Interferon-induced transmembrane protein 3 blocks fusion of sensitive but not resistant viruses by partitioning into virus-carrying endosomes

Suddala, Krishna C.; Lee, Christine C.; Meraner, Paul; Marin, Mariana; Markosyan, Ruben M.; Desai, Tanay M.; Cohen, Fredric S.; Brass, Abraham L.; Melikyan, Gregory B.

The effector Cos7 cells were transfected with the Lassa virus GPc expression vector. Briefly, cells were grown on 35 mm culture dishes to~60% confluency and transfected with 4 μg GPc expression vector using a calcium-phosphate protocol [22] . After 48 hours following transfection, cells were loaded with 1.3 μM of the green cytoplasmic Calcein-AM dye (Invitrogen). In parallel, 293T cells or their derivatives stably expressing human IFITM1, IFITM2 or IFITM3 [22] were labeled with 30 μM of the blue cytoplasmic dye CMAC (Invitrogen). Effector and target cells were washed, detached from the culture dishes using a non-enzymatic solution, resuspended in PBS++, mixed at a 1:1 ratio and co-plated onto 8-well chamber slides. After incubating for 30 min at room temperature, cells were exposed to a pH 5.0 buffer at 37˚C for 20 min, and the resulting cell-cell fusion was measured by visual inspection under a fluorescent microscope, as described in [22] . Ten fields of view each containing 10-12 heterologous cell pairs were examined in each well.