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 delineate the functional domain within PALS1 that mediates the interaction with E, we performed a GST pull-down assays with the longest PALS1 cDNA clone identified through the yeast-two-hybrid screen, clone 131, and a series of truncated mutants (Figure 2A). To test the biochemical interaction of E with functional domains of PALS1 in vitro, purified GST-PALS1 fusion proteins (0.5 μg and 1.0 μg) bound to sepharose beads were incubated overnight at 4°C with cell lysate from Vero E6 cells transiently expressing the E protein. Pulled-down proteins were analyzed by SDS-PAGE and immunoblotting. The GST-PALS1 (clone 131), GST-PALS1 (L27, PDZ, SH3), and GST-PALS1 (PDZ) proteins efficiently pulled down E protein from the cell lysate (Figure 2B, panel b, lanes 1–4, and panel d, lanes 1–2, respectively). The GST-PALS1 (L27, PDZ) fusion protein could pull down detectable levels of E only when 1.0 μg of fusion protein was used for the assay (Figure 2B, panel c, lanes 1–2). Conversely, the GST-fusion proteins consisting only of either the L27 or the SH3 domain could not precipitate the E protein (Figure 2B, panels c and d, lanes 3–4). No nonspecific binding to either sepharose beads or GST protein was detected (Figure 2B, panel a, lanes 3–4). Taken together, these results show that human PALS1 protein binds E in vitro and that the PDZ domain of PALS1 is both necessary and sufficient to mediate the interaction with E.