Figure 6. CPZ does not rescue the restriction of IFITMs on cell-cell fusion of viral fusion proteins.

COS7 cells expressing JSRV Env or HAB2 cells expressing IAV HA (images not shown) were loaded with calcein-AM (green) and bound to target cells (unlabeled), either parental 293/LH2SN (Mock) or derivatives expressing IFITM1 (IFITM1). Cells were treated with a pH 5.0 buffer at 4°C for 1 min to create a cold arrested state (CAS), at which aqueous dye had not transferred. Cells were then switched to 37°C or treated with CPZ, cell-cell fusion were monitored under a fluorescence microscope. (A) In mock cells (JSRV Env-mediated fusion): Upon raising temperature from the 4°C of CAS to 37°C, the two target cells of the image became labeled by calcein-AM (arrows), illustrating that fusion was now extensive. Similarly, adding CPZ to cells at CAS also led two target cells receiving calcein-AM, illustrating that fusion was as extensive upon addition of CPZ as upon raising temperature. (B) In IFITM1-expressing cells (JSRV Env-mediated fusion): Raising temperature led to calcein transfer to only one (arrow) of the four target cells. Addition of CPZ did not lead to calcein-AM transfer to any of the three target cells. (C–D) The quantifications of these phenomena are presented in JSRV Env (C) and for IAV HA (D). Similar experimental procedures were applied to IFITM2 and 3-expressing cells, and the data were plotted as show in (C) and (D).