the indicated constructs. The number of single lumen cysts with preserved polarity is expressed as percentage of total count of cysts scored. Results are shown as means ± SEM of the specified number of replicates from three independent experiments. For each cell line, we have counted total of 870–885 cysts. ***p < 0.001 by the unpaired Student's t test.

Figure 6.

SARS-E (wt), but not ΔPBM, delays the development of transepithelial electrical resistance (TER) in polarized monolayers of MDCKII epithelial cells. MDCKII and eGFP-PALS1 cells were seeded onto Transwell filters and grown to confluence for five days. Cells were then incubated at a low Ca²⁺ concentration (5 μM) for twenty-four hours to disrupt cell–cell junctions, and subsequently switched to normal growth medium (1.8 mM Ca²⁺). The restoration of cell junctions was monitored by measuring TER (Ohms/cm²) as a function of time. Cells expressing SARS-E (wt) significantly perturbed the establishment of TJ. The maximal TER value was reached two hours post-calcium switch for both control and SARS-E (ΔPBM) cells and only after seventeen hours for SARS-E (wt) cells. Results are shown as means ± SEM of nine observations from one representative experiment and have been corrected for background.

Figure 7.

Tight junction formation is delayed in MDCKII cells expressing HA-E (wt) protein, but not in cells expressing the HA-E (ΔPBM) mutant protein. MDCKII, eGFP-PALS1 cells grown to confluence on polyester membrane filter were transferred into low calcium medium (5 μM) for twenty-four hours to disrupt cell–cell junctions and then switched to normal growth medium (1.8 mM Ca²⁺). Cells were fixed at different time points (t = 0, 1, 2, 4, 6, 8 h) post-calcium switch, permeabilized and stained with antibodies against ZO-1 (a marker of tight junctions), E-cadherin (a marker of adherens junction), the apical protein GP135 and hemagglutinin (HA) tag at the amino terminus of HA-E (wt/ΔPBM) proteins, as indicated. Images were acquired with a ZEISS LSM 510 Axiovert 200M confocal microscope. All images shown in this figure were taken from cells fixed at 2 h post-calcium switch. For A–C, two representative fields are shown. (A) PALS1 is localized at the tight junction, as confirmed by colocalization of ZO-1 (panel b, black color arrowhead). E-cadherin is at the lateral membrane of two adjacent cells, which marks the adherens junction. (B) SARS-E (wt) containing a hemagglutinin (HA) tag at its amino terminus position is localized at the perinuclear region (panels a and b). In these cells, PALS1 is partially localized at the cell–cell periphery, with little overlap with ZO-1 (panel b). Tight junction formation is disrupted, as indicated by discontinuous ZO-1 staining (panel b, white arrows). Interestingly, eGFP-PALS1 protein is partially colocalized with HA-E (wt) protein at the perinuclear region (panels a and b, white arrowhead). (C) HA-E (ΔPBM) mutant protein is diffused in the cytoplasm and localized at the subapical region (underneath GP135), as confirmed with the apical protein GP135 (panel a). This mutant protein did not colocalize with PALS1 in any subcellular compartment. PALS1 is distributed at the tight junction, as indicated by colocalization with ZO-1 (panel b, black color arrowhead). XZ and YZ are Z-section series along the X- and Y-axis, respectively.

Figure 8.

E expression causes mis-localization of PALS1 and alters the structure of MDCKII monolayers in a PBM-dependent manner. MDCKII and eGFP-PALS1 cells grown to confluence on polyester membrane filter were transferred into low calcium medium (5 μM) for 24 h to disrupt cell–cell junctions and then switched to normal growth medium (1.8 mM Ca²⁺). (A) The restoration of cell junctions was monitored by measuring TER (Ohms/cm²) as a function of time. Cells expressing SARS-E (wt) perturbed the establishment of TJ. The maximal TER value was reached 3 h post-calcium switch for both control and SARS-E (ΔPBM) cells, whereas TER kept rising moderately for SARS-E (wt) cells. At 24 and 120 h post-calcium switch TER values had stabilized at low levels for all cell lines. Results are shown as means ± SEM of nine observations from one representative experiment and have been corrected for background. (B) Cells were fixed at different time points (t = 2, 8, 24, and 120 h) post-calcium switch, permeabilized, and stained with antibodies against Giantin (a cis-Golgi marker) and HA tag, as indicated. HA-E (wt) colocalized with Giantin (white arrows). HA-E (ΔPBM) was present in the apical region of the cytoplasm. Round cells were observed in HA-E (wt) but not HA-E (ΔPBM) expressing cells (red arrows). At all time points and for both cell lines, a significant fraction of eGFP-PALS1 was observed at cell–cell contact, except in E (wt) expressing rounding cells. Thickness of the monolayer is indicated on the right for each condition (μm). (a and b) At 2 h, eGFP-PALS1 was enriched at the apical region of the cell–cell junctions in HA-E (ΔPBM) expressing cells (black arrowhead) while only partially distributed at cell-cell junctions in HA-E (wt) expressing cells. In these later cells, eGFP-PALS1 was often present in the cell cytosol and round cells were found (panel a, red arrow). (c and d) At 8 h, complete loss of junctions were occasionally observed for round cells with high cytoplasmic eGFP-PALS1 expression for the MDCKII eGFP-PALS1 HA-E (wt) cell line. (e and f) At 24 h, round cells frequently showed a higher expression of E (wt) and a portion of eGFP-PALS1 colocalized with E (white arrowhead). EGFP-PALS1 was predominantly at apical cell-cell contacts in MDCKII eGFP-PALS1 HA-E (ΔPBM) cells, although cells had an irregular shape. (g and h) eGFP-PALS1 was mainly present at cell–cell contacts, although cells have irregular shape in both cell lines at 120 h post-calcium switch. Round cells are found for the E(wt) expressing cellline (panel g, red arrow). Scale bar, 5 μm.

