Late Endosomal/Lysosomal Cholesterol Accumulation Is a Host Cell-Protective Mechanism Inhibiting Endosomal Escape of Influenza A Virus

Abstract

To transfer the viral genome into the host cell cytoplasm, internalized influenza A virus (IAV) particles depend on the fusion of the IAV envelope with host endosomal membranes. The antiviral host interferon (IFN) response includes the upregulation of interferon-induced transmembrane protein 3 (IFITM3), which inhibits the release of the viral content into the cytosol. Although IFITM3 induction occurs concomitantly with late endosomal/lysosomal (LE/L) cholesterol accumulation, the functional significance of this process is not well understood. Here we report that LE/L cholesterol accumulation itself plays a pivotal role in the early antiviral defense. We demonstrate that inducing LE/L cholesterol accumulation is antiviral in non-IFN-primed cells, restricting incoming IAV particles and impairing mixing of IAV/endosomal membrane lipids. Our results establish a protective function of LE/L cholesterol accumulation and suggest endosomal cholesterol balance as a possible antiviral target.

Targeting the endolysosomal host-SARS-CoV-2 interface by clinically licensed functional inhibitors of acid sphingomyelinase (FIASMA) including the antidepressant fluoxetine

“…Because the pronounced decrease in SARS-CoV-2 titers observed upon treatment with U18666A hinted at an antiviral capacity of dysregulated endolysosomal cholesterol contents in SARS-CoV-2 infection, we next determined whether fluoxetine treatment of Vero and Calu-3 cells was associated with increased endolysosomal cholesterol pools. We acquired z-stacks from confocal microscopy using CD63 as the specific endolysosomal marker protein (20) (12,13). In line with the images, this quantitative colocalization analysis ( Fig 2B), revealed that fluoxetine at a low concentration of 5 µM caused slightly, yet not significantly elevated cholesterol levels in CD63-positive LELs, whereas treatment with 20 µM fluoxetine induced a prominent and significant endolysosomal cholesterol accumulation.…”

“…Because our previously published results had revealed that increased endolysosomal cholesterol levels act as an effective antiviral barrier for enveloped viruses, including influenza viruses (12)(13)(14)19), we also tested the impact of a post-infection treatment with U18666A, a small molecule inhibitor of the endolysosomal cholesterol transporter NPC1(17) on SARS-CoV-2 infection. As shown in Fig.…”

“…A key feature of the late endosomal compartment is the acidic pH. We, therefore, assessed whether fluoxetine treatment impacted endolysosomal pH values in Calu-3 cells, utilizing a quantitative ratiometric fluorescence microscopy assay (12,13). In control cells, the measured pH was at a value of 4.7, whereas cells exposed to 10 µg/mL U18666A displayed a significantly reduced acidification, well in agreement with our previously reported data of the impact of higher U18666A concentrations on pH values in LELs (19).…”

Functional remodeling of lysosomes by type I interferon modifies host defense

“…Although IFN-I-induced lysosomal acidification sensitizes cells to an intracellular bacterial pathogen, our finding that several known ISGs with antiviral properties, such as Ifitm3, Slc15a3 and Cnp , participate in this process leads us to speculate that this mechanism may be protective against viral threats. IFN-I-mediated lysosomal remodeling may also play a role in non-infectious pathologies, such as lysosomal cholesterol accumulation (Kuhnl et al, 2018) and other lysosome-related disorders. It remains unclear whether these effects might be driven by the tonic IFN-I signaling that occurs in many tissues (Schoggins et al, 2014), or instead require pathogenic elevations of IFN-I.…”

In vitro and in vivo RNAi screening with a West Nile virus library encoding artificial microRNAs identifies novel host restriction factors

“…Additionally IFITM3 binds to vesicle-associated membrane proteinassociated protein A (VAPA), a master regulator of endosome-ER lipid transport and alters endosomal cholesterol levels to inhibit virus entry [300]. Further research has shown that cholesterol accumulates in the late endosome upon IFITM3 expression [274,309].…”