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 lipid mixing (hemifusion) experiments, influenza virus surface proteins and membrane were co-labeled with AF647 and with the lipophilic dye SP-DiIC 18 , respectively. Briefly, 100 μg of the purified IAV A/PR/8/34 virus (2 mg/ml, Charles River, CT) was mixed with 50 μM AF647 in 150 mM freshly prepared sodium bicarbonate buffer, pH 9.0. The labeling reaction was allowed to proceed at room temperature with tumbling in the dark for 30 min. Next, 5.8 μL of 1.75 mM SP-DiI 18 was added to this reaction, while gently vortexing and viruses further incubated at room temperature in the dark for 1 hr with shaking. The AF647 was quenched by adding 2 μL of 1 M Tris-buffer, pH 7.0. The labeled viruses were purified from excess dyes on a Nap-5 gel filtration column (GE Healthcare) that was equilibrated with 50 mM HEPES, pH 7.4, 145 mM NaCl at room temperature. The fractions containing labeled viruses were passed through a 0.45 μm filter to remove any large lipid and/or virus aggregates. The purified viruses were bound to poly-L-lysine coverslips and imaged to quantify their colabeling efficiency which was at least 55%, determined as the percentage of AF647 labeled viruses that showed detectable signal (under the high self-quenching concentrations) of SP-DiI 18 . The viruses were aliquoted into tubes, flash-frozen, and stored at -80˚C until use.