Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis

Area of Science:

• Virology
• Cell Biology
• Biochemistry

Background:

• Hepatitis C virus (HCV) infection induces extensive intracellular membrane rearrangements, forming a membranous web (MW) with double-membrane vesicles (DMVs) crucial for RNA replication.
• Cholesterol enrichment in DMVs is observed, but its precise sourcing and recruitment mechanisms to the viral replication organelle remain incompletely understood.

Purpose of the Study:

• To investigate the role of specific lipid transfer proteins (LTPs) in cholesterol recruitment to the HCV replication organelle.
• To identify novel HCV dependency factors involved in membrane trafficking and lipid metabolism.

Main Methods:

• RNA interference (RNAi)-mediated knockdown of selected LTPs, including STARD3, OSBPL1A/B, and NPC1.
• Focus on NPC1: knockdown and pharmacological inhibition.
• Analysis of cholesterol localization, NS5A (viral replicase factor) localization, and MW integrity in HCV-infected cells.

Main Results:

• RNAi screening identified STARD3, OSBPL1A/B, and NPC1 as novel HCV dependency factors, localized to late endosome/lysosome membranes.
• NPC1 knockdown or inhibition led to cholesterol accumulation in lysosomes and reduced cholesterol at NS5A-positive sites.
• Impaired NPC1-dependent cholesterol transport disrupted MW integrity and reduced HCV RNA replication, while not affecting dengue virus replication.

Conclusions:

• HCV usurps LTPs, particularly NPC1 at ER-late endosome/lysosome contact sites, to facilitate cholesterol transport to the replication organelle.
• Cholesterol is essential for the structural integrity and functionality of the membranous web (MW) in HCV replication.
• These findings reveal a critical role for endosomal-lysosomal lipid transport in HCV pathogenesis and may offer insights into replication organelle biogenesis for other positive-strand RNA viruses.