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.

For the lipid mixing experiments, SP-DiI 18 dequenching curves for the individual viruses were obtained by tracking particles, either using the AF647 channel or the DiI channel. The dequenching ratios and times were manually obtained from time traces that show at least 4-fold increase in SP-DiI 18 intensity. The dequenching ratio was calculated as ratio of the mean SP-DiI 18 intensity before the rise of the signal and the mean intensity after completion of dequenching. The dequenching time was measured as time taken to reach an intensity plateau from the time of initial rise in intensity. The traces showing multi-phase increase in SP-DiI 18 signal in IFITM3 expressing cells were excluded from the calculation of dequenching ratios and times. IAVpp was labeled with mCherry-2xCL-YFP-Vpr and added to A549-IFIT-M3-imTFP1 cells treated with 1 μM AmphoB. Movie depicts a viral particle entering an IFITM3+ endosome at around 33 min (white arrow) and co-trafficking until fusion at 42 min (loss of mCherry signal, white arrow). Movie is slowed to 0.2x speed at 34-46 and 42-44min. Scale bar 2.1 μm. (Related to Fig 4C and 4D ). (AVI) S9 Movie. IAV pseudovirus fusion in a cell expressing inactive IFITM3 mutant. IAVpp was prepared with mCherry-2xCL-YFP-Vpr and added to cells expressing the 2M-IFITM3-imTFP1 mutant. The viral particle does not co-traffic with any local IFITM3-positive maxima, and fuses at around 19 min (white arrow). Movie is slowed to 0.2x speed from 18-21 min. Scale bar 4.1 μm. (Related to Fig 4E and 4F ). (AVI) S10 Movie. LASV pseudovirus fusion in A549 cells. LASVpp was labeled with mCherry-2xCL-YFP-Vpr and added to A549 Vector cells. The particle undergoes YFP quenching at around 32 min (white arrow) and fusion at around 44 min, as indicated by the sudden loss of mCherry and de-quenching of YFP (white arrow). Movie is slowed to 0.2x speed at 32-35 min and 43-45 min. Scale bar 4.0 μm. (Related to Fig 5B and 5C ). (AVI) S11 Movie. LASV pseudovirus fusion in A549 cells expressing IFITM3-imTFP1. LASVpp does not co-traffic with IFITM3-imTFP1-positive endosomes and subsequent viral fusion occurs at sites without any local IFITM3-imTFP1 maxima. YFP quenching occurs at around 14 min (white arrow) and fusion occurs at around 46 min (white arrow), as indicated by the sudden loss of mCherry and dequenching of YFP. Movie is slowed to 0.2x speed at 13-16 min and 45-46 min. Scale bar 3.1 μm. (Related to Fig 6A and 6B ). (AVI) S12 Movie. LASV pseudovirus content release from within an IFITM3 containing endosome. LASVpp co-trafficking with an IFITM3-imTFP1-positive endosome and fusion occurring at 14:49, as indicated by the white arrow. Movie is slowed to 0.2x speed from 14-15 min. Scale bar 1.6 μm. (Related to Fig 6E and 6F ). (AVI)