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 mechanism by which IFITM3 impedes IAV infection was divergent in previous studies, and inhibition was mostly described upon IFITM3 over-expression [37, 38, 51] . It was demonstrated that an IFITM3 knockdown in A549 cells increases infection rates [37] . To determine whether endogenous IFITM3 is relevant for IAV infection in A549 cells under the experimental conditions of our study, we performed IAV infection in A549 IFITM3 knock-down cells ( Figure S1A ,B). The IFITM3 expression was reduced by a factor of 10 (on the mRNA level) in these cells ( Figure S1C ). The IAV infection rate was significantly increased in the knock-down situation compared to the A549 wildtype cells ( Figure 2A ). The increase in the IFITM3 signal intensity in A549 cells upon IAV infection ( Figure 1A ) and interferon treatment ( Figure 1B ) could be caused either by higher expression levels or by IFITM3 clustering, yielding Viruses 2019, 11, 548 7 of 18 a higher density of the signal. To distinguish between these possibilities, we determined the IFITM3 abundance by an immunoblot analysis of the A549 cells at different time points after IAV infection or interferon treatment, respectively. No significant increase in the IFITM3 expression was observed during the first 6 h of IAV infection ( Figure 2B) , indicating that the signal changes observed by fluorescence microscopy (examples are shown in Figure 3 ) during this period were caused by the re-localization of constitutively expressed IFITM3 rather than by an induced IFITM3 expression. A strong increase in the IFITM3 expression levels was observed at late time points (24 h p.i.; Figure S2B ), in accordance with previous studies [32] . An increased IFITM3 expression was also observed following the interferon treatment, but starting already at 6 h after the interferon addition ( Figure S2 ), as previously reported [32] . Accordingly, the immunofluorescence phenotype after the interferon treatment ( Figure 1B) is likely to be caused by an increased expression rather than by clustering. The increase in the IFITM3 signal intensity in A549 cells upon IAV infection ( Figure 1A ) and interferon treatment ( Figure 1B ) could be caused either by higher expression levels or by IFITM3 clustering, yielding a higher density of the signal. To distinguish between these possibilities, we determined the IFITM3 abundance by an immunoblot analysis of the A549 cells at different time points after IAV infection or interferon treatment, respectively. No significant increase in the IFITM3 expression was observed during the first 6 h of IAV infection ( Figure 2B) , indicating that the signal changes observed by fluorescence microscopy (examples are shown in Figure 3 ) during this period were caused by the re-localization of constitutively expressed IFITM3 rather than by an induced IFITM3 expression. A strong increase in the IFITM3 expression levels was observed at late time points (24 h p.i.; Figure S2B ), in accordance with previous studies [32] . An increased IFITM3 expression was also observed following the interferon treatment, but starting already at 6 h after the interferon addition ( Figure S2 ), as previously reported [32] . Accordingly, the immunofluorescence phenotype after the interferon treatment ( Figure 1B) is likely to be caused by an increased expression rather than by clustering.