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.

We next probed the ability of single IAVpp to fuse with IFITM3+ compartments under conditions that rescue bulk IAVpp fusion. A549-IFITM3-imTFP1 cells were infected with IAVpp labeled with mCherry-2xCL-YFP-Vpr, as above, in the presence of 1 μM AmphoB. As seen with a bulk fusion assay (Fig 4A) , single IAVpp fuses with IFITM3+ endosomes in the are marked with an asterisk and an arrow, respectively. (See S6 Movie). (I) 3D trajectories of IAVpp and an endosome shown in panel G confirm that, prior to fusion (yellow trace), the virus-carrying endosome only transiently encounters the IFITM3 + endosome (blue). Neither before (dark yellow trace) nor after fusion (green trace) of the particle co-trafficking with the IFITM3+ endosome was observed. (J) Single IAVpp fusion efficiency in A459 Vector and IFITM3-imTFP1 (IFITM3+) cells. presence of AmphoB. Representative single virus images show the entry and subsequent cotrafficking of an IAV particle with an IFITM3+ compartment and fusion within the compartment, as indicated by the sudden loss of mCherry (Fig 4B and 4C , S8 Movie). Of the 23 total fusion events that occur in IFITM3-imTFP1 cells treated with AmphoB, 7 particles co-traffic and fuse with IFITM3+ compartments (Fig 4G) . In contrast to IFITM3-imTFP1 expressing cells in the presence of AmphoB, IAVpp exclusively fused at sites devoid of the mutant IFITM3 in 2M-IFITM3-imTFP1 cells (Fig 4D and 4E , S9 Movie). None of the IAVpp fusion events of the total 4442 particles annotated in 2M-IFITM3 cells co-traffic with IFITM3+ compartments. Fig 4D and 4E illustrates this phenomenon, whereby an IAVpp particle fuses within a 2M-IFITM3-imTFP1 cell but does not co-traffic with appreciable local 2M-imTFP1 maxima. These results suggest that loss of antiviral activity of the F75/78A IFITM3 mutant may be due to its altered subcellular distribution that prevents co-trafficking with IAV. This is in contrast to AmphoB, which renders wild-type IFITM3-imTFP1 inactive without affecting its trafficking pathways. Of note, both conditions that rescued the IAVpp fusion delayed the fusion kinetics relative to untreated cells expressing IFITM3-imTFP1 (S5 Fig), indicating a global effect on the rate of virus endocytosis and entry into permissive compartments. Together, the above results support the notion that IFITM3 inhibits IAV fusion through a proximity-based mechanism-by co-trafficking with the virus and accumulating in compartments that are otherwise permissive for IAV fusion.