Supplementary Information - HAP1 cells and rVSV-GP-EboV susceptibility

Oct 20, 2026

Supplementary information for a study using haploid HAP1 cells and gene-trap mutagenesis to map host factors required for Ebola virus entry. HAP1 near-haploid KBM7 cells are engineered with retroviral vectors expressing OCT4, SOX2, c-MYC and KLF4, then infected with gene-trap virus and expanded for insertion-site sequencing. Resistance to rVSV-GP-EboV identifies enriched mutations across the genome, with downstream validation using clonal lines targeting NPC1, VPS11 and VPS33A, and characterization of cytotoxicity classes and viral infectivity.

LanguageEnglish

CategoryResearch & Report

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Pages19

Reading Time48 min

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Supplemental Figure 1

Generation of HAP1 cells and rVSV-GP-EboV susceptibility

Supplemental Figure 2

Haploid genetic screen outline for Ebola entry host factors

Supplemental Figure 3

Gene classes required for rVSV-GP-EboV induced cytotoxicity

Supplemental Figure 4

Characterization of NPC1, VPS11 and VPS33A gene-trap cell lines

Supplemental Figure 5

Susceptibility of NPC1 and HOPS mutant lines to influenza and VSV

Supplemental Figure 6

NPC1, VPS11, VPS33A loss and rescue by host gene expression

SUPPLEMENTARY INFORMATION doi:10.1038/nature10348 a b cells ( percentage of maximum) c viability (compared to un infected) karyotype HAP1 (27/39) KBM7; non-adherent HAP1; adherent KLF4 SOX2 c-MYC OCT4 KBM7 HAP1 0 102 103 104 105 0 102 103 104 105 fluorescence 1 0.5 0 0 50 100 1 0.5 0 virus concentration (MOI) Supplemental Figure 1. Generation of HAP1 cells and susceptibility to rVSV-GP-EboV. a, Near-haploid KBM7 cells were coinfected with retroviral vectors expressing OCT4/ SOX2/c-MYC and KLF4 and an adherently growing subclone was identified (HAP1 cells) . Karyotypic analysis of HAP1 cells indicates that the majority of cells (27 out of 39 analyzed) is haploid for all chromosomes b, Staining of KBM7 cells and HAP1 cells with pan-hematopoietic markers CD43 and CD45 . Stained cells were examined by flow-cytometry. The unstained control is indicated in grey. c. Susceptibility of HAP1 and KBM7 cells to cell-killing by rVSV-GP-EboV. W W W . N A T U R E . C O M / N A T U R E | 1 RESEARCH SUPPLEMENTARY INFORMATION HAP1 cells mutagenized with gene trap (complexity ~ 100 x 106) unselected control deep sequence insertion sites selected with rVSV-GP-Ebola deep sequence insertion sites isolate clonal derivatives count independent insertions per gene control dataset~430,000 insertions count independent insertions per gene experimental dataset~700 insertions functional studies calculate per gene the significance of gene with multiple insertions Genes in experimental dataset Supplemental Figure 2. Outline of the haploid genetic screen to identify host factors for Ebola virus entry. 100 million early passage HAP1 cells were infected with gene-trap virus and further expanded. A subset of cells was used to characterize the distribution of gene-trap insertion across the human genome. Sequences flanking the gene-traps were amplified, sequenced in parallel and aligned to the human genome. Independent insertion events into annotated genes were counted. 100 million cells were infected with rVSV-GP-EboV virus and resistant clones were pooled and expanded. Most of these cells were used to amplify sequences flanking the gene-traps, sequence the insertion sites in parallel, and align these sequences to the human genome. A subset of the cells was used to obtain NPC1GT and VPS33GTcells through subcloning. Gene disruption events in the selected population were compared to the unselected cell population and genes that were significantly enriched for mutations were identified. 2 | W W W . N AT U R E . C O M / N AT U R E SUPPLEMENTARY INFORMATION RESEARCH Niemann-Pick disease C1 Components of (6 subunit) HOPS complex Cathepsin B Involved in lysosome function/biogenesis or vesicle formation No known function -Log 10 p-value NPC1 VPS33A VPS11 VPS18 SETDB1 VPS41 PIKFYVE CTSB BLOC1S1 GNPTAB VPS39 VPS16 MAGEF1 FIG4 BRI3 BLOC1S2 ARHGAP23 Genes by chromosomal position Supplemental Figure 3. Classes of genes that are required for rVSV-GP-EboV induced cytotoxicity. Significant hits were labeled according to the following annotation: Niemann-Pick disease C1, lysosomal cholesterol transporter (blue), component of 6-subunit HOPS complex (red), cathepsin B (orange), involved in lysosome function /biogenesis (green) or having no known function (purple) . W W W . N A T U R E . C O M / N A T U R E | 3 RESEARCH SUPPLEMENTARY INFORMATION a | |
VPS33A
| | --- | --- | b c NPC1 endogenous locus NPC1 gene trap locus VPS11 endogenous locus VPS11 gene trap locus VPS33 endogenous locus VPS33 gene trap locus WTVPS11GTVPS33GT NPC1GT1 NPC1GT2 Supplemental Figure 4. Identification and characterization of HAP1 cells carrying gene-trap insertions in the NPC1, VPS11 and VPS33A loci. a, Schematic outline of the positions of gene-trap insertions in the corresponding genes. Gene-traps were located in the sense orientation

FAQs

How are HAP1 cells generated for the host-factor screen?

Near-haploid KBM7 cells are coinfected with retroviral vectors expressing OCT4/SOX2/c-MYC and KLF4, followed by identification of an adherently growing subclone designated as HAP1 cells.

What is the core workflow of the haploid genetic screen for Ebola virus entry factors?

Early passage HAP1 cells are infected with a gene-trap virus, expanded, and insertion sites are sequenced and aligned to the human genome. Resistant clones after rVSV-GP-EboV infection are pooled, amplified, sequenced, and compared with an unselected control to find genes enriched for mutations.

Which host genes are highlighted and how are their roles validated?

NPC1, VPS11, and VPS33A carry gene-trap insertions in clonal lines, which are challenged with rVSV-GP-EboV. Loss of these factors confers resistance, and functional restoration can be achieved by exogenous expression of the corresponding host gene.

Oct 20, 2026

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