The SARS Coronavirus E Protein Interacts with PALS1 and Alters Tight Junction Formation and Epithelial Morphogenesis

Teoh, Kim-Tat; Siu, Yu-Lam; Chan, Wing-Lim; Schlüter, Marc A.; Liu, Chia-Jen; Peiris, J. S. Malik; Bruzzone, Roberto; Margolis, Benjamin; Nal, Béatrice

To confirm this finding in human epithelial cells, coimmunoprecipitation experiments were performed with lysates of HEK 293T cells transfected with the appropriate combination of plasmid vectors pcDNA-Flag-PALS1, pcDNA-HA-E (wt) and pcDNA-HA-E (ΔPBM). As negative control, the cells were mock-transfected and individually transfected with these plasmids (Figure 4B, panels a and b). All proteins were expressed in transfected cells, although we observed that Flag-PALS1 protein levels were slightly lower in cells coexpressing HA E (wt) (Figure 4B, panel a, compare lanes 4, 5, and 6). Proteins were immunoprecipitated from cell lysates with anti-Flag M2 agarose resin and analyzed by immunoblotting. Membranes were hybridized with either the mouse monoclonal anti-Flag M2 HRP-conjugated or anti-HA tag antibody. As expected, the HA-E (wt) protein was efficiently coprecipitated with Flag-PALS1 (Figure 4B, panels c and d, lane 5). In contrast, only trace amounts of HA-E (ΔPBM) mutant protein were coimmunoprecipitated by Flag-PALS1 (Figure 4B, panels c and d, lane 6). The binding ability of SARS-E protein to PALS1 was reduced by ∼85% by deleting the putative PBM formed by the last four amino acids of the CT tail. Neither HA-E (wt) nor HA-E (ΔPBM) mutant protein was coimmunoprecipitated from the negative control cell lysates (Figure 4B, panels c and d, lanes 2–3). Altogether, these findings strongly suggest that the D-L-L-V motif is a PDZ domain-binding motif, which mediates E binding to the PALS1 PDZ domain, both in vitro and in vivo. It also indicates that internal motifs other than the PBM sequence could mediate binding to either PALS1 or other proteins that interact with PALS1 in human epithelial cells, albeit with lower affinity.