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.

We obtained HEK 293T/17, Cos7 and human lung epithelial A549 cells from ATCC (Manassas, VA). TZM-bl cells were obtained from NIH AIDS Research and Reference Reagent Program. 293T cells stably expressing IFITM1, IFITM2 or IFITM3 were a gift from Dr. Shan-Lu Liu, Ohio State University [22] ). Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Cellgro, Mediatech, Masassas, VA) containing 10% heat-inactivated Fetal Bovine Serum (Hyclone Laboratories, Logan, UT or Atlanta Biologicals, Flowery Branch, GA) and 1% penicillin/streptomycin from Gemini Bio-products (West Sacramento, CA). For HEK 293T/ 17 cells the growth medium was supplemented with 0.5 mg/ml G418 (Genesee Scientific, San Diego, CA). DMEM without phenol red was purchased from Life Technologies (Grand Island, NY). Live Cell Imaging Buffer (LCIB) and Fluorobrite TM DMEM were purchased from Life Technologies (Grand Island, NY). Stable cell lines expressing fluorescently-tagged IFITM3 used for imaging analysis were obtained by transducing with VSV-G pseudotyped viruses encoding wild-type or the 2M mutant (F75/78A) IFITM3 [21] or with the Vector pQCXIN (Clontech) and selecting with 1 mg/ml G418. Following selection, cells were maintained in 0.5 mg/ml G418. Stable cell lines expressing unlabeled and fluorescently-tagged IFITM3 used for bulk fusion assays were obtained by transducing with VSV-G pseudotyped viruses encoding wild-type IFITM3 with the Vector pQXCIP (Clontech) and selecting with 1.5 μg/ml puromycin.