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.

A549 cells expressing IFITM3-imNG were cultured on collagen-coated 8-chamber coverslips (Lab-Tek, NY #1.5 glass) in Fluorobrite DMEM to~60-80% confluency. The cells were chilled by placing on ice for 10 min, followed by aspirating the media and washing with cold phosphate buffered saline with calcium and magnesium (PBS+/+). Cells were inoculated with a 5-fold dilution of the mCherry-labeled IAV or LASV pseudoviruses in 100 μL of cold LCIB supplemented with 2% FBS, and viruses were allowed to bind to cells by incubation at 4˚C for 90 min. Unbound viruses were removed by washing with cold PBS+/+, and virus entry was initiated by adding 200 μL pre-warmed (37˚C) LCIB. The slides were incubated at 37˚C for varied times followed by fixation with 4% paraformaldehyde (PFA) in PBS-/-for 10 min at 37˚C. For the zero time-point, cells were fixed with PFA immediately following the initial virus binding step at 4˚C. After fixation, PFA was washed away with PBS-/-a few times and cells were imaged.