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. 2011 Aug 24;477(7364):340-3.
doi: 10.1038/nature10348.

Ebola Virus Entry Requires the Cholesterol Transporter Niemann-Pick C1

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Free PMC article

Ebola Virus Entry Requires the Cholesterol Transporter Niemann-Pick C1

Jan E Carette et al. Nature. .
Free PMC article

Abstract

Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann-Pick C1 (NPC1). Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann-Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.

Conflict of interest statement

Competing financial interests

J.E.C., M.R., S.P.W., K.C. and T.R.B. have filed a patent on filovirus host factors identified in this study and T.R.B. is a co-founder of Haplogen, an early-stage company involved in haploid genetic approaches.

Figures

Figure 1
Figure 1. A haploid genetic screen identifies the HOPS complex and NPC1 as host factors for filovirus entry
a, Genes enriched for gene-trap insertions in the rVSV-GP-EboV-selected cell population compared to unselected control cells. Circles represent genes and their size corresponds to the number of independent insertions identified in the rVSV-GP-EboV selected population. Genes are ranked on the X-axis based on chromosomal position. b, RT-PCR analysis of the expression levels of NPC1, VPS33A and VPS11 in mutant clones. c, Infectivity of VSV pseudotyped with the indicated filovirus glycoproteins. Means ± standard deviation (SD) (n=3) are shown. EboV, Ebola virus (Zaire), MarV, Marburg virus. *below detection limit. d, HAP1 clones were infected with viruses including recombinant VSV viruses carrying rabies or Borna disease virus glycoproteins (rVSV-G-RABV and rVSV-GP-BDV) and stained with crystal violet.
Figure 2
Figure 2. Viral infection mediated by filovirus glycoproteins requires NPC1 but not NPC2
a, Primary skin fibroblasts from a healthy individual and patients carrying homozygous mutations in NPC1 or NPC2 were stained with filipin, or challenged with rVSV-G or rVSV-GP-EboV. Filipin-stained (black) and infected cells (green) were visualized by fluorescence microscopy. Filipin-stained images were inverted for clarity. Blue indicates Hoechst nuclear counterstain. b, Infectivity of VSV pseudotyped with the indicated viral glycoproteins in control and Niemann-Pick fibroblasts. *below detection limit. c, NPC1 patient fibroblasts expressing empty vector or human NPC1 were stained with filipin or challenged with rVSV-GP-EboV. d, Infectivity of rVSV-G and rVSV-GP-EboV in Vero cells preincubated for 30 minutes with the indicated concentrations of U18666A. Scale bars, 200 μm (a, c). Means ±SD (n=3 to 6) are shown (b, d).
Figure 3
Figure 3. Ebola virus entry is arrested at a late step in cells deficient for the HOPS complex and NPC1
a, Viral particles attach and internalize into HOPS-and NPC1-deficient cells. Indicated HAP1 clones were infected with Alexa 647-labeled rVSV-GP-EboV (blue) at 4°C. Non-internalized, bound viral particles (blue) were also stained with a GP-specific antibody (green) and the plasma membrane with Alexa 594-wheat germ agglutinin (red) (top panels). To assess viral internalization, cells were warmed to 37°C (bottom panels). Internalized viral particles (blue punctae) are resistant to acid-stripping and inaccessible to a GP antibody. b, Cells were inoculated with rVSV-GP-EboV and examined by transmission electron microscopy. Representative images of early entry steps are shown. c, In vitro-cleaved rVSV-GP-EboV cannot bypass the infection block observed in VPS11GT, VPS33AGT and NPC1GT cells. Infectivity of thermolysin-cleaved rVSV-GP-EboV in the indicated HAP1 clones. *below the limit of detection. d, Viral escape into the cytoplasm is blocked in HOPS complex- and NPC1-deficient cells. Wild type HAP1 cells treated with U18666A (10 μg/ml), and the indicated mutant clones were infected with VSV or rVSV-GP-EboV virus for 3h and processed for VSV M staining (red). Punctuate staining is indicated by arrows. e, Electron micrographs of rVSV-GP-EboV-infected VPS33A- and NPC1-deficient HAP1 cells and NPC1-deficient fibroblasts showing agglomerations of bullet-shaped VSV particles in vesicular compartments. All images were taken at 3 h post-inoculation. Asterisks highlight rVSV-GP-EboV particles in cross-section.
Figure 4
Figure 4. NPC1 function is required for infection by authentic Ebola and Marburg viruses
a, NPC1 patient fibroblasts were exposed to EboV or MarV at a multiplicity of infection (MOI) of 0.1. Supernatants were harvested and yields of infectious virus were measured. *below detection limit. b, Vero cells treated with DMSO or U18666A (20 μM) were infected with EboV or MarV at an MOI of 0.1 and yields of infectious virus were measured. c, Human peripheral blood monocyte-derived dendritic cells (DC) and umbilical-vein endothelial cells (HUVEC) were infected in the presence or absence of U18666A at an MOI of 3 and the percentage of infected cells was determined by immunostaining. d, HUVEC were transduced with lentiviral vectors expressing a non-targeting shRNA (Ctrl) or an shRNA targeting NPC1, infected with EboV or MarV at an MOI of 3 and the percentage of infected cells was determined. Representative images of cells are also shown: green, viral antigen; blue, nuclear counterstain. For panels a–d, Means ±SD are shown (n=2 to 3). In panels a–b, error bars are not visible because they are within the symbols. For panels c–d, ** p-value < 0.01, *** p-value < 0.001. e, Survival of NPC1+/+ and NPC1−/+ mice (n=10 for each group) inoculated i.p. with ~1000 pfu of mouse-adapted EboV or MarV. f, A proposed hypothetical model for the roles of CatB, the HOPS complex, and NPC1 in Ebola virus entry.

Comment in

  • Achilles heel of Ebola viral entry.
    Flemming A. Flemming A. Nat Rev Drug Discov. 2011 Sep 30;10(10):731. doi: 10.1038/nrd3568. Nat Rev Drug Discov. 2011. PMID: 21959282 No abstract available.

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