The Niemann Pick C1 (NPC1) protein is an intracellular cholesterol transporter located in the late endosome/lysosome (LE/Ly) that is involved in the mobilization of endocytosed cholesterol. Loss-of-function mutations in the NPC1 gene lead to the accumulation of cholesterol and sphingolipids in LE/Ly, resulting in severe fatal NPC1 disease. Cellular alterations associated with NPC1 inactivation affect the integrity of lipid rafts and the endocytic pathway. Because the angiotensin-converting enzyme 2 (ACE2) and type 2 serine transmembrane protease (TMPRSS2), interactors of the SARS-CoV-2 Spike protein also localize to lipid rafts, we sought to investigate the hypothesis that NPC1 inactivation would generate an intrinsically unfavorable barrier to SARS-CoV-2 entry. In this study, we show that inhibition of the cholesterol transporter activity of NPC1 in cells that express both ACE2 and TMPRSS2, considerably reduces SARS-CoV-2 infectivity, evaluated as early as 4 h post-infection. Mechanistically, treatment with NPC1-specific inhibitor U18666A relocalizes ACE2 from the plasma membrane to the autophagosomal/lysosomal compartment, thereby reducing SARS-CoV-2 entry into treated cells. Reduction of viral entry was observed for both fully infectious SARS-CoV-2 virus and with a pseudotyped VSV-Spike-GFP virus. For instance, U18666A-treated Caco-2 cells infected with the pseudotyped VSV-Spike-GFP showed a > threefold and > 40-fold reduction in virus titer when infectivity was measured at 4 h or 24 h post-infection, respectively. A similar effect was observed in CRISP/R-Cas9-edited Caco-2 cells, which were even more resistant to SARS-CoV-2 infection as indicated by a 97% reduction of viral titers. Overall, this study provides compelling evidence that the inhibition of NPC1 cholesterol transporter activity generates a cellular environment that hinders SARS-CoV-2 entry. ACE2 depletion from the plasma membrane appears to play a major role as limiting factor for viral entry.

The inactivation of the Niemann Pick C1 cholesterol transporter restricts SARS-CoV-2 entry into host cells by decreasing ACE2 abundance at the plasma membrane / LA ROSA, Piergiorgio; Tiberi, Jessica; Palermo, Enrico; Stefanelli, Roberta; Maria Luigia Tiano, Sofia; Canterini, Sonia; Cortese, Mirko; Hiscott, John; Fiorenza, Maria Teresa. - In: CELL & BIOSCIENCE. - ISSN 2045-3701. - 14:1(2024). [10.1186/s13578-024-01331-4]

The inactivation of the Niemann Pick C1 cholesterol transporter restricts SARS-CoV-2 entry into host cells by decreasing ACE2 abundance at the plasma membrane.

Piergiorgio La Rosa;Jessica Tiberi;Enrico Palermo;Roberta Stefanelli;Sonia Canterini;Maria Teresa Fiorenza
2024

Abstract

The Niemann Pick C1 (NPC1) protein is an intracellular cholesterol transporter located in the late endosome/lysosome (LE/Ly) that is involved in the mobilization of endocytosed cholesterol. Loss-of-function mutations in the NPC1 gene lead to the accumulation of cholesterol and sphingolipids in LE/Ly, resulting in severe fatal NPC1 disease. Cellular alterations associated with NPC1 inactivation affect the integrity of lipid rafts and the endocytic pathway. Because the angiotensin-converting enzyme 2 (ACE2) and type 2 serine transmembrane protease (TMPRSS2), interactors of the SARS-CoV-2 Spike protein also localize to lipid rafts, we sought to investigate the hypothesis that NPC1 inactivation would generate an intrinsically unfavorable barrier to SARS-CoV-2 entry. In this study, we show that inhibition of the cholesterol transporter activity of NPC1 in cells that express both ACE2 and TMPRSS2, considerably reduces SARS-CoV-2 infectivity, evaluated as early as 4 h post-infection. Mechanistically, treatment with NPC1-specific inhibitor U18666A relocalizes ACE2 from the plasma membrane to the autophagosomal/lysosomal compartment, thereby reducing SARS-CoV-2 entry into treated cells. Reduction of viral entry was observed for both fully infectious SARS-CoV-2 virus and with a pseudotyped VSV-Spike-GFP virus. For instance, U18666A-treated Caco-2 cells infected with the pseudotyped VSV-Spike-GFP showed a > threefold and > 40-fold reduction in virus titer when infectivity was measured at 4 h or 24 h post-infection, respectively. A similar effect was observed in CRISP/R-Cas9-edited Caco-2 cells, which were even more resistant to SARS-CoV-2 infection as indicated by a 97% reduction of viral titers. Overall, this study provides compelling evidence that the inhibition of NPC1 cholesterol transporter activity generates a cellular environment that hinders SARS-CoV-2 entry. ACE2 depletion from the plasma membrane appears to play a major role as limiting factor for viral entry.
2024
spike-ACE2 interaction, ACE2, virus entry, lipid dyshomeostasis
01 Pubblicazione su rivista::01a Articolo in rivista
The inactivation of the Niemann Pick C1 cholesterol transporter restricts SARS-CoV-2 entry into host cells by decreasing ACE2 abundance at the plasma membrane / LA ROSA, Piergiorgio; Tiberi, Jessica; Palermo, Enrico; Stefanelli, Roberta; Maria Luigia Tiano, Sofia; Canterini, Sonia; Cortese, Mirko; Hiscott, John; Fiorenza, Maria Teresa. - In: CELL & BIOSCIENCE. - ISSN 2045-3701. - 14:1(2024). [10.1186/s13578-024-01331-4]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/1730044
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