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 evaluate whether lipid mixing between IAV and IFITM3-containing compartments occurs, AF647 and SP-DiI 18 labeled viruses were incubated with A549 cells expressing IFIT-M3-imNG, and virus entry/fusion monitored by three-color live cell microscopy ( Fig 2D) . As expected, fluorescent IFITM3 primarily localized to endosomes that exhibited retrograde and anterograde movement in living cells. The dynamics of IFITM3-imTFP1 intracellular transport is illustrated by S2 Movie. Snapshots of a single IAV show the hemifusion event that occurs within an IFITM3-imNG endosome (IFITM3), with the onset of SP-DiI 18 dequenching detected at around 32 min (Fig 2E, S3 Movie) . The overall kinetics of onset of lipid mixing was not affected by IFITM3 expression (S2A Fig). Fluorescence traces with levels of fluorescence intensity from IFITM3 (blue), SP-Dil 18 (green), and AF647 (red) are shown with the time to SP-DiI dequenching (Δt) and dequenching ratio of the final and initial mean intensities ( Fig 2F) . It is clear that IAV HA-mediated lipid mixing is not inhibited by accumulation of IFITM3 in endosomes.