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

Bait cloning and yeast-two-hybrid screening were performed by Hybrigenics, S.A., Paris, France. In brief, the cDNA coding for the CT domain of SARS-CoV E was amplified by PCR and cloned into the pB27 vector enabling fusion with the LexA binding domain. The bait construct was checked by sequencing the entire insert and was subsequently introduced into the L40ΔGAL4 yeast strain (Fromont-Racine et al., 1997). A random-primed human placenta cDNA library that consists of ten million independent fragments was transformed into the Y187 yeast strain, and both strains were allowed to mate. In the screening, 70 millions interactions were tested with E CT. After selection on medium lacking leucine, tryptophane, and histidine, 146 positive clones were picked, and the corresponding prey fragments were amplified by PCR and sequenced at their 5′ and 3′ terminal ends. Sequences were then filtered and contiged as described previously (Formstecher et al., 2005) and compared with the latest release of the GenBank database using BLASTN. A Predicted Biological Score (PBS) was attributed to assess the reliability of each interaction, as described earlier (Rain et al., 2001; Formstecher et al., 2005).