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.

The time-lapse Z-stack movies are visually inspected as maximum intensity projections, using ImageJ, and the ROI manager tool was used to annotate the single fusion or hemifusion events (observed as color change or DiI intensity increase). The sets of waiting times for hemi/fusion obtained from multiple movies are combined from experiments done on different days, sorted and plotted as cumulative probability curves that show the kinetics of the event. Acquired image series were converted to maximum intensity projections and annotated particles were tracked using either Volocity (GE Healthcare) or ICY image analysis software (icy.bioimageanalysis.org). Fluorescently labeled viral particles were identified using the spot detection algorithm and tracked in 3D to determine fluorescence intensities at every time point. With three-color imaging to track double-labeled viral particles that co-traffic with fluorescentlylabeled IFITM3 compartments, single Z-planes in which the viral particle trafficked were used so that background subtraction could be performed using the ICY spot tracking algorithm. The local background was determined by dilating the identified objects corresponding to viral particles by two pixels. The difference between the integrated intensities of the particle and the dilated surrounding gave the intensity surrounding the particle, from which an average per pixel local background was calculated. This was used to obtain the background-corrected intensity of the particle at every time point, which is plotted in the time traces as shown in the figures.