Amphotericin B Increases Influenza A Virus Infection by Preventing IFITM3-Mediated Restriction

Lin, Tsai-Yu; Chin, Christopher R.; Everitt, Aaron R.; Clare, Simon; Perreira, Jill M.; Savidis, George; Aker, Aaron M.; John, Sinu P.; Sarlah, David; Carreira, Erick M.; Elledge, Stephen J.; Kellam, Paul; Brass, Abraham L.

IFITM3's mechanism of restriction is not fully understood. Given that AmphoB reverses IFITM3's inhibition of viral replication, we set about using it as a molecular probe to investigate IFITM3's actions. IAV fusion requires endosomal acidification, and IFITM3 both blocks IAV fusion and increases intracellular acidity by expanding the late endosomal and lysosomal compartments (Feeley et al., 2011) . Therefore, IFITM3 might inhibit IAV by altering endosomal pH. To test this idea, HeLa-vector and HeLa-IFITM3 cells were stably transduced with a RAB7red fluorescent protein (RAB7-RFP) to identify their late endosomes and lysosomes. The cell lines were then incubated with a mixture of two dextrans, conjugated to either a pH-sensitive or pH-insensitive fluor, in the presence or absence of AmphoB. Calculating the ratio of these two signals allowed us to determine that IFITM3 increased the acidity in the RAB7 compartments ( Figure S2A ). In addition, AmphoB modestly decreased the relative acidity of both the vector and IFITM3 cell lines. We next evaluated the consequences of AmphoB's altering pH on acid-induced HA activation. At the indicated times after the initiation of a synchronized infection, we immunostained for the presence of HA that has undergone an acid-induced conformational change required for fusion ( Figure S2B ). These experi-ments showed more acid-induced HA signal in the IFITM3 cells, consistent with their higher acidity. However, we observed no appreciable change in the levels of acid-induced HA upon AmphoB treatment of either cell line. Along with the dextran and NP translocation studies, these results suggest that AmphoB did not prevent IFITM3-mediated restriction by decreasing endosomal acidity. These studies also showed that the levels and location of IFITM3 were not altered by AmphoB treatment with or without viral infection.