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.

Considering that the overwhelming majority of the IAV lipid mixing events occur in IFITM3-containing endosomes (Fig 2) and based upon our previous observation that IFITM3 expression inhibits single IAVpp fusion [30] , we hypothesized that a sufficiently high local IFITM3 concentration is required for restriction of IAV fusion. To test this hypothesis, we synchronized IAVpp entry into A549-IFITM3-imTFP1 cells, as described above, and monitored mCherry/YFP-Vpr-labeled viral particles using three-color live cell imaging. Time series images show the IAVpp entering the IFITM3+ compartment and co-trafficking without undergoing fusion ( Fig 3D) . The fluorescence traces obtained by single virus tracking confirm entry (indicated by the red arrow) into an IFITM3+ endosome. At a later time, the virus-carrying endosome encounters and merges with another IFITM3+ compartment, as indicated by the second red arrow around 36 min (Fig 3E, S5 Movie) . This pseudovirus did not fuse (release mCherry) for as long as the time-lapse imaging was performed. Co-trafficking of IAVpp with an IFITM3+ compartment can be visualized by examining 3D trajectories of the virus and relevant endosomes, which shows IAVpp co-trafficking with the first and then the second endosomal compartments containing IFITM3-imTFP1 ( Fig 3F) . Importantly, analysis of 6309 particles did not reveal a single viral fusion event occurring after extensive IAVpp co-trafficking with an IFITM3+ compartment.