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

The following plasmids were previously described: pEYFP-PALS1 (Kamberov et al., 2000), pSecTag2B-Myc-CRB3 (Makarova et al., 2003), pHIT/G and pHIT/gag-pol (Soneoka et al., 1995; Fouchier et al., 1997), and pcDNA-E (wt) (Siu et al., 2008). The pcDNA-Flag-PALS1 construct was generated by cloning the full-length human PALS1 cDNA derived from purified RNA of wd-NHBE cells (Chan et al., 2010). Wd-NHBE cells were lysed and total RNA was extracted using RNeasy Mini kit (Qiagen, Valencia, CA). The total RNA was retro-transcribed to generate single-stranded cDNAs using random hexamer oligonucleotides and the ThermoScript RT-PCR system (Invitrogen, Carlsbad, CA). The cDNAs were further amplified by PCR using specific sense and antisense primers (Supplemental Table 1) for human PALS1 allowing fusion of a Flag tag in 5′ position and containing the BamHI and XhoI restriction sites for subcloning into the pcDNA3.1 vector (Invitrogen). The pcDNA plasmids containing either the wild-type HA-E (wt) or a truncated mutant with a deletion of the last four amino acids (ΔPBM) were prepared using a similar strategy. The pPCRScript-E (codon optimized) plasmid (GeneArt, Regensburg, Germany) was used as template for PCR amplifications with specific sense and antisense primers, and hemagglutinin (HA) tag was introduced in frame upstream of E sequences (Supplemental Table 1).