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

To visualize the subcellular localization of endogenous IFITM3, we performed immunofluorescence microscopy of the uninfected and IAV infected A549 cells. The basal IFITM3 levels of the uninfected cells showed weak cytosolic signals upon immunofluorescence staining. Following infection with IAV A/Hong Kong/1/1968 (HK/1/68) for 24 h, most cells exhibited strong and clustered IFITM3 signals in the extranuclear space ( Figure 1A ). The IFITM3 signal increase and clustering correlated with the overall dim NP signals in the extranuclear space and no nuclear NP signals in the respective cells. In contrast, the productively IAV infected A549 cells (exhibiting a strong nuclear NP signal) in the same field of view did not exhibit an increased IFITM3 signal intensity or clustering ( Figure 1A , right panels). Given that we used a relatively high MOI of one and that dim NP signals were generally detected in these cells, these observations strongly argue that IAV infection was abortive in the cells exhibiting an early accumulation of IFITM3. IFITM3 is induced by type 1 interferon [34] . Therefore, we analyzed its distribution upon the treatment of A549 cells with human interferon alpha (hIFNα) for 24 h and compared this to A549 cells which had been pretreated with hIFNα and subsequently infected with IAV ( Figure 1B ). The cells treated for 24 h with hIFNα exhibited an increased IFITM3 signal intensity in the extranuclear IFITM3 is induced by type 1 interferon [34] . Therefore, we analyzed its distribution upon the treatment of A549 cells with human interferon alpha (hIFNα) for 24 h and compared this to A549 cells which had been pretreated with hIFNα and subsequently infected with IAV ( Figure 1B ). The cells treated for 24 h with hIFNα exhibited an increased IFITM3 signal intensity in the extranuclear space, both in the uninfected A549 cells and in the IAV-infected cells with a dim or absent NP signal. Importantly, the productively infected cells exhibiting strong nuclear NP signals again exhibited a weak IFITM3 signal, even after the pretreatment with hIFNα. Comparing the non-treated and hIFNα-pretreated IAV-infected A549 cells lacking a strong nuclear NP signal, we observed an increased IFITM3 signal in both cases, partially colocalizing with vesicular structures. Vesicular localization appeared more obvious in the absence of a hIFNα pretreatment with a less diffuse cytosolic signal. Blocking IFNα with a neutralizing antibody during the IAV infection of A549 cells resulted in increased infection rates, and a concomitant minimal increase of the IFITM3 signal ( Figure 1C ).