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 visualize single IAV fusion in A549 cells, we pseudotyped the HIV-1 core with H1N1 HA and NA glycoproteins, as previously described [30] . IAVpp was labeled with a bi-functional mCherry-2xCL-YFP-Vpr construct, with a 2xCL tandem cleavage site for the viral protease that is cleaved during virus maturation, generating a free mCherry and a core-associated YFP-Vpr [50] . Virus fusion is detected based upon the release of mCherry into the cytoplasm through a fusion pore, while the YFP-Vpr marker remains in the viral core ( Fig 3A) . Labeled IAVpp were bound to A549 Vector cells in the cold by spinoculation (see Methods). Virus fusion was synchronously initiated by adding pre-warmed LCIB and visualized by two-color live cell microscopy for 2 hours with mCherry and YFP signals acquired every 6 seconds. Single IAVpp entered and fused with A549 cells, in agreement with our published data [30] . Time series images show the initial trafficking of a representative mCherry/YFP-Vpr labeled virus until fusion occurs, as evidenced by the loss of mCherry fluorescence, while the YFP-Vpr signal remains relatively constant (Fig 3B and 3C , S4 Movie).