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 further study the functional consequences of E protein expression on PALS1-dependent TJ formation, we measured the establishment of transepithelial electrical resistance (TER) in confluent monolayers of MDCKII cells grown on membrane filters, using a calcium-switch assay. MDCKII eGFP-PALS1 control cells could rapidly form TJ (Figure 6), as indicated by a maximum TER value of ∼400 Ω/cm2 reached at 2 h post-calcium switch in the experiment shown. A similar time course of TJ formation was also recorded with MDCKII cells expressing eGFP-PALS1 together with HcRed fluorescent proteins (data not shown). At later time points, TJ were gradually loosing strength, with lower TER values of 100 Ω/cm2 measured after 17 h post-calcium switch, as previously reported in two different studies (Straight et al., 2004; Latorre et al., 2005). Interestingly, ectopic expression of HA-E protein led to a significant delay in TJ formation, which started only 4 h post-calcium switch, with TER values gradually raising to attain a maximum of 700 Ω/cm2 recorded at 17 h post-calcium switch (Figure 6). Conversely, monolayer cells of the MDCKII eGFP-PALS1 expressing HA-E (ΔPBM) reached maximum TER 2 h post-calcium switch with a maximum of 700 Ω/cm2 (Figure 6). Interestingly, two opposite effects were observed for cells expressing E (wt) and E (ΔPBM) when compared with control cells. Whereas E (wt) expressing cells presented a significant delay in establishment of TER, E (ΔPBM) cells could reach higher TER values more rapidly than control cells. These data suggest that both expression of E (wt) and E (ΔPBM) affect the kinetics of TJ formation and that the DLLV C-terminal motif of E is responsible for the delay in TJ establishment in MDCKII epithelial cells. A similar pattern was observed for clones of MDCKII cells expressing E (wt) or E (ΔPBM) on an endogenous PALS1 background (Supplemental Figure 4).