Figure 8.

E expression causes mis-localization of PALS1 and alters the structure of MDCKII monolayers in a PBM-dependent manner. MDCKII and eGFP-PALS1 cells grown to confluence on polyester membrane filter were transferred into low calcium medium (5 μM) for 24 h to disrupt cell–cell junctions and then switched to normal growth medium (1.8 mM Ca²⁺). (A) The restoration of cell junctions was monitored by measuring TER (Ohms/cm²) as a function of time. Cells expressing SARS-E (wt) perturbed the establishment of TJ. The maximal TER value was reached 3 h post-calcium switch for both control and SARS-E (ΔPBM) cells, whereas TER kept rising moderately for SARS-E (wt) cells. At 24 and 120 h post-calcium switch TER values had stabilized at low levels for all cell lines. Results are shown as means ± SEM of nine observations from one representative experiment and have been corrected for background. (B) Cells were fixed at different time points (t = 2, 8, 24, and 120 h) post-calcium switch, permeabilized, and stained with antibodies against Giantin (a cis-Golgi marker) and HA tag, as indicated. HA-E (wt) colocalized with Giantin (white arrows). HA-E (ΔPBM) was present in the apical region of the cytoplasm. Round cells were observed in HA-E (wt) but not HA-E (ΔPBM) expressing cells (red arrows). At all time points and for both cell lines, a significant fraction of eGFP-PALS1 was observed at cell–cell contact, except in E (wt) expressing rounding cells. Thickness of the monolayer is indicated on the right for each condition (μm). (a and b) At 2 h, eGFP-PALS1 was enriched at the apical region of the cell–cell junctions in HA-E (ΔPBM) expressing cells (black arrowhead) while only partially distributed at cell-cell junctions in HA-E (wt) expressing cells. In these later cells, eGFP-PALS1 was often present in the cell cytosol and round cells were found (panel a, red arrow). (c and d) At 8 h, complete loss of junctions were occasionally observed for round cells with high cytoplasmic eGFP-PALS1 expression for the MDCKII eGFP-PALS1 HA-E (wt) cell line. (e and f) At 24 h, round cells frequently showed a higher expression of E (wt) and a portion of eGFP-PALS1 colocalized with E (white arrowhead). EGFP-PALS1 was predominantly at apical cell-cell contacts in MDCKII eGFP-PALS1 HA-E (ΔPBM) cells, although cells had an irregular shape. (g and h) eGFP-PALS1 was mainly present at cell–cell contacts, although cells have irregular shape in both cell lines at 120 h post-calcium switch. Round cells are found for the E(wt) expressing cellline (panel g, red arrow). Scale bar, 5 μm.

Figure 9.

Model of the potential consequences of SARS-CoV infection on polarity and intercellular junctions formed by alveolar epithelial cells. (A) The interior surface of human lung alveolae is lined with a monolayer of polarized epithelial cells that organize themselves spherically around a central lumen. CRB and PAR polarity complexes are clustered to the apical domain to maintain and regulate apical polarity. Green, PALS1, a tight junction-associated protein. (B) A scheme illustrating a working model of sequential events that occur during SARS-CoV infection in alveolar epithelial cells. (a) Infection of alveolar epithelial cells by incoming viruses. The SARS-CoV virions attach to ACE2 receptors, which are localized at the apical surface. Virions are internalized into endosomes where the acidic pH triggers envelope fusion. The viral RNA (vRNA) is released into the cytoplasm and is transcribed to a set of subgenomic (sgmRNA) strands that encode for structural proteins S, M, N, E, and other accessory proteins. S, M, N, and E accumulate in the ERGIC compartment where virions assemble. At this stage, SARS-E could bind to PALS1 and disrupt its trafficking to TJ. (b) Disruption of TJ and virus dissemination. Loss of PALS1 at TJ results in a progressive disruption of TJ, which leads to leakage between adjacent epithelial cells, loss of barrier function, and infiltration of SARS-CoV virions into underlying tissues. Eventually, viruses reach the systemic circulation and disseminate to distant organs. Hijacking of PALS1 by SARS-E in infected pneumocytes may explain the severe alveolar damages observed in post-mortem lung biopsies from SARS-CoV–infected patients.