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.

To probe the fusion activity of IFITM3-containing pseudoviruses, A549 cells were incubated with IAVpp/IFITM3 (or control viruses lacking IFITM3) at 4ËšC for 30 min, followed by incubation for 2 hours at 37ËšC in either drug-free medium or medium supplemented with AmphoB (which rescues IAV fusion in A549-IFITM3-imTFP1 cells, Fig 4A and [35] ). Compared to the control IAVpp, fusion of IAVpp containing IFITM3 was potently inhibited (p<0.001, Fig 8B) . Interestingly, AmphoB rescued IAVpp/IFITM3 fusion (Fig 8B) , suggesting a direct effect of this antibiotic on IFITM3 or the viral membrane that is independent of cellular processes, including endocytic transport. The diminished ability of IAVpp produced in the presence of IFITM3 to fuse with target cells was not caused by IFITM3-containing extracellular vesicles present in the viral preparations, as have been suggested in [59] . Viral fusion Further evidence supporting the proximity-based antiviral activity of IFITM3 was obtained by measuring LASV GPc-mediated cell-cell fusion. In this model, cell fusion is triggered by exposure to low pH, which bypasses the need for endocytic trafficking that may sort LASVpp away from IFITM3+ compartments. Cos7 cells transiently expressing LASV GPc were brought in contact with 293T cells stably expressing IFITM1, IFITM2 or IFITM3 [22] or, in control experiments, with parental 293T cells. Cos7 and 293T cells were pre-loaded with different cytosolic fluorescent dyes to monitor fusion initiated by an acidic buffer, as previously described [22] . In stark contrast to LASVpp fusion with IFITM3-expressing cells (Fig 6) , GPcmediated cell-cell fusion was markedly inhibited by all three IFTIMs expressed in target cells (Fig 8C) . This result supports the notion that LASV GPc is not inherently resistant to IFITM restriction and that the reason LASV is insensitive to IFITM3 expression is through its usage of trafficking pathways that are distinct from those used by IFITM3.