IFITM3 Clusters on Virus Containing Endosomes and Lysosomes Early in the Influenza A Infection of Human Airway Epithelial Cells

Kummer, Susann; Avinoam, Ori; Kräusslich, Hans-Georg

The ring-like appearance of IFITM3 clusters suggested the vesicular recruitment of this protein to IAV-carrying endosomal structures. We therefore analysed whether IFITM3 clusters co-localize with early endosomal (EEA1), late endosomal (Rab7), or lysosomal (Lamp1) marker proteins at early stages of IAV infection in A549 cells ( Figure 6 , Figure S3 ). We observed typical staining for early and late endosomes and for lysosomes with a weak IFITM3 signal in uninfected cells. The IFITM3 signal intensity increased at 1 h and 6 h p.i., as seen in the previous experiments, with some apparent co-localization with the endosomal marker proteins. To determine the degree of co-localization, we calculated the Mander's Correlation Coefficient (MCC) [64] . Only IFITM3 clusters defined as objects with a size of > 10 5 nm 2 were included in this analysis. We observed a highly significant increase of IFITM3 cluster co-localization with early endosomes at 1 h p.i., which was subsequently lost in the course of infection ( Figure 6A ). In contrast, a significant increase of co-localization with the late endosomal marker ( Figure 6B ) and some increased co-localization with the lysosomal marker ( Figure 6C ) was seen at 6 h, but not at 1 h p.i. These results suggest that IFITM3 clustering on IAV carrying endosomes already occurs early after endocytosis, prior to the pH-induced fusion and release from late endosomes, and probably persists as the endosomes mature.