ATG16L1 WD domain and linker regulates lipid trafficking to maintain plasma membrane integrity to limit influenza virus infection

The non-canonical functions of autophagy protein ATG16L1 are dependent on a C-terminal WD domain. Recent studies show that the WD domain is required for conjugation of LC3 to single membranes during endocytosis and phagocytosis, where it is thought to promote fusion with lysosomes. Studies in cells lacking the WD domain suggest additional roles in the regulation of cytokine receptor recycling and plasma membrane repair. The WD domain also protects mice against lethal influenza virus <i>in vivo</i>. Here, analysis of mice lacking the WD domain (ΔWD) shows enrichment of cholesterol in brain tissue suggesting a role for the WD domain in cholesterol transport. Brain tissue and cells from ΔWD mice showed reduced cholesterol and phosphatidylserine (PS) in the plasma membrane. Cells from ΔWD mice also showed an intracellular accumulation of cholesterol predominantly in late endosomes. Infection studies using IAV suggest that the loss of cholesterol and PS from the plasma membrane in cells from ΔWD mice results in increased endocytosis and nuclear delivery of IAV, as well as increased <i>Ifnb</i>/<i>Ifnβ</i> and <i>Isg15</i> gene expression. Upregulation of <i>Il6</i>, <i>Ifnb</i> and <i>Isg15</i> mRNA were observed in “ex vivo” precision cut lung slices from ΔWD mice both at rest and in response to IAV infection. Overall, we present evidence that regulation of lipid transport by the WD domain of ATG16L1 may have downstream implications in attenuating viral infection and limiting lethal cytokine signaling. <b>Abbreviations</b>: BMDM: bone marrow-derived macrophages, CASM: conjugation of ATG8 to single membranes, CCD: coil-coil domain, IAV: influenza virus A, IFIT1: interferon-induced protein with tetratricopeptide repeats 1, IFITM3: interferon induced transmembrane protein 3, IFN: interferon, ISG15: ISG15 ubiquitin-like modifier, LANDO: LC3-associated endocytosis, LAP: LC3-associated phagocytosis, LDL: low density lipoprotein, NP: nucleoprotein, PS: phosphatidylserine, WD: WD40-repeat‐containing C‐terminal domain, WT: wild type.