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 β-lactamase (BlaM) assay for virus-cell fusion were performed as described previously [30, 43] . Briefly, pseudoviruses containing a β-lactamase-Vpr chimera (BlaM-Vpr) were bound to target cells by centrifugation at 4˚C for 30 min at 1550xg. Unbound viruses were removed by washing with DMEM without phenol red supplemented with 20 mM HEPES (GE Healthcare Life Sciences). Fusion was initiated by shifting to 37˚oC for 2 hours, after which cells were placed on ice and loaded with the CCF4-AM substrate (Life Technologies) and incubated overnight at 11˚C. The cytoplasmic BlaM activity (ratio of blue to green fluorescence) was measured using a SpectraMaxi3 fluorescence plate reader (Molecular Devices, Sunnyvale, CA).