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.

Live cell imaging experiments were performed at least 6 times independently, and on average, 1.64% and 1.87% of double-labeled LASVpp particles bound to cells fused in A549 Vector and A549-IFITM3-imTFP1 cells, respectively (p = 0.551) (Fig 6C) . These data confirm previous reports that the expression of IFITM3 does not affect LASV fusion [15, 30] . In addition, the kinetics of LASVpp fusion in control and A549-IFITM3-imTFP1 cells were not significantly different (S6A Fig). LASVpp fusion kinetics were the same regardless of IFIT-M3-imTFP1 expression, as observed using the BlaM assay and stopping fusion at varied times by NH 4 Cl addition (S6B Fig). We note that in one or two rare examples, LASVpp fusion appears to occur in an endosome containing detectable IFITM3-imTFP1 signal. Representative images and fluorescence traces show co-trafficking of a LASV particle within an IFITM3+ endosome until fusion occurs around 14 min post-infection (Fig 6E and 6F and Inset, S12 Movie). This unique event is atypical of the majority of tracked particles due to several reasons: (1) there is an apparent colocalization with IFITM3+ beginning at time 0; and (2) LASVpp rarely fuse as early as 14 min postinfection. Most importantly, the fusion event in Fig 6E and 6F appears to represent transient fusion pore opening, as indicated by the sudden re-quenching of YFP, or re-acidification of the virus interior following pore closure (Fig 6E-6G) . We report this instance to illustrate that, while LASVpp typically avoids IFITM3+ endosomes, miniscule levels of fusion may occur within IFITM3+ compartments. Overall, LASVpp exhibit a strong tendency to bypass IFITM3+ endosomes and this important feature likely represents the mechanism by which this virus escapes restriction.