Reovirus infection is regulated by NPC1 and endosomal cholesterol homeostasis

Abstract

Cholesterol homeostasis is required for the replication of many viruses, including Ebola virus, hepatitis C virus, and human immunodeficiency virus-1. Niemann-Pick C1 (NPC1) is an endosomal-lysosomal membrane protein involved in cholesterol trafficking from late endosomes and lysosomes to the endoplasmic reticulum. We identified NPC1 in CRISPR and RNA interference screens as a putative host factor for infection by mammalian orthoreovirus (reovirus). Following internalization via clathrin-mediated endocytosis, the reovirus outer capsid is proteolytically removed, the endosomal membrane is disrupted, and the viral core is released into the cytoplasm where viral transcription, genome replication, and assembly take place. We found that reovirus infection is significantly impaired in cells lacking NPC1, but infection is restored by treatment of cells with hydroxypropyl-β-cyclodextrin, which binds and solubilizes cholesterol. Absence of NPC1 did not dampen infection by infectious subvirion particles, which are reovirus disassembly intermediates that bypass the endocytic pathway for infection of target cells. NPC1 is not required for reovirus attachment to the plasma membrane, internalization into cells, or uncoating within endosomes. Instead, NPC1 is required for delivery of transcriptionally active reovirus core particles into the cytoplasm. These findings suggest that cholesterol homeostasis, ensured by NPC1 transport activity, is required for reovirus penetration into the cytoplasm, pointing to a new function for NPC1 and cholesterol homeostasis in viral infection.

A CRISPR-Cas9 screen reveals a role for WD repeat-containing protein 81 (WDR81) in the entry of late penetrating viruses

“…Our work is congruent with a parallel study, which used CRISPR-Cas9 and RNA interference screens to identify Niemann-Pick C1 (NPC1) as a putative host factor for reovirus entry [ 52 ]. NPC1 mediates the transport of cholesterol from late endosomes and lysosomes to the endoplasmic reticulum [ 64 ].…”

“…Our data indicate that the requirements for the entry of in vitro generated ISVPs may be different. This idea is supported by another study demonstrating that endosomal cholesterol levels impact infection initiated by virions but not infection initiated by ISVPs [ 52 ]. These results counter the generally accepted assumption that once disassembled particles encounter a membrane, the remainder of the entry process (i.e., ISVP-to-ISVP* conversion, pore formation, and core delivery) is similar regardless of whether the infection was initiated by virions or ISVPs [ 53 – 56 ].…”

“…NPC1 mediates the transport of cholesterol from late endosomes and lysosomes to the endoplasmic reticulum [ 64 ]. NPC1-deficient cells are unable to support the delivery of transcriptionally active reovirus core particles to the host cytoplasm, whereas treatment with hydroxypropyl-β-cyclodextrin, which binds and solubilizes cholesterol, restores infection [ 52 ]. Thus, reovirus cannot launch infection from endocytic vesicles that are disrupted by the loss of WDR81 or NPC1.…”

Proteasome activity is required for reovirus entry into cells

“…Successful ISVP-to-ISVP* and membrane penetration allows delivery of the cores into the cytoplasm. Following removal of the altered δ fragment present on the cores, the particles begin transcribing viral RNA using particle-associated transcriptional machinery (49,50). Based on reduced ISVP-to-ISVP* conversion and membrane penetration, we reasoned that viral mRNA transcription following core delivery will be reduced by MG132.…”