SARS-CoV-2 ORF3a blocks lysosomal cholesterol egress by disrupting VPS39-regulated NPC2 trafficking and BMP...

Area of Science:

• Virology
• Molecular Biology
• Cell Biology

Background:

• SARS-CoV-2 infection alters host lipid metabolism, impacting cholesterol homeostasis.
• The precise mechanisms by which viral proteins influence cellular cholesterol are not fully understood.

Purpose of the Study:

• To investigate how SARS-CoV-2 infection affects cellular cholesterol transport.
• To identify specific viral proteins involved in cholesterol dysregulation.
• To elucidate the molecular mechanisms underlying SARS-CoV-2-induced cholesterol sequestration.

Main Methods:

• Cell-based assays to monitor cholesterol localization and transport.
• Co-immunoprecipitation and mutagenesis to study protein-protein interactions (ORF3a and VPS39).
• Analysis of lysosomal lipid content (BMP) and protein trafficking (NPC2, CI-MPR).
• Inhibition studies using U18666A to assess the role of lysosomal cholesterol egress in viral infection.

Main Results:

• SARS-CoV-2 infection leads to cholesterol accumulation within lysosomes.
• The viral protein ORF3a is identified as a key driver of lysosomal cholesterol sequestration.
• ORF3a interacts with VPS39, disrupting the trafficking of CI-MPR and NPC2, which impairs cholesterol egress.
• Reduced levels of bis(monoacylglycero)phosphate (BMP), essential for lysosomal cholesterol transport, were observed.
• Inhibiting lysosomal cholesterol egress with U18666A decreased SARS-CoV-2 infection rates.

Conclusions:

• SARS-CoV-2 ORF3a disrupts cellular cholesterol transport by interfering with lysosomal protein trafficking and reducing BMP levels.
• The virus manipulates lysosomal cholesterol homeostasis, potentially to enhance its replication or modulate host cell susceptibility.
• These findings provide novel insights into virus-host interactions and lipid dysregulation during SARS-CoV-2 infection.