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 then hypothesized that E CT could compete with CRB3 CT, a natural ligand of PALS1 PDZ domain, and affect CRB3-PALS1 interaction. To verify this hypothesis, we designed two peptides corresponding to the CT tails of E (amino acids 34-76) and CRB3 (amino acids 80-120) and tested their capacity to interfere with the interaction between CRB3 and PALS1 PDZ domain in a GST-pull down assay (Figure 4C). Briefly, the peptides (0.2–1 mM in DMSO) were incubated with 1 μg of GST-PDZ fusion protein for six hours at 4°C. As control, GST-PDZ fusion proteins were incubated with DMSO in absence of peptide. The inhibitory complexes (GST-PDZ fusion proteins bound with E or CRB3 peptides) formed were not washed and cell lysate containing either myc-CRB3 or HA-E (wt) proteins were added immediately, followed by overnight incubation at 4°C. The precipitated interacting proteins were determined by immunoblotting using a rabbit anti-CRB3 serum (Figure 4C, panel a) and a mouse IgG1 monoclonal anti-HA antibody (Figure 4C, panel b), respectively.