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.

Pseudoviruses were produced by transfecting HEK 293T/17 cells with JetPRIME transfection reagent (Polyplus-transfection, Illkirch-Graffenstaden, France). For all pseudovirus productions the transfection reagent/DNA containing medium was replaced with fresh phenol red-free medium after~14 hrs. Viruses were harvested~48 hrs post-transfection, and cellular debris were removed by centrifuging at 230xg for 10 min. The collected viruses were passed through a 0.45 μm polyethersulfone filter (PES, VWR) to further clear cellular debris and virus aggregates, aliquoted and stored at -80˚C. The infectious titers (~10 6 IU/ml) were determined using serial dilutions of the inoculum in TZM-bl cells using a β-galactosidase assay. To produce the pseudoviruses for co-localization analysis, HEK293T/17 cells were grown to~60-70% confluency in a 6-well culture dish and transfected with 0.8 μg pR8ΔEnv, 0.4 μg pcRev, 0.5 μg HIV-1 Gag-mCherry (Not cleaved by protease) and 0.8 μg GPc-Lassa or 0.4 μg each of WSN HA-and NAexpressing plasmids, respectively. For single viral fusion experiments in live cells, dual-labeled LASVpp was made by transfecting the HEK293T/17 cells grown to~60-70% confluency in a 6-well culture dish using 0.8 μg pR9ΔEnv, 0.2 μg pcRev, 0.2 μg mCherry-2pxCLYFP-Vpr and 1 μg GPc-Lassa plasmids. Similarly, dual-labeled IAVpp were produced using 4 μg pR9ΔEnv, 1 μg pcRev, 1 μg mCherry-2xCL-YFP-Vpr and 2.5 μg each of WSN HA-and NA-expressing plasmids for transfection of~60% confluent cells in a 100 mm dish. The purified viruses were diluted 10-fold in PBS without calcium or magnesium (PBS-/-, Cellgro, Mediatech), bound to poly-L-lysine coated 8-well chamber cover slips (LabTek, MA), and imaged to estimate the colabeling efficiency which was over 90% for all pseudoviruses used for this study.