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 produce MDCKII stable cell lines, early passage MDCKII cells were seeded into six-well plates and grown to 30–40% confluence before infection by VSV-Gpp-eGFP-C1 and VSV-Gpp-eGFP-PALS1. Three days after infection, cells were replated into culture media supplemented by 600 μg/ml G418 (Sigma-Aldrich) for selection. Cell culture medium was changed every 2–3 d. After 10–12 d of antibiotic selection, the surviving clones were propagated in 25-cm2 flasks, and expression of eGFP and eGFP-PALS1 were verified by fluorescence microscopy (data not shown). Subsequently, the MDCKII-eGFP-PALS1 cell line was further infected with an additional retroviral vector to produce monoclonal cell lines stably expressing either HA-E (wt), HA-E (ΔPBM), or HcRed (control). Cells were maintained in medium supplemented with both 600 μg/ml G418 and 200 μg/ml Hygromycin B (Invitrogen). Expression of EGFP-PALS1, HA-E (wt), HA-E (ΔPBM), and HcRed fluorescent proteins were verified by flow cytometry and immunoblotting (Supplemental Figure S2). Alternatively, MDCKII cells were also infected with retroviral vectors to produce monoclonal cell lines stably expressing either HA-E (wt), HA-E (ΔPBM), or HcRed and endogenous PALS1 (Supplemental Figures S3 and S4).