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.

(legend continued on next page) cell line, A549, and antibody against VAPA as the seminal report, we estimate that our overexpression of VAPA met or surpassed the published levels. Modulation of cholesterol levels had no appreciable effect on IFITM3-mediated restriction, suggesting that cholesterol mislocalization does not contribute to VAPA's antagonism of IFITM3. Furthermore, cells expressing either of three IFITM3 proteins containing mutations that span IM2, the reported interaction domain of IFITM3 with VAPA, exhibited WT levels of restriction and intracellular cholesterol. These results suggest that a direct interaction between VAPA and IFITM3 does not underlie the subtle effect of VAPA on restriction. We mention, however, that our alanine scanning strategy may have been too conservative because IM2 is a hydrophobic domain. We also addressed the role of oleic acid in overcoming IFITMmediated inhibition of cell-to-cell fusion. Specifically, we found that oleic acid cannot overcome IFITM1-or IFITM3-mediated restriction of IAV in vitro under the conditions tested. Furthermore, oleic acid did not overcome IFITM1-mediated inhibition of cell-to-cell fusion assays by IAV particles (data not shown). Therefore, while these recent reports have improved our understanding of IFITM3-mediated restriction, we conclude that additional mechanistic studies are required.