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 from endosomes 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.

Cholesterol-Dependent Membrane Deformation by Metastable Viral Capsids Facilitates Entry

“…4C). This non-monotonic dependence of GUV rupture on the cholesterol content is qualitatively consistent with previous reports that either depletion [30,31] or overly accumulation [32] of cholesterol blocked the infectivity of non-enveloped viruses. Further, we found that when the ISVP concentration was increased to 300 pM, both T1L and T1L/T3DM2 strains caused the rupture of nearly all GUVs containing 7.7 mol% cholesterol.…”

“…It is known that cholesterol levels play a significant role in viral infections [29] . Depleting cholesterol was shown to block cell entry of non-enveloped viruses such as adenovirus and poliovirus [30,31] , while cholesterol accumulation in endosomes also impairs reovirus escape [32] . It was proposed that membrane cholesterol level might influence either ISVP-to-ISVP* conversion or membrane disruption by reovirus [32] .…”

Cholesterol 25-hydroxylase suppresses avian reovirus replication by its enzymatic product 25-hydroxycholesterol

“…Another example is the Niemann-Pick C1 (NPC1), which is a transmembrane protein that mediates the exportation of cholesterol from late endosomes and lysosomes. According to a recent study, NPC1-mediated cholesterol transport is required for MRV penetration from the late endosome into the cytoplasm, but not for MRV attachment, internalization, or uncoating ( Ortega-Gonzalez et al, 2022 ). In the present study, we preliminarily investigated the potential inhibition mechanism of 25HC against ARV at the viral entry stage.…”

Cholesterol-Dependent Membrane Deformation by Metastable Viral Capsids Facilitates Entry

“…36,37 It remains unclear how cholesterol affects reovirus interaction with membranes, but studies suggested that membrane cholesterol level influences either ISVP-to-ISVP* conversion or membrane disruption by reovirus. 38 In our experiments, we prepared GUVs with varying amounts of cholesterol, from 0% (DOPC: DOPE = 2:1 molar ratio), 7.7 mol % (DOPC:DOPE: cholesterol = 8:4:1), 14.3 mol % (DOPC:DOPE: cholesterol = 4:2:1), to 25.0 mol % (DOPC:DOPE: cholesterol = 2:1:1), comparable to the endosomal cholesterol level. 39−41 After adding 10 pM of either T1L or T1L/T3DM2 ISVPs to the GUVs, we observed more ISVPs adhered to the membranes containing cholesterol and formed larger aggregates compared to that on membranes without cholesterol.…”