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

We next verified the E-PALS1 biochemical interaction in mammalian epithelial cells by coimmunoprecipitation assay. Briefly, plasmid vectors encoding Flag-PALS1 or E were cotransfected into Vero E6 epithelial cells and expressed proteins were detected by specific antibodies (Figure 1C, panels a and b, lane 1). As negative controls, Vero E6 cells were mock-transfected or individually transfected with plasmids encoding either Flag-PALS1 or E. Forty-eight hours posttransfection, Flag-PALS1 proteins were immunoprecipitated from cell lysates with anti-Flag M2-conjugated agarose resin and coimmunoprecipitation of Flag-PALS1 and E was analyzed by immunoblotting using the anti-Flag M2 (Figure 1C, panel a) and a rabbit serum against the E carboxy-terminal region (Figure 1C, panel b). Flag-PALS1 was efficiently immunoprecipitated from cell lysates of transfected Vero E6 cells (Figure 1C, panel a, lane 4–5). The E protein was only found in immunoprecipitates from cells transiently expressing both Flag-PALS1 and E (Figure 1C, panels a and b, lane 5), thus confirming an interaction between these two proteins in mammalian epithelial cells.