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 facilitate multi-color live cell imaging of IFITM3 with fluorescently labeled viruses, we replaced the internal EGFP tag with cyan mTFP1 or bright green mNeonGreen protein. We next examined the ability of fluorescent IFITM3 constructs to inhibit IAV fusion using HIV-1 particles pseudotyped with influenza HA and NA proteins from the H1N1 A/WSN/33 strain (designated as IAVpp) and carrying the β-lactamase-Vpr (BlaM-Vpr) chimera, as described in [30] . A549 cells transduced with an empty vector, unlabeled IFITM3, IFITM3-imNG, or IFIT-M3-imTFP1 were inoculated with IAVpp, and the extent of viral fusion was measured after 2 h at 37˚C based on the resulting cytosolic BlaM activity [43, 44] . Compared to the Vector control, IFITM3 severely restricts IAVpp fusion, as shown previously [30] . IFITM3-imNG and IFITM3-imTFP1 proteins were expressed in A549 cells at levels comparable to that of untagged IFITM3, as determined by Western blot analysis of respective cell lysates (Fig 1F) , and also potently inhibited IAVpp fusion (Fig 1E) . Comparable expression of the two fluorescent constructs is further supported by live cell fluorescence microscopy analysis (S1 Fig). Taken together, our results demonstrate both the appropriate subcellular localization and antiviral activity of fluorescently labeled IFITM3 constructs